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EP-4735611-A1 - OTOFERLIN GENE TRANSFER

EP4735611A1EP 4735611 A1EP4735611 A1EP 4735611A1EP-4735611-A1

Abstract

The invention relates to a first fusion protein comprising N-terminally an N-terminal fragment of otoferlin and C-terminally an N-terminal fragment of an intein, and a second fusion protein comprising N-terminally a C-terminal fragment of an intein and C-terminally a C-terminal fragment of intein.

Inventors

  • DE VIN, Filip
  • HENCKAERTS, ELS

Assignees

  • Katholieke Universiteit Leuven

Dates

Publication Date
20260506
Application Date
20240701

Claims (10)

  1. 1. A first fusion protein comprising N-terminally an N-terminal fragment of otoferlin and C-terminally an N-terminal fragment of an intein, and a second fusion protein comprising N-terminally a C-terminal fragment of an intein and C-terminally a C-terminal fragment of intein, wherein said N terminal and C terminal fragment of otoferlin form the complete sequence of otoferlin, and wherein respectively the N terminal fragment of otoferlin and the C terminal fragment of otoferlin are selected from the group consisting of: -amino acids 1-797 of SEQ. ID NO: 3 and amino acids 798-1997 of SEQ. ID NO:3, -amino acids 1-710 of SEQ. ID NO: 3 and amino acids 711-1997 of SEQ. ID NO:3, - amino acids 1-1219 of SEQ. ID NO: 3 and amino acids 1220-1997 of SEQ. ID NO:3, - amino acids 1-1258 of SEQ. ID NO: 3 and amino acids 1259-1997 of SEQ. ID NO:3, - amino acids 1-1262 of SEQ. ID NO: 3 and amino acids 1263-1997 of SEQ. ID NO:3, - amino acids 1-770 of SEQ. ID NO: 3 and amino acids 771 -1997 of SEQ. ID NO:3, wherein Cys772 of otoferlin is modified into His772, -amino acids 1-930 of SEQ. ID NO: 3 and amino acids 931-1997 of SEQ. ID NO:3, -amino acids 1-994 of SEQ. ID NO: 3 and amino acids 995-1997 of SEQ. ID NO:3, -amino acids 1-1250 of SEQ. ID NO: 3 and amino acids 1251-1997 of SEQ. ID NO:3, -amino acids 1- 784 of SEQ. ID NO: 3 and amino acids 785-1997 of SEQ. ID NO:3, -amino acids 1- 785 of SEQ. ID NO: 3 and amino acids 786-1997 of SEQ. ID NO:3, - amino acids 1-1259 of SEQ. ID NO: 3 and amino acids 1260-1997 of SEQ. ID NO:3, and - amino acids 1-1254 of SEQ. ID NO: 3 and amino acids 1255-1997 of SEQ. ID NO:3.
  2. 2. The fusion proteins according to claim 1, wherein respectively the N terminal fragment of otoferlin and the C terminal fragment of otoferlin are selected from the group consisting of: -amino acids 1-797 of SEQ. ID NO: 3 and amino acids 798-1997 of SEQ. ID NO:3, -amino acids 1-710 of SEQ. ID NO: 3 and amino acids 711-1997 of SEQ. ID NO:3, - amino acids 1-1219 of SEQ. ID NO: 3 and amino acids 1220-1997 of SEQ. ID NO:3, - amino acids 1-1259 of SEQ. ID NO: 3 and amino acids 1260-1997 of SEQ. ID NO:3, - amino acids 1-1262 of SEQ. ID NO: 3 and amino acids 1263-1997 of SEQ. ID NO:3,
  3. 3. The fusion proteins according to claim 1 or 2, wherein the N terminal fragment of otoferlin and the C terminal fragment of otoferlin are amino acids 1-797 of SEQ. ID NO:3 and amino acids 798-1997 of SEQ. ID NO:3.
  4. 4. The fusion proteins according to any one of claims 1 to 3, wherein the N terminal and C terminal fragments of intein, are: the intein N terminal polypeptide (102AA) with SEQ. ID NO: 1 CLSYETE I LT VEYGLLPI GK IVEKRIECTV YSVDNNGNIY TQPVAQWHDR 50 GEQEVFEYCL EDGSLIRATK DHKFMTVDGQ MLPIDE I FER ELDLMRVDNL 100 PN 102 and the intein C terminal polypeptide (36AA) with SEQ. ID NO:2 MIKIATRKYL GKQNVYDI GV ERDHNFALKN GFIASN 36 .
  5. 5. The fusion proteins according to any one of claims 1 to 4, comprising at the N terminus of otoferlin and/or at the C terminus of otoferlin a peptide tag for purification and or detection (e.g. epitope for Ab recognition or GFP protein).
  6. 6. A set of nucleic acids encoding the fusions proteins according to any one of claims 1 to 5.
  7. 7. The set of nucleic acids according to claim 6, wherein the nucleic acids are AAV vectors.
  8. 8. The set of nucleic acids according to claim 7, wherein expression of the proteins is under control of a tissue specific promotor.
  9. 9. The set of nucleic acids according to claim 8, wherein the tissue specific promoter is a Myol5a promoter.
  10. 10. A set of AAV vectors according to any one of claims 7 to 9, for use in the treatment of hearing loss.

Description

OTOFERLIN GENE TRANSFER FIELD OF THE INVENTION The invention relates to gene therapy with otoferlin to treat hearing loss. The invention further relates to intein based splicing of otoferlin. BACKGROUND OF THE INVENTION Congenital hearing loss is a common sensory impairment affecting 1-4/1,000 newborns. A genetic etiology accounts for more than half of these. To date, more than 120 genes have been shown to be able to cause non-syndromic genetic hearing loss. Several of these have been investigated for their potential regarding gene therapy. Murine research has shown that genes with an important role in structural inner ear development, such as the prevalent deafness genes GJB2 and SLC26A4, are poor gene therapy targets as they must be administered in a very early murine stage, corresponding to prenatal treatment in humans Of main interest are the genes provoking congenital hearing loss without early structural damage though, and late- onset progressive hearing loss. The most promising results have been obtained for the otoferlin (OTOF) gene, which is the estimated 12th most prevalent deafness gene. Otoferlin acts as a calcium sensor for vesicle fusion and replenishment at the level of the auditory hair cell ribbon synapses. Mutations in the OTOF gene have long been known to cause a form of deafness named DFNB9. DFNB9 is a specific type of congenital profound hearing loss, auditory neuropathy, which is characterized by initial structural preservation but functional impairment. The estimated prevalence in the US and EU is 20,000 patients. Current treatment is early cochlear implantation, which results in an acceptable hearing and speech understanding, but is still far away from natural hearing. Gene therapy based upon adeno-associated virus (AAV) is increasingly being recognized as a powerful and safe technology to cure monogenic diseases. Given its specific profile and commercial potential, several murine OTOF studies using AAV have been performed [Akil et al. (2019) Proc Natl Acad Sci USA 116, 4496-4501; Al-Moyed et al. (2019) EMBO Mol Med 11, 201809396; Tertrais et al. (2019) J. Neurosc. 39, 3394; Rankovic et al. (2020) Front Mol Neurosci 13, 600051; Tang et al. (2023) Hum Genet. 142, 289-304]. While these studies are promising, the clinical translation is questionable. Indeed, most studies using OTOF gene replacement that report rescue of auditory brainstem responses (ABR, measure for hearing) and a high transduction efficiency of the inner hair cells (IHC) in DFNB9 mice, focus on early injection for their proof of concept, ranging from postnatal day (P)0-2 to PIO, which is well before the end of hearing maturation (P12) in this species [Song et al. (2006) J. Acoust Soc Am. 119, 2242-2257]. However, this early stage is clinically irrelevant as this would imply a prenatal intervention long before otoferl in -related deafness is diagnosed. The few studies that did include AAV injection at a time point at which the cochlea had reached full maturation (P30) [Song et a/., cited above], showed good but incomplete restoration of hearing parameters. Another hurdle in otoferlin gene therapy is the size of the coding sequence of full- length otoferlin, which exceeds the cargo capacity of AAV (4.7 kb). Several approaches have been used for AAV otoferlin delivery: single overloaded AAVs or the use of (hybrid) dual AAV approaches delivering split otoferlin relying on (geneindependent) homologous recombination [Akil et al. cited above; Al-Moyed, cited above] . A major drawback of single overloaded AAVs is that the cDNA of the oversized gene becomes fragmented in such a way that AAVs contain either the 5' part of the coding sequence or the 3' part of it. This results in vector genomes that are highly variable in size, thereby limiting clinical application potential. In dual-vector strategies, this is solved by splitting the transgene in two parts, each part being delivered via a different AAV vector. Upon transduction of target cells with both parts of the transgene, a functional construct is formed. The approach based on homologous recombination relies on the formation of a functional, full-length mRNA through a variety of mechanisms, depending on the dual-AAV-vector design. Protein expression levels achieved with dual-AAV vector strategies are typically much lower than from single AAV and tend to vary considerably depending on the design, the transgene and the targeted cell type. In general, lower expression levels achieved by a dual-AAV approach will not be problematic when only a few copies of protein are required for normal function but optimization efforts will be necessary for proteins that are required at high expression levels to fulfil their function, such as otoferlin. Indeed, otoferlin is essential for two phases of vesicle release from IHC synapses: for exocytosis of the readily releasable pool of vesicles, taking place during the first ~15 ms of a strong depolarizing stimulus, and for vesicle replenishme