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WO2025051758A1 - Utilisation d'intéine divisée pour le traitement d'une maladie associée à scn1a - Google Patents

Utilisation d'intéine divisée pour le traitement d'une maladie associée à scn1a Download PDF

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WO2025051758A1
WO2025051758A1 PCT/EP2024/074640 EP2024074640W WO2025051758A1 WO 2025051758 A1 WO2025051758 A1 WO 2025051758A1 EP 2024074640 W EP2024074640 W EP 2024074640W WO 2025051758 A1 WO2025051758 A1 WO 2025051758A1
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protein
seq
intein
terminal fragment
residue
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Silvia FRUTOS
Gerard CAELLES
Sandra MOTAS
Miquel VILA-PERELLO
Sergio POUS
Belen Garcia
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Splicebio SL
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/40—Systems of functionally co-operating vectors

Definitions

  • the present invention relates to the use of split inteins for expressing Na v l.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.
  • Dravet syndrome 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 Na v l .1.
  • AAVs have been shown to be highly efficient when treating neurological disease.
  • 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.
  • 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.
  • split intein-mediated protein trans-splicing can be used to reconstitute efficiently complex Na v l.l protein encoded by SCN1A gene in a cell.
  • the Na v l.l channel sodium is localized correctly in the cell membrane and is active as a sodium channel.
  • Na v l. l reconstitution by protein trans-splicing is efficient and mediates efficacy in Dravet syndrome mouse model.
  • Na v l. 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 Na v l.
  • SCN1A Sodium channel protein type 1 subunit alpha
  • said N-split intein is a N-Cfa-intein of SEQ ID NO: 1 or any functional variant thereof having at least 90% identity to SEQ ID NO: 1; and said C-split intein is a C-Cfa intein of SEQ ID NO: 2 or any functional variant thereof having at least 90% identity to SEQ ID NO: 2, preferably wherein amino acid residues 20 to 22 of SEQ ID NO: 2 are GEP, more preferably C-Cfa mu t intein of SEQ ID NO: 13 or any functional variant thereof having at least 90% identity to SEQ ID NO: 13.
  • said Na v l. l protein is human Na v l. 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.
  • the N-terminal fragment of Na v l .1 protein ends up to residue 925 and the C-terminal fragment of Na v l.l protein starts from residue 926 respectively
  • the N- terminal fragment of Na v l.l protein ends up to residue 1059 and the C-terminal fragment of Na v l. l protein starts from residue 1060 respectively
  • the N-terminal fragment of Na v l. l protein ends up to residue 1178 and the C-terminal fragment of Na v l. l protein starts from residue 1179 respectively, wherein said residue is numbered according to SEQ ID NO: 15.
  • 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.
  • 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.
  • 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.
  • 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
  • CMV Cy
  • 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.
  • 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.
  • AAV adeno associated viral
  • 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:
  • a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : a C-Cfa intein of SEQ ID NO: 2 or C-Cfa mu t 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 Na v 1.1 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:
  • 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.
  • Figure 1 Splicing efficiency of Na v l.l protein at position 926 monitored by Western blot.
  • Na v l . 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 Na v l . 1 human amino acid sequence (SEQ ID NO: 15).
  • FIG. 2 Splicing efficiency of Na v l.l protein at position 948 monitored by Western blot.
  • Na v l . 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 Na v l . 1 serve as control.
  • Split position is numbered according to Na v l . 1 human amino acid sequence (SEQ ID NO: 15).
  • FIG. 3 Splicing efficiency of Na v l.l protein at position 1060 and 1179 monitored by Western blot.
  • Na v l . 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 and splicing efficiency monitored by Western blotting.
  • HEK293T cells transfected with a full-length Na v l . 1 serve as control.
  • Split position is numbered according to Na v l . 1 human amino acid sequence (SEQ ID NO: 15).
  • FIG. 4 HEK293T cells transfected with SCNIA-intein plasmids comprising Na v l.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 Na v l.l protein. DAPI was used to counterstain nuclei.
  • FIG. 5 Patch Clamp assay on HEK293T cells transfected with SCNIA-intein plasmids comprising Na v l.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-Na v l.l stable cell line were used as controls.
  • Figure 6 Schematic representation of the constructs according to the present disclosure including degron (DD).
  • DD degron
  • 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 Na v l.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 Na v l.l protein in hippocampus (A) and cortex (B) samples of WT mice treated at 4-5 weeks-of-age.
  • 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.
  • Figure 9 Detection of Na v l.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.
  • CNS Central Nervous System
  • Figure 10 Reconstituted Na v l.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.
  • A monoclonal antibody anti -HA tag
  • B monoclonal antibody anti-Na v l.l protein
  • FIG. 11 Co-localization signal of reconstituted Na v l.l protein and endogenous protein in hippocampus sample of WT treated mice.
  • 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.
  • SCNlA N -CfaN or SCNlA c -CfaC mut respectively
  • dual N- and C- AAV9 vectors PTS split site 3, 4.4E+10 vg/mouse
  • Figure 13 Long-term expression of reconstituted Na v l.l protein in CNS.
  • 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 Na v l. l protein expression was sustained 3-mohts post injection in CNS.
  • Figure 14 Efficacy of dual AAV approach in Dravet syndrome mouse model.
  • Saline-injected DS littermate mice were used as controls.
  • Analysis of survival (A) by Logrank test, p 0.0056.
  • the limited cargo capacity of the AAV vectors precludes its use for delivering large genes such as SCN1A gene in gene therapy.
  • the inventors took the advantage of the intrinsic ability of split inteins to mediate protein trans-splicing to reconstitute large Na v l .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 Na v l. 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 Na v l. l protein, fused directly or indirectly via a linker, wherein expression of first and second polynucleotides in a cell generates full length Na v l.
  • the combination of polynucleotides comprises a first polynucleotide encoding a first fusion protein and a second polynucleotide encoding a second fusion protein.
  • first and second polynucleotides according to the present disclosure can be formulated in a single or as separate formulations.
  • nucleic acid sequence may be used interchangeably to refer to any molecule composed of or comprising monomeric nucleotides.
  • a polynucleotide may be a DNA or RNA.
  • peptide oligopeptide
  • polypeptide polypeptide
  • protein protein
  • 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.
  • amino acid and “amino acid residue” are used interchangeably throughout.
  • fusion protein 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.
  • 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.
  • said first fusion protein comprises a N-terminal fragment of Na v l.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 Na v l. l protein, fused directly or indirectly via a linker.
  • linker refers to a chemical group or a molecule linking two adjacent molecules or moieties.
  • 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.
  • a linker is not used.
  • 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.
  • first and second polynucleotides encoding said first and second fusion proteins in a cell generates Na v l.l protein by protein splicing.
  • 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).
  • intein refers to a protein that is capable of ligating the flanking sequences (exteins) into a new protein.
  • 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.
  • 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).
  • N-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.
  • C-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.
  • 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.
  • DriaE DNA polymerase III
  • 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.
  • 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).
  • 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
  • N- and/or C- split inteins according to the present disclosure comprised in the first and second fusion proteins respectively can be engineered N- and/or C- split inteins.
  • Said engineered N- and/or C- split inteins can be engineered by introducing mutations in natural N- and/or C- split intein sequences, in particular to enhance protein splicing activity.
  • the N-split intein and/or C-split intein sequences comprised in the first and second fusion proteins respectively comprise or consist of amino acid sequences selected from any of N- and C-split inteins listed in Table 1 below:
  • Table 1 Examples of pairs of N- and C-split inteins that can be used according to the present disclosure. Amino acids in bold can be replaced by GEP amino acids to improve extein tolerance.
  • the N- and C-split inteins comprised in the first and second fusion proteins respectively according to the present disclosure are N- and C-split inteins comprising or consisting of amino acid sequences of SEQ ID No: 1 (Cfa-N split intein) and SEQ ID No: 2 (Cfa-C-split intein:), SEQ ID No: 3 (Npu-N) and 4 (Npu-C), SEQ ID No: 5 (Cat-N) and 6 (Cat- C), SEQ ID No: 7 (Gp41-N) and 8 (Gp41-C), SEQ ID No: 9 (ConN) and 10 (ConC) or SEQ ID No: 11 (Nrdj 1-N) and 12 (Nrdj 1-C) or any functional variant(s) thereof.
  • variant refers to a polypeptide sequence that is derived from N- and/or C-split inteins as described above and comprises an alteration, i.e., a substitution, insertion, and/or deletion, at one or more positions, but retain the capacity when bound to form a functional enzyme to catalyze a protein splicing reaction that excises the N and C-intein sequences and joins flanking sequences (N- and C-exteins) with a peptide bond.
  • the variant may be obtained by various techniques well known in the art. Examples of techniques for altering the nucleotide sequence encoding the native protein, include, but are not limited to, site-directed mutagenesis, random mutagenesis and synthetic oligonucleotide construction.
  • the protein splicing efficiency of N- and/or C-split intein functional variants may be assessed for instance by measuring the protein reconstitution efficiency in a cell.
  • the protein reconstitution efficiency can be measured by expressing in a cell a combination of polynucleotides, said first polynucleotide encodes a N-terminal fragment of a reporter protein (e.g., GFP) fused to N-split intein and a second polynucleotide encodes a C-terminal fragment of said gene reporter fused to the C-split intein.
  • the reconstitution efficiency of the reporter protein can then be monitored by determining the level of expression of reconstituted protein.
  • the 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.
  • the quantity of protein may be measured by flow cytometry or fluorescence microscopy.
  • 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.
  • a reporter protein e.g., GFP
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100.
  • the identity is 60%.
  • the % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and/or biological modification.
  • variants 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • splicing buffer e.g., lOOmM sodium phosphates, 150 mM NaCl, ImM EDTA, pH 7.2
  • TCEP tris(2-carboxyethyl)phosphine
  • a major caveat to splicing-based methods is that all characterized inteins exhibit a sequence preference at extein residues adjacent to the splice site.
  • 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.
  • the C-split intein is selected from any C-split inteins disclosed in Table 2.
  • C-split intein is a mutated C-split intein having GEP amino acids in positions 20, 21 and 22 wherein said residue is numbered according to SEQ ID NO: 2 or any functional variant thereof, preferably retaining the functional splicing activity of split intein as described above, and more preferably having improved extein tolerance.
  • the C-split intein may be functional variants of the C-split inteins as described in Table 2, preferably comprising or consisting of amino acid sequence SEQ ID NO: 13 or 14, or any functional variants thereof having 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity to any one of sequences SEQ ID NO: 13 or 14 and preferably retaining the functional splicing activity of C-split intein as described above, and more preferably having improved extein tolerance.
  • the extein tolerance can be assessed for instance in kanamycine resistance assay as described in WO2017/132580 p. 60, paragraphs [00735] - [00738] in which a nucleic acid construct coding for a fragmented aminoglycoside phosphotransferase fused to a split intein with F, G, R or E present at the position +2 of the C-extein was transformed in DH5a competent cells and cultured at various concentrations of kanamycin. The cell density at 650 nm at 24 hours end point is measured and IC50 value is determined and compared with Cfa-Cmut.
  • a functional variant having improved extein tolerance is a C-split intein having a similar IC50 than the split intein having GEP amino acids in positions 20, 21 and 22 wherein said residue is numbered according to SEQ ID NO: 2.
  • 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.
  • 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).
  • 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 Na v l.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 Na v l.l protein, fuse
  • the inventors took the advantage of the intrinsic ability of split inteins as described above to mediate protein trans-splicing to reconstitute large Na v l .1 protein following their fragmentation into two split-intein flanked polypeptides.
  • Sodium channel protein type 1 subunit alpha (Na v l. 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.
  • Na v l.l has four homologous domains, each of which contains six transmembrane regions. Na v l.l plays a key role in brain, probably by regulating the moment when neurotransmitters are released in neurons.
  • Na v l.l protein is a human Na v l. l protein (UniProtKB accession number: P35498; updated on May 03, 2023, SEQ ID NO: 15 (human Na v l. 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.
  • Na v l. l protein can be a human Na v l. l protein as disclosed above or any functional variant thereof.
  • 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 Na v l. l protein, preferably human Na v l .1 protein (SEQ ID NO: 15), in particular sodium channel activity of the human Na v l .1 protein.
  • Na v l. 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 Na v l.
  • the current amplitude of Na v l. 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.
  • the value is higher than the negative control and preferably varies by less than 40%, 30%, 20% or 10% of the positive control value.
  • Na v l. l protein is human Na v l. 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.
  • 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.
  • 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 Na v l. 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.
  • a polynucleotide encoding the Na v l .1 protein as described above is split into at least two nucleic acid sequences encoding N- and C-terminal Na v l. 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 Na v l. l fragment flanking sequences (N- and C-terminal Na v l. l fragments, also named N- and C- exteins) with a peptide bond to reconstitute the full-length of Na v l .1 protein as described above in a cell.
  • the Na v l. l protein or any functional variant thereof as described above can be split at any positions into a N- and C-terminal Na v l. l fragments.
  • the inventors have showed that some specific split positions are particularly advantageous to increase the efficiency of Na v l. l protein reconstitution.
  • the Na v l .1 protein or any functional variant thereof as described above can be split into N- and C- terminal Na v l.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 Na v l.l, in particular by sequence alignment.
  • the N-terminal Na v l. l fragment ends up to residue 925 and the C- terminal fragment of Na v l.l protein starts from residue 926 respectively wherein said residue is numbered according to SEQ ID NO: 15.
  • the N-terminal fragment of Na v l.1 protein ends up to residue 1059 and the C-terminal fragment of Na v l.l protein starts from residue 1060 respectively wherein said residue is numbered according to SEQ ID NO: 15.
  • the N-terminal fragment of Na v l.1 protein ends up to residue 1178 and the C-terminal fragment of Na v l.l protein starts from residue 1179 respectively wherein said residue is numbered according to SEQ ID NO: 15.
  • the first and second fusion proteins according to the present disclosure comprise a N-terminal Na v l.l fragment and a C-terminal Na v l.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 Na v l.l protein retains the function of said Na v l. l, in particular sodium channel activity of the human Na v l.l protein.
  • Table 3 Examples of N-and C-terminal Na v l.l fragments. Highlighted amino acids in Full- length Na v l.l represent preferred split positions. The first methionine of the Na v l. 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 Na v l .1 full-length sequence without methionine.
  • 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 Na v l.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 Na v l. l protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:
  • N-terminal Na v l .1 protein up to residue 1178 and the C-terminal fragment of Na v l. l protein from residue 1179 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Na v l .1 fragment and the C-terminal Na v l .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.
  • 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 Na v l.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 Na v l.
  • first and second fusion proteins comprises: - the N-terminal fragment of Na v l .1 protein up to residue 925 and the C-terminal fragment of Na v l.l protein from residue 926 respectively,
  • residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Na v l .1 fragment and the C-terminal Na v l .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.
  • 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 Na v l. l full-length sequence without methionine.
  • 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.
  • the degron targets protein to the ubiquitin-proteasome pathway.
  • 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.
  • 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.
  • said degron is fused directly or indirectly via a linker to the N-split intein and C-split intein comprised in the first and second fusion proteins, respectively, preferably said degron is located at the 3’-end of the N-Split intein and at the 5’end of the C- split intein.
  • the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Na v l. 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 Na v l.
  • 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.
  • 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 Na v l.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 Na v l.
  • 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.
  • 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 Na v l.
  • 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.
  • 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 Na v l.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 Na v l.
  • 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.
  • 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 Na v l. 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 Na v l. l protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:
  • the N-terminal Na v l .1 protein up to residue 1178 and the C-terminal fragment of Na v l. l protein from residue 1179 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Na v l .1 fragment and the C-terminal Na v l .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 ’
  • 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 Na v l. 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 Na v l. l protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:
  • the N-terminal Na v l .1 protein up to residue 1178 and the C-terminal fragment of Na v l. l protein from residue 1179 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Na v l .1 fragment and the C-terminal Na v l .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 ’
  • 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 Na v l.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 Na v l. l protein
  • the N-terminal Na v l .1 protein up to residue 1178 and the C-terminal fragment of Na v l. l protein from residue 1179 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Na v l .1 fragment and the C-terminal Na v l .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 ’
  • 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 Na v l.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 Na v l. l protein
  • the N-terminal Na v l .1 protein up to residue 1178 and the C-terminal fragment of Na v l. l protein from residue 1179 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal Na v l .1 fragment and the C-terminal Na v l .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 ’
  • 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.
  • 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.
  • 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.
  • 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 (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 (SEQ ID NO: 28), Degl (SEQ ID NO: 29), PEST (SEQ ID NO: 30), DD1 (SEQ ID NO: 31), DD2 (SEQ ID NO: 32),
  • T1 SEQ ID NO: 56
  • T2 SEQ ID NO: 57
  • T3 SEQ ID NO: 58
  • DD1, 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., Na v l.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 protein of interest i.e., Na v l.l protein
  • 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., Na v l.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.
  • 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.
  • nucleic acid construct 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.
  • the nucleic acid construct comprises a coding sequence and regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence that are required for expression of the selected gene product.
  • a nucleic acid construct typically comprises a promoter sequence, a coding sequence and a 3' untranslated region that usually contains a polyadenylation site and/or transcription terminator.
  • said 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).
  • bGH bovine growth hormone polyadenylation signal
  • SynpA synthetic polyadenylation signal
  • SV40 simian virus 40 late or full-length polyadenylation signal
  • 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.
  • said nucleic acid construct may comprise a SV40 intron.
  • 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.
  • 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.
  • 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.
  • 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).
  • operably linked refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically but not necessarily contiguous; where it is necessary to join two protein encoding regions, they are contiguous and in reading frame.
  • each polynucleotide or nucleic acid construct according to the present disclosure may be comprised in an expression vector.
  • expression vector refers to a nucleic acid molecule used as a vehicle to transfer genetic material, and in particular to deliver a nucleic acid into a host cell, either in vitro or in vivo.
  • Expression vector also refers to a nucleic acid molecule capable of effecting expression of a gene (transgene) in host cells or host organisms compatible with such sequences.
  • Expression vectors typically include at least suitable transcription regulatory sequences and optionally 3 ’-transcription termination signals.
  • Vectors include, but are not limited to, plasmids, phasmids, cosmids, transposable elements, viruses, and artificial chromosomes (e.g., YACs).
  • the vectors of the disclosure is vectors suitable for use in gene or cell therapy, and in particular is suitable to target neuronal cells.
  • 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.
  • lentiviral vectors e.g. derived from human immunodeficiency virus (
  • suitable sequences should be introduced in the vector of the disclosure for obtaining a functional viral vector, such as AAV ITRs for an AAV vector, or LTRs for lentiviral vectors.
  • said vector is an AAV vector.
  • AAV has arisen considerable interest as a potential vector for human gene therapy.
  • 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.
  • the polynucleotides, nucleic acid constructs or expression vectors according to the present disclosure thereof further comprises a 5’ITR and a 3TTR sequences, preferably a 5’ITR and a 3’ ITR sequences of an adeno-associated virus.
  • inverted terminal repeat refers to a nucleotide sequence located at the 5’-end (5’ITR) and a nucleotide sequence located at the 3’-end (3’ITR) of a virus, that contain palindromic sequences and that can fold over to form T-shaped hairpin structures that function as primers during initiation of DNA replication. They are also needed for viral genome integration into the host genome; for the rescue from the host genome; and for the encapsidation of viral nucleic acid into mature virions. The ITRs are required in cis for the vector genome replication and its packaging into the viral particles.
  • the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure further comprises a 5’ITR and a 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”.
  • the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure is packaged into an AAV-derived capsids to generate an "adeno- associated viral particles" or "AAV particles”.
  • the present disclosure relates to viral particles comprising the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure and preferably comprising capsid proteins of adeno-associated virus.
  • AAV viral particles The construction of recombinant AAV viral particles is generally known in the art and has been described for instance in US 5,173,414 and US5,139,941; WO 92/01070, WO 93/03769, Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533- 539; Muzyczka, N. (1992) Current Topics in Microbiol, and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.
  • the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins as described above including ITR(s) of a given AAV serotype can be packaged, for example, into: a) a viral particle constituted of capsid proteins derived from the same or different AAV serotype [e.g.
  • AAV2 ITRs and AAV5 capsid proteins AAV2 ITRs and AAV8 capsid proteins; AAV2 ITRs and Anc80 capsid proteins; AAV2 ITRs and AAV9 capsid proteins];
  • a mosaic viral particle constituted of a mixture of capsid proteins from different AAV serotypes or mutants [e.g. AAV2 ITRs with AAV1 and AAV5 capsid proteins];
  • a chimeric viral particle constituted of capsid proteins that have been truncated by domain swapping between different AAV serotypes or variants e.g. AAV2 ITRs with AAV5 capsid proteins with AAV3 domains.
  • the 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.
  • 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.
  • adeno-associated virus such as capsid proteins from AAV9 and AAV-PHP.B, AAV2 and AAV5.
  • 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.
  • 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.
  • treatment 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.
  • 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.
  • subject refers to mammals.
  • Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, humans, nonhuman primates such as apes, chimpanzees, monkeys, and orangutans, domesticated animals, including dogs and cats, as well as livestock such as horses, cattle, pigs, sheep, and goats, or other mammalian species including, without limitation, mice, rats, guinea pigs, rabbits, hamsters, and the like.
  • said subject is human patient, preferably children under the age of 18.
  • compositions or medicament comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure
  • 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.
  • the term "pharmaceutically acceptable” means approved by a regulatory agency or recognized pharmacopeia such as European Pharmacopeia, for use in animals and/or humans.
  • excipient refers to a diluent, adjuvant, carrier, or vehicle with which the therapeutic agent is administered.
  • 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.
  • 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.
  • 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 (Na v ) channels such as Na v l. 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).
  • 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.
  • 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
  • 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.
  • a "therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result.
  • the therapeutically effective amount of the products of the disclosure may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the product or pharmaceutical composition to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • a therapeutically effective amount is also typically one in which any toxic or detrimental effect of the product or pharmaceutical composition is outweighed by the therapeutically beneficial effects. According to the present disclosure, a therapeutically effective amount allows to reduce for example the sudden unexpected death in epilepsy (SUDEP) and both spontaneous and thermic inducible seizures.
  • SUVP sudden unexpected death in epilepsy
  • 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.
  • such administration also encompasses use of each polynucleotide in a sequential or separate manner, either at approximately the same time or at different times.
  • the first and second polynucleotides are administered to the same subject as part of the same course of therapy.
  • the treatment regimen will provide beneficial effects in treating the diseases described herein.
  • 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.
  • 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 Na v l.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 Na v l.
  • 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
  • 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.
  • the kit comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Na v l.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 Na v l.
  • 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 comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Na v l.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 Na v l. l protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprises:
  • 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
  • the kit comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of Na v l.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 Na v l. l protein, fused directly or indirectly via a linker, wherein the first polynucleot
  • 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
  • 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
  • the kit may include instructions or packaging materials that describe how to administer the polynucleotides contained within the kit to a patient.
  • Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration.
  • the kits may include one or more ampoules or syringes that contain the products of the invention in a suitable liquid or solution form.
  • Oligonucleotides were purchased from Eurofins genomics. Synthetic genes were purchased from GENEWIZ. Pfu Ultra fusion polymerase for cloning and all restriction enzymes were purchased from Thermofisher Scientific. High-competency cells used for cloning were generated from XLIO-Gold chemically competentE. coli. HEK293T cells were purchased from ATCC. DNA purification kits were purchased from Thermofisher Scientific. All plasmids were sequenced by Macrogen. Luria Bertani (LB) media, and all buffering salts were purchased from Thermofisher Scientific.
  • LB Luria Bertani
  • Coomassie brilliant blue, NH4HCO3, DTT, formic acid, fetal bovine serum and asolectin from soybean were purchased from Sigma-Aldrich.
  • Acetonitrile (ACN) was purchased from Carlo-Erba.
  • EDTA-free complete protease inhibitors were purchased from Roche.
  • Lipofectamine 2000 transfection reagent, DMEM high glucose GlutaMAX supplement, RPMI 1640 medium GlutaMAX supplement, RIPA lysis and extraction buffer, BCA protein assay kit, MES-SDS running buffer, pre-stained protein ladder and SDS-PAGE (Bis-tris and Tris-acetate gels) were purchased from Thermofisher Scientific.
  • the primary antibodies used were anti-flag tag mouse monoclonal antibody (Sigma), anti-Na v l.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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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- Na v l. l to detect the Na v l. l protein and anti-P-tubulin as loading control.
  • the quantification of Na v l .1 bands detected by Western blot was performed using LLCOR Odyssey Infrared Imager.
  • 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.
  • 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 Na v l.l protein. DAPI was used to counterstain nuclei.
  • 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).
  • 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
  • intracellular solution 120 mM CsF, 10 mM NaCl,
  • 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.
  • a Boltzmann curve was fitted to the plots to obtain the activation V50.
  • 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.
  • 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.
  • 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).
  • IP intraperitoneal
  • ketamine 50-75 mg/kg
  • medetomidine 0.5 -1.0 mg/kg
  • 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).
  • mice 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.
  • inteins have been engineered based on consensus design (Stevens et al., 2016; Stevens, Sekar, Gramespacher, Cowburn, & Muir, 2018) and shown to have superior properties than naturally occurring inteins.
  • One of these inteins termed Cfa
  • Cfa have faster kinetics, higher expression levels and high tolerance to extreme conditions such as high temperature and concentration of denaturing agents.
  • Cfa variants with degrees of homology from 90% or higher display similar properties.
  • a major caveat to splicing-based methods is that all characterized inteins exhibit a sequence preference at extein residues adjacent to the splice site.
  • Deviation from this preferred sequence context leads to a marked reduction in splicing activity, limiting the applicability of protein trans-splicing (PTS)-based methods.
  • PTS protein trans-splicing
  • Recently 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.
  • Na v l. l which are sodium channels
  • a study using different split sites was performed.
  • Na v l .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.
  • 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 SCN1A at position 1179 (site 4) (SEQ ID NO: 65 and 66).
  • Sites were selected based on the topological structure of Na v l.l and taking into consideration the presence of folded domains. Sites were selected outside of well-defined folded domains in the intracellular side of Na v l. 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).
  • 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.
  • a stable cell line that expressed Na v l.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).

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Abstract

La présente invention concerne l'utilisation d'intéines divisées pour exprimer la protéine Nav1,1 codée par le gène SCN1A chez un sujet en ayant besoin pour une thérapie génique en particulier pour le traitement d'une maladie associée à SCN1A, de préférence le syndrome de Dravet.
PCT/EP2024/074640 2023-09-04 2024-09-04 Utilisation d'intéine divisée pour le traitement d'une maladie associée à scn1a Pending WO2025051758A1 (fr)

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