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KR-20260068082-A - Use of Split Intein for the Treatment of SCN1A-Related Diseases

KR20260068082AKR 20260068082 AKR20260068082 AKR 20260068082AKR-20260068082-A

Abstract

The present invention relates to the use of split intene for expressing the Na v 1.1 protein encoded by the SCN1A gene in subjects requiring it for gene therapy, particularly for the treatment of SCN1A-related diseases, preferably Dravet syndrome.

Inventors

  • 프루토스, 실비아
  • 카예스, 제라르
  • 모타스, 산드라
  • 빌라-페렐로, 미켈
  • 포우스, 세르히오
  • 가르시아, 벨렌

Assignees

  • 스플라이스바이오 에스.엘.

Dates

Publication Date
20260513
Application Date
20240904
Priority Date
20230904

Claims (15)

  1. A polynucleotide combination for use in the treatment of sodium channel protein type 1 subunit alpha (SCN1A)-related disease in subjects, wherein the combination comprises: iii) A first polynucleotide encoding a first fusion protein comprising an N-terminal fragment of a Na v 1.1 protein and an N-split intein fused directly from 5' to 3' or indirectly through a linker, iv) A second polynucleotide encoding a second fusion protein comprising a C-split intene fused directly from 5' to 3' or indirectly through a linker and a C-terminal fragment of Na v 1.1 protein, Herein, a polynucleotide combination in which the expression of the first and second polynucleotides in the subject above produces a Na v 1.1 protein by protein splicing.
  2. In paragraph 1, - The above N-split integra is the N-Cfa-integra of SEQ ID NO: 1 or any functional variant thereof having at least 90% identity with SEQ ID NO: 1; and - The above C-split intein is the C-Cfa intein of SEQ ID NO: 2 or any functional variant thereof having at least 90% identity with SEQ ID NO: 2, preferably a polynucleotide combination in which amino acid residues 20 to 22 of SEQ ID NO: 2 are GEP.
  3. A polynucleotide combination according to claim 1 or 2, wherein the C-split intane is the C-Cfa mut intane of SEQ ID NO: 13 or any functional variant thereof having at least 90% identity with SEQ ID NO: 13.
  4. A combination according to any one of claims 1 to 3, wherein the Na v 1.1 protein is a human Na v 1.1 protein, preferably comprising or composed of SEQ ID NO: 15 or any functional variant thereof having at least 90% identity with SEQ ID NO: 15.
  5. In any one of paragraphs 1 to 4, the first and second fusion proteins comprise the following: - The N-terminal fragment of the Na v 1.1 protein up to residue 925 and the C-terminal fragment of the Na v 1.1 protein starting from residue 926, respectively - The N-terminal fragment up to residue 1059 of the Na v 1.1 protein and the C-terminal fragment from residue 1060 of the Na v 1.1 protein, respectively; or - The N-terminal fragment up to residue 1178 of the Na v 1.1 protein and the C-terminal fragment from residue 1179 of the Na v 1.1 protein, respectively Here, the above residue is a combination numbered according to SEQ ID NO: 15.
  6. A combination according to any one of claims 1 to 5, wherein the first and second fusion proteins comprise an amino acid sequence selected from any one of the following pairs SEQ ID NO: 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 with any one of SEQ ID NO: 22-27.
  7. A combination according to any one of claims 1 to 6, wherein the first fusion protein or the second fusion protein further comprises a degron.
  8. In any one of paragraphs 1 through 7, i) The first fusion protein further comprises a degron located at the 3' end of the N-split-intein, and/or ii) The second fusion protein is a combination that additionally includes a degron located at the 5' end of the C-split-intein.
  9. A combination according to claim 7 or 8, wherein the degron is selected from a group consisting of any functional variant having at least 90% identity with any one of SEQ ID NO: 28 to 58 or SEQ ID NO: 28 to 58.
  10. A combination according to any one of claims 1 to 9, wherein each polynucleotide further comprises a promoter selected from the group consisting of a cytomegalovirus (CMV) promoter, a chimeric reduced version promoter of CMV and chicken beta-actin (CEBA), a human phosphoglycerate kinase (hPGK) promoter, a chimeric CMV enhancer and human phosphoglycerate kinase (ePGK) promoter, a chimeric synapsin I and CMV (Syn-CMV) promoter, a minimal neuron-specific enolase (NSEmin) promoter, a chimeric murine SCN1A E2 enhancer, and a minimal CMV (E2min) promoter.
  11. A combination according to any one of claims 1 to 10, wherein each polynucleotide is included in an expression vector.
  12. A combination according to claim 11, wherein the expression vector is a viral vector, preferably an adeno-associated virus (AAV) vector, and preferably the AAV vector comprises a capsid protein of an AAV selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or rh10.
  13. In any one of claims 1 to 12, the SCN1A-related disease is selected from the group consisting of Dravet syndrome, SCN1A-related non-Dravet syndrome epilepsy such as generalized epilepsy with febrile seizure-plus, malignant transitional focal seizures of infancy, West syndrome, Lennox-Gastaut syndrome, Rett syndrome, developmental and epileptic, and SCN1-related non-epileptic diseases such as familial hemiplegic migraine, autism spectrum disorder, and arthrogryposis multiplex congenita, and A combination preferably in which the disease is Dravet syndrome, preferably administered to a subject by a parenteral route, and more preferably administered by an intravenous, intraarterial, intracerebral, intracerebral spinal fluid, intrathecal, intra-cisterna magna, or intrarebroventricular route.
  14. Kit including the following: (i) Fusion from 5' to 3' directly or indirectly through a linker - N-terminal fragment of Na v 1.1 protein and - A first polynucleotide encoding a first fusion protein comprising the N-Cfa integrin of SEQ ID NO: 1 or any functional variant having at least 90% identity with SEQ ID NO: 1, (ii) fused directly from 5' to 3' or indirectly through a linker - C-Cfa integrin of SEQ ID NO: 2 or C-Cfa mut integrin of SEQ ID NO: 13 or any functional variant thereof having at least 90% identity with SEQ ID NO: 2 or SEQ ID NO: 13 and - A second polynucleotide encoding a second fusion protein containing a C-terminal fragment of Na v 1.1 protein, Herein, the first and second fusion proteins comprise the following: - The N-terminal fragment of the Na v 1.1 protein up to residue 925 and the C-terminal fragment of the Na v 1.1 protein starting from residue 926, respectively - The N-terminal fragment up to residue 1059 of the Na v 1.1 protein and the C-terminal fragment from residue 1060 of the Na v 1.1 protein, respectively, or - The N-terminal fragment up to residue 1178 of the Na v 1.1 protein and the C-terminal fragment from residue 1179 of the Na v 1.1 protein, respectively Here, the above residue is numbered according to SEQ ID NO: 15, and A combination comprising, preferably, a pair of N-terminal Na v 1.1 fragments and C-terminal Na v 1.1 fragments consisting of SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21 or any functional variant thereof, wherein the amino acid sequence is selected from having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity with any one of SEQ ID NO: 16 to 21.
  15. In claim 14, the kit, wherein the first and second fusion proteins comprise an amino acid sequence 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.

Description

Use of Split Intein for the Treatment of SCN1A-Related Diseases The present invention relates to the use of a split intein to express the Na v 1.1 protein encoded by the SCN1A gene in subjects requiring it for gene therapy, particularly for the treatment of SCN1A-related diseases, preferably Dravet syndrome. Dravet syndrome (DS) is a severe developmental and epileptic encephalopathy characterized by the onset of long-term febrile and non-febrile seizures in infancy, progression to drug-resistant epilepsy, and accompanying cognitive, behavioral, and motor impairments. It is currently known that most cases are caused by pathogenic mutations in the sodium channel gene SCN1A, which encodes the α-subunit of the voltage-dependent (gated) ion channel Na v 1.1. AAVs have proven to be highly efficient in the treatment of neurological diseases. However, the limited cargo capacity of AAV vectors restricts their use in gene therapy for delivering large genes such as SCN1A. In fact, due to the large size of the SCN1A gene, it cannot be encapsulated by a single AAV, and there is still a need to develop new strategies to deliver the SCN1A gene into target cells. Inteins are genetic elements that perform trans-splicing, where two protein fragments combine to form a catalytically active enzyme, which then catalyzes self-cleavage and the ligation of flanking sequences. Split inteins have primarily been used to fuse different functional protein domains in protein purification systems and labeling steps. In recent years, several new inteins have been developed based on consensus design (Stevens et al., J Am Chem Soc. 2016 Feb 24;138(7):2162-5; Stevens, Sekar, Gramespacher, Cowburn, & Muir, J Am Chem Soc. 2018 Sep 19;140(37):11791-117999), and have been shown to exhibit superior properties compared to naturally occurring inteins. The use of split-intein-mediated protein trans-splicing to reconstitute therapeutic proteins in vivo has recently been studied for only a few proteins (WO2021/191447 and WO2020/079034). However, the reconstitution of complex functional proteins, such as voltage-dependent ion channels, has not yet been shown in vivo. In this application, the inventors have demonstrated for the first time that the complex Na v 1.1 protein encoded by the SCN1A gene can be efficiently reconstituted in cells using split-intein-mediated protein trans-splicing. In vitro, Na v 1.1 channel sodium is correctly localized to the cell membrane and exhibits activity as a sodium channel. Furthermore, in vivo, the reconstitution of Na v 1.1 by protein trans-splicing is efficient and mediates efficacy in a mouse model of Dravet syndrome. The reconstitution of Na v 1.1 using split-intein-mediated protein trans-splicing represents a viable strategy for treating SCN1A-related diseases. The present specification relates to polynucleotide combinations for use in the treatment of sodium channel protein type 1 subunit alpha (SCN1A)-related diseases, preferably selected from the group consisting of Dravet syndrome, SCN1A-related non-Dravet syndrome epilepsy such as generalized epilepsy with febrile seizures-plus, malignant transitional focal seizures of infancy, West syndrome, Lennox-Gastaut syndrome, Rett syndrome, developmental and epileptic, and SCN1-related non-epileptic diseases such as familial hemiplegic migraine, autism spectrum disorder, and arthrogryposis multiplex congenita, more preferably the disease is Dravet syndrome, and the combinations are for treatment in subjects requiring treatment, and the combinations comprise: i) A first polynucleotide encoding a first fusion protein comprising an N-terminal fragment of a Na v 1.1 protein and an N-split intein fused directly from 5' to 3' or indirectly through a linker, ii) a second polynucleotide encoding a second fusion protein comprising a C-split intene fused directly from 5' to 3' or indirectly through a linker and a C-terminal fragment of Na v 1.1 protein, Here, the expression of the first and second polynucleotides in the above-mentioned subject produces Na v 1.1 protein by protein splicing. In a preferred embodiment, the N-split entain is the N-Cfa-entain of SEQ ID NO: 1 or any functional variant thereof having at least 90% identity with SEQ ID NO: 1; and the C-split entain is the C-Cfa entain of SEQ ID NO: 2 or any functional variant thereof having at least 90% identity with SEQ ID NO: 2, preferably amino acid residues 20 to 22 of SEQ ID NO: 2 are GEP, and more preferably, the C-split entain is the C-Cfa mut entain of SEQ ID NO: 13 or any functional variant thereof having at least 90% identity with SEQ ID NO: 13. In a preferred embodiment, the Na v 1.1 protein is a human Na v 1.1 protein, preferably comprising or composed of SEQ ID NO: 15 or any functional variant thereof having at least 90% identity with SEQ ID NO: 15. In a more preferred embodiment, each N-terminal fragment of Na v 1.1 protein ends at residue 925 and the C-terminal fragment of Na v 1.1 protein begins at resid