EP4735611A1

EP4735611A1 - Otoferlin gene transfer

Info

Publication number: EP4735611A1
Application number: EP24739141.0A
Authority: EP
Inventors: Filip DE VIN; Els Henckaerts
Original assignee: Katholieke Universiteit Leuven
Current assignee: Katholieke Universiteit Leuven
Priority date: 2023-06-29
Filing date: 2024-07-01
Publication date: 2026-05-06
Also published as: WO2025003513A1

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.

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 12^th 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 replenishment, i.e. for the reformation, recruitment, and priming of vesicles that fuse with the active zone membrane during longer depolarization. While otoferlin levels as low as 30% of wildtype values are sufficient for the release of the readily releasable pool, vesicle replenishment requires presumably at least 70% of wild-type levels, like in heterozygous Otof^+/_ mice, which do not have a measurable hearing impairment. The observation that in the profoundly hearing impaired Pachanga mouse model (Otof^Pga/Pga), which carries a point mutation in otoferlin only sustained exocytosis is impaired, while fast exocytosis is not, corroborates this assumption. Additionally, Al- Moyed et al., cited above; noted similarities in otoferlin protein levels (~30% of wildtype), sustained IHC exocytosis levels, ABR thresholds, and ABR wave amplitudes between their dual-AAV treated Otof ^/_ mice and mildly hearing impaired otof^I515T/I515T mice, which indicates that treatment of DFNB9 patients with vectors that fail to sufficiently restore otoferlin protein levels might result in almost normal auditory thresholds but also in impaired speech comprehension and auditory fatigue similar to the patients with the p.Ile515Thr mutation. Since sustained exocytosis and auditory function seem to scale with otoferlin protein levels, increasing otoferlin protein expression levels via a split-intein approach will improve vesicle replenishment rates and cochlear function.

In contrast to (hybrid) dual AAV vectors designed for (gene-independent) homologous recombination, dual AAV vectors designed for protein splicing using split inteins contain all required elements to obtain high levels of the N- and C-terminal parts of the protein to allow efficient splicing of the full-length protein [Tornabene et al. (2019) Sci Transl. Med 11, AAV4523] . Protein trans-splicing is a post-translational process during which an intervening sequence (intein) auto-catalytically excises itself from the precursor protein, and concomitantly ligates the two flanking sequences (exteins) with a peptide bond (Fig. 1) [Gramespacher et al. (2018) Protein Sci. 27, 614-619] .

Tang et al. (2023) cited above disclose a dual-AAV-mediated gene therapy system of otoferlin based on the principles of protein trans-splicing using inteins. The authors of this paper also published Chinese patent application CN116925239.

However, splicing efficiency of these constructs is not efficient enough.

DETAILED DESCRIPTION

Figure legends

Figure 1. Schematic depicting trans-splicing of a naturally split intein to generate the spliced extein product.

Figure 2. Evaluation of splicing efficiency after transfection of HEK293T cells with split-EGFP constructs. A) Comparison of co-transfection of equimolar amounts of p- EGFP^N-Int^N and p-Int^c-EGFP^c with equimolar amount of p-EGFP using filler pDNA to keep the total amount of pDNA constant during transfections. B) Demonstration that EGFP signal after co-transfection of p-EGFP^N-Int^N & p-Int^c-EGFP^c is intein-mediated by co-transfection of p-EGFP^N-Int^N with p-EGFP^c, which lacks the C-terminal part of the intein.

Figure 3. Evaluation of splicing efficiency after transduction of HEK293T cells with AAV9 split-EGFP vectors.

Figure 4. Verification of trans-splicing of split otoferlin at two different positions.

Figure 5. AAV2-GFP transduction levels in wild-type adult mice. A: immune staining against MYO7A (responsible for both syndromic hearing loss; transduction efficiency in inner hair cells (B) and outer hair cells (C).

Figure 6. Schematic of the transgene cassettes of the dual AAV vector sets for otoferlin delivery. A) Representative dual AAV vector set (LVCBT1) for intein- mediated otoferlin delivery. B) Akouos hybrid dual AAV vectors adapted for benchmarking purposes. C) Decibel Tx hybrid dual AAV vectors adapted for benchmarking purposes. CMV cytomegalovirus promoter, 5'Otof_vl 5' fragment of mouse otoferlin isoforml, Int^N N-terminal part of the intein, pA polyA signal, Int^c C- terminal part of the intein, 3'Otof_vl 3' fragment of mouse otoferlin isoforml, SD splice donor sequence, AK Fl phage genome recombinogenic sequence, SA splice acceptor sequence, AP human alkaline phosphatase recombinogenic sequence.

Figure 7. SDS-PAGE analysis of hOtofv5 trans-splicing efficiency in combination with intein 1 at eight different split sites. N-terminal hOtoferlin is FLAG-tagged, C-terminal hOtoferlin is HA-tagged. Positive control is a double-tagged full-length otoferlin, Negative control is non-transfected control.

Figure 8. shows a schematic diagram of A c/s-splicing Firefly luciferase plasmids and B trans-splicing Firefly luciferase plasmids for fast screening of the impact of hOTOFv5 extein sequences on intein splicing. Int^N and Int^c are the N- and C-terminal part of the intein under investigation, respectively. hOTOF EXT N (3AA) and hOTOF EXT C (3AA) are the 6 AA of the extein junction for the split site under investigation, whereby the first AA of the C-extein (+1 position) contains a mandatory catalytic Cysteine or Serine residue.

Figure 9. Firefly trans-splicing efficiency of intein 1 at hOtofv5 Cysteine split sites. Split sites with Firefly luciferase activity > 2-fold higher than native control are indicated with an arrow. Figure 10. Firefly trans-splicing efficiency of intein 1 at hOtofv5 Serine split sites. Split sites with Firefly luciferase activity > 2-fold higher than native control are indicated with an arrow.

Figure 11. Firefly trans-splicing efficiency of intein 2 at hOtofv5 Cysteine split sites. Split sites with Firefly luciferase activity > 2-fold higher than native control are indicated with an arrow.

Figure 12. Firefly trans-splicing efficiency of intein 2 at hOtofv5 Serine split sites. Split sites with Firefly luciferase activity > 2-fold higher than native control are indicated with an arrow.

Figure 13. shows a schematic diagram of split otoferlin plasmids used for SDS-PAGE analysis. Int^N and Int^c are the N-terminal and C-terminal part of the intein under investigation, respectively. FLAG-N-hOTOFv5 and C-hOTOFv5-HA are the tagged N- and C-terminal fragments of otoferlin, respectively. Otoferlin is split between AA 700 and 1300 at the split sites listed in Table 1 and Table 2.

Figure 14. SDS-PAGE analysis of hOtofv5 trans-splicing efficiency in combination with intein 1 at Serine split sites identified in the Firefly luciferase screen (Fig. 10). N-terminal hOtoferlin is FLAG-tagged, C-terminal hOtoferlin is HA-tagged. Positive control is a double-tagged full-length otoferlin.

Figure 15. SDS-PAGE analysis of hOtofv5 trans-splicing efficiency in combination with intein 2 at Serine split sites identified in the Firefly luciferase screen (Fig. 12). N-terminal hOtoferlin is FLAG-tagged, C-terminal hOtoferlin is HA-tagged. Positive control is a double-tagged full-length otoferlin, negative control is non-transfected control.

Figure 16. SDS-PAGE analysis of murine otoferlin trans-splicing efficiency.

Figure 17. SDS-PAGE analysis of murine and corresponding human otoferlin trans- splicing efficiency.

SUMMARY OF THE INVENTION

The invention relates to fusion protein of fragments of otoferlin fused to fragments of intein.

A first fusion protein comprises N-terminally an N-terminal fragment of otoferlin and C-terminally an N-terminal fragment of intein.

A second fusion protein has N-terminally a C-terminal fragment of intein and C- terminally a C-terminal fragment of intein. In a set of first and second fusion protein, the fragments of intein are chosen to obtain an effective intein-mediated protein splicing. Suitable inteins for this purpose are known in the art.

A specific embodiment of intein fragments is: intein N terminal protein (102AA) [ SEQ. ID NO: 1]

CLSYETE I LT VEYGLLPI GK IVEKRIECTV YSVDNNGNIY TQPVAQWHDR 50

GEQEVFEYCL EDGSLIRATK DHKFMTVDGQ MLPIDE I FER ELDLMRVDNL 100

PN 102 intein C terminal protein (36AA) [ SEQ. ID NO:2]

MIKIATRKYL GKQNVYDI GV ERDHNFALKN GFIASN 36

Embodiments of N-terminal and C-terminal fragments of otoferlin in a set of first and second fusion protein are :

N terminal fragment C terminal fragment

AA 1-797 AA 798-1997

AA 1-994 AA 995-1997

AA 1-1254 AA 1255-1997

AA 1-770 AA 771-1997 with an optional 772 C to H mutation

For the numbering of these polypeptides reference is made to the below otoferlin sequence [ SEQ. ID NO:3].

The end of the N terminal fragment and the beginning of the C terminal fragments of selected splicing sites is indicated for a representative number of splicing sites (bold and underlined):

MALLIHLKTV SELRGRGDRI AKVTFRGQS F YSRVLENCED VADFDETFRW 50 PVASS IDRNE MLE IQVFNYS KVFSNKLI GT FRMVLQKVVE ESHVEVTDTL 100 IDDNNAI IKT SLCVEVRYQA TDGTVGSWDD GDFLGDESLQ EEEKDSQETD 150 GLLPGSRPSS RPPGEKS FRR AGRSVFSAMK LGKNRSHKEE PQRPDE PAVL 200 EMEDLDHLAI RLGDGLDPDS VSLASVTALT TNVSNKRSKP DIKME PSAGR 250 PMDYQVS ITV IEARQLVGLN MDPVVCVEVG DDKKYTSMKE STNCPYYNEY 300 FVFDFHVS PD VMFDKI IKI S VIHSKNLLRS GTLVGS FKMD VGTVYSQPEH 350 QFHHKWAI LS DPDDI SSGLK GYVKCDVAVV GKGDNIKTPH KANETDEDDI 400 EGNLLLPEGV PPERQWARFY VKIYRAEGLP RMNTSLMANV KKAFI GENKD 450 LVDPYVQVFF AGQKGKTSVQ KSSYE PLWNE QVVFTDLFPP LCKRMKVQIR 500 DSDKVNDVAI GTHFIDLRKI SNDGDKGFLP TLGPAWVNMY GSTRNYTLLD 550 EHQDLNEGLG EGVS FRARLL LGLAVE IVDT SNPELTSSTE VQVEQATPI S 600 ESCAGKMEE F FLFGAFLEAS MIDRRNGDKP ITFEVTI GNY GNEVDGLSRP 650 QRPRPRKE PG DEEEVDLIQN ASDDEAGDAG DLASVSSTPP MRPQVTDRNY 700 FHLPYLERKP CIYIKSWWPD QRRRLYNANI MDHIADKLEE GLNDIQEMIK 750]l 710 711 785 786

TEKSYPERRL RGVLEELSCG CCRFLSLADK DQGH S SRTRL DRERLKS CMR 800 770 771 784 7 85 797 798

ELENMGQQAR MLRAQVKRHT VRDKLRLCQN FLQKLRFLAD EPQHS I PDI F 850

IWMMSNNKRV AYARVPSKDL LFS IVEEETG KDCAKVKTLF LKLPGKRGFG 900 900

SAGWTVQAKV ELYLWLGLSK QRKE FLCGLP CGFQEVKAAQ GLGLHAFPPV 950 901 930 931

SLVYTKKQAF QLRAHMYQAR SLFAADSSGL SDPFARVFFI NQSQ CTEVLN 1000 980 981 994 995

ETL CPTWDQM LVFDNLELYG EAHELRDDPP I IVIE IYDQD SMGKADFMGR 1050 1003 1004

TFAKPLVKMA DEAYCPPRFP PQLEYYQIYR GNATAGDLLA AFELLQI GPA 1100 GKADLPPING PVDVDRGPIM PVPMGIRPVL SKYRVEVLFW GLRDLKRVNL 1150 AQVDRPRVDI E CAGKGVQSS LIHNYKKNPN FNTLVKWFEV DLPENELLHP 1200 1161 1162

1250 PLNIRVVDCR AFGRYTLVG SHAVSSLRRFI YRPPDRSAPS WNTTVRLLRR 1250 1219 1220 12591260 1251 12581259 CRVL CNGGSS SH STGEVVVT ME PEVPIKKL ETMVKLDATS EAVVKVDVAE 1300 1254 1255 1262 1263 EEKEKKKKKK GTAEE PEEEE PDESMLDWWS KYFAS IDTMK EQLRQQE PSG 1350 IDLEEKEEVD NTEGLKGSMK GKEKARAAKE EKKKKTQSSG SGQGSEAPEK 1400 KKPKIDELKV YPKELESE FD NFEDWLHTFN LLRGKTGDDE DGSTEEERIV 1450 GRFKGSLCVY KVPLPEDVSR EAGYDSTYGM FQGI PSNDPI NVLVRVYWR 1500 ATDLHPADIN GKADPYIAIR LGKTDIRDKE NYI SKQLNPV FGKS FDIEAS 1550 FPMESMLTVA VYDWDLVGTD DLI GETKIDL ENRFYSKHRA TCGIAQTYST 1600 HGYNIWRDPM KPSQI LTRLC KDGKVDGPHF GPPGRVKVAN RVFTGPSEIE 1650 DENGQRKPTD EHVALLALRH WEDI PRAGCR LVPEHVETRP LLNPDKPGIE 1700 QGRLELWVDM FPMDMPAPGT PLDI S PRKPK KYELRVI IWN TDEVVLEDDD 1750 FFTGEKSSDI FVRGWLKGQQ EDKQDTDVHY HSLTGEGNFN WRYLFPFDYL 1800 AAEEKIVI SK KESMFSWDET EYKI PARLTL QIWDADHFSA DDFLGAIELD 1850 LNRFPRGAKT AKQCTMEMAT GEVDVPLVS I FKQKRVKGWW PLLARNENDE 1900 FELTGKVEAE LHLLTAEEAE KNPVGLARNE PDPLEKPNRP DTS FIWFLNP 1950 LKSARYFLWH TYRWLLLKLL LLLLLLLLLA LFLYSVPGYL VKKI LGA 2000

The invention further relates to nucleic acids encoding said first fusion protein and set second fusion protein. These nucleic acids can encode further polypeptides fused to the otoferlin-intein fusion proteins such as tags for purification and/or tags for detection (e.g. epitope for Ab recognition or GFP protein)

Nucleic acids encoding said first fusion protein and set second fusion protein are typical AAV vectors.

In these AAV vectors the expression of the first and second fusion protein can be under the control of a tissue specific promotor such as hair cell-type specific promoter (e.g. Myol5a promoter). Another aspect of the present invention is the use of AAV vectors encoding said first fusion protein and said second fusion protein in the treatment of hearing loss.

The invention is further summarized in the following statements:

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- 900 of SEQ. ID NO: 3 and amino acids 901-1997 of SEQ. ID NO:3

-amino acids 1-710 of SEQ. ID NO: 3 and amino acids 711-1997 of SEQ. ID NO:3 ammo 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 ammo acids 1259-1997 of SEQ. ID

NO:3

- amino acids 1-1262 of SEQ. ID NO: 3 and ammo 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 1-1003 of SEQ. ID NO: 3 and amino acids 1004-1997 of SEQ. ID NO:3

-amino acids 1-1161 of SEQ. ID NO: 3 and amino acids 1162-1997 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 1-980 of SEQ. ID NO: 3 and amino acids 981-1997 of SEQ. ID NO:3

-amino acids 1-992 of SEQ. ID NO: 3 and amino acids 993-1997 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. The fusion proteins according to statement 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- 900 of SEQ. ID NO: 3 and amino acids 901-1997 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. The fusion proteins according to statement 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. The fusion proteins according to any one of statements 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. The fusion proteins according to any one of statements 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. A set of nucleic acids encoding the fusions proteins according to any one of statements 1 to 5.

7. The set of nucleic acids according to statement 6, wherein the nucleic acids are AAV vectors.

8. The set of nucleic acids according to statement 7, wherein expression of the proteins is under control of a tissue specific promotor.

9. The set of nucleic acids according to statement 8, wherein the tissue specific promoter is a Myol5a promoter. 10. A set of AAV vectors according to any one of statements 8 or 9, for use in the treatment of hearing loss.

DETAILED DESCRIPTION

The present invention provides highly specific, potent, efficacious and high-quality dual AAV vectors that rely on otoferlin splicing at the protein level for increased otoferlin expression. Therefore, several AAVs with optimized vector design are generated and their ability to specifically transduce IHC and to recover auditory functions in relevant animal models is benchmarked with AAV vectors to be applied in clinical trials for otoferlin hearing loss. The development of inner ear gene therapy products, will aid to improve the quality of life of patients suffering from hearing loss. The dual AAV approach based on protein trans-splicing of otoferlin results in a superior gene therapy product with higher clinical potential for several reasons:

1) Examples shows near 100% splicing efficiency of otoferlin in vitro.

2) The present invention aims to obtain complete rescue of hearing loss via AAV administration in DFNB9 mice between P20-P30, which roughly corresponds with a 1-year-old human patient. This timing is clinically relevant, as deafness can be fully diagnosed at that time and treatment is feasible.

3) The AAV therapy can be optionally be used in a combination therapy (cochlear implantation and meanwhile injecting the therapeutic using the same surgical route).

Otoferlin in the context of the present invention relates to human otoferlin variant 5 (hOtofv5) (NCBI Reference Sequence NM_001287489.2) as depicted in SEQ. ID NO:3.

Numbering of splice site relates to the 1997 AA sequence of SEQ. ID NO:3.

Sequences with more than 95, more than 97, or more that 99 sequence identity and maintaining the function of otoferlin equally fall under the concept of otoferlin.

Sequence having truncations at the aminoterminus and/or carboxyterminus and maintaining the function of otoferlin equally fall under the concept of otoferlin.

For the use of split inteins for otoferlin delivery using a dual AAV approach, the splicing efficiency of a naturally occurring split intein was first evaluated in vitro, using split-EGFP (enhanced green fluorescent protein) experiments. This intein has extein preferences with minimal sensitivity to N-extein residues but more stringent requirements for the C-extein. In these experiments, EGFP was split in two fragments: The N-terminal part of the intein (Int^N) was then fused to the C-terminal of the N-terminal EGFP fragment (EGFP^NInt^N), while the C-terminal part of the intein (Int^c) was fused to the N-terminal of the C-terminal EGFP fragment (Int^cEGFP^c). Human embryonic kidney (HEK)293T transfection experiments with split EGFP constructs (Fig. 2) showed that EGFP splicing was highly efficient (Fig. 2A) and intein- mediated (Fig. 2B). As successful EGFP trans-splicing relies on both the presence of the N-terminal and C-terminal EGFP fragment fused to the corresponding part of the intein in each cell, the transfection/transduction efficiency plays an important role in the overall splicing efficiency obtained. Given the generally much lower copy number after transduction compared to transfection per cell, corresponding vectors were made to allow assessment of EGFP trans-splicing efficiency in transduction context (Fig. 3). AAV9 vectors expressing either EGFP^NInt^N or Int^cEGFP^c were transduced at a multiplicity of infection (MOI) of 8E4 (vector genome copies/cell) and imaged 48h later.

Splicing efficiency is dependent on the choice of the intein, the secondary structure of the split site favoring structures that allow sufficient degrees of freedom (such as a-helices, p-sheets, loop structures), and the stability of the two split parts. Another important factor for efficient intein splicing is the substrate specificity or junction dependency of the intein i.e. the extein sequence surrounding the split site. Overall, it seems that the N-extein sequence has little impact on intein splicing while the catalytic +1 residue and the +2 residue of the C-extein are crucial for efficient splicing. The catalytic + 1 residue consists of a Cysteine, Serine, or Threonine, which can act as a nucleophile in the splicing reaction [Nanda et al. (2020) Microorganism 8, 2004]. Regarding the requirements of the +2 residue of one of the best characterized inteins contradicting reports exist, which could be the result of the different methods used between the studies to assess splicing efficiency [Iwai et a/., (2006) FEBS Lett. 580, 1853-1858; Oeemig etal. (2020) FEBS Lett. 594, 3338-3355; Stevens et al. (2017) Proc Natl Acad Sci USA 114, 8538-8543). Whereas for many other inteins data on substrate specificity is either scarce or lacking. For the development of an efficacious DFNB9 gene therapy, however, consistently high otoferlin expression levels are crucial to improve vesicle replenishment rates and cochlear function. Our data (Fig. 7) clearly shows that optimization of the splice site in combination with careful transgene cassette design for AAV gene therapy can result in highly efficient splicing, which stands in stark contrast to the results of e.g. Tang et al. 2023 (cited above) (Fig.lOB), where the signal of spliced otoferlin is only a fraction of the signal of the split otoferlin halves.

To identify and investigate the potential split sites in human otoferlin variant 5 (hOtofv5) CDS (NCBI Reference Sequence NM_001287489.2), the limited cargo capacity of rAAV ~5kb was considered, which means that in practice the otoferlin CDS is split between AA position 700 and 1300 to prevent the DNA length from exceeding 3.9 kb, thereby providing the minimal space required for regulatory elements. The search was limited to sites with either a Cysteine (Table 1) or a Serine (Table 2) at the +1 position as these catalytic residues seem to be preferred by many inteins, over Threonine. Initially, splicing efficiency was evaluated by SDS-PAGE analysis (Fig. 13) for eight split sites in combination with intein 1 (Fig. 7) yielding one excellent (C6), three decent (Cll, C12, C17), and four poor split sites. In order to evaluate 50 split sites for two inteins, these split sites in combination with either intein 1 or intein 2 were first evaluated in a split-luciferase assay to increase the screening throughput (Fig. 8). Otoferlin split sites with extein junctions that resulted in highly efficient luciferase trans-splicing, corresponding split-otoferlin constructs were generated to evaluate otoferlin splicing by SDS-PAGE.

Table 1. Potential split sites for hOtofv5 between AA position 700 and 1300 with Cys at + 1 position. Split site at position 771 is listed twice as split site C3 is the original split site as present in hOtofv5 while split site C4 is at the same location but followed by a C772H mutation that was introduced in an attempt to increase the splicing efficiency at position 771 by altering the extein junction. In mouse otoferlinvl, the C-extein at corresponding location is CHR instead of CCR.

Table 2. Potential split sites for hOtofv5 between AA position 700 and 1300 with Ser at + 1 position. Confirmation of successful and highly efficient splicing of EGFP in both transfection as transduction context led us to generate split otoferlin constructs using the current intein. Split sites were chosen based on the C-extein requirements of the intein and the secondary structure wherein the split site is located, which can impact the splicing efficiency. The Protein Structure Prediction Server (PSIPRED) was used for this purpose. As prediction of the optimal splice site for a given coding sequence is not trivial, and can best be determined empirically.

Aiming to split otoferlin in roughly equal sized parts, six potentially favorable split sites were identified. Currently, two of these split sites for otoferlin have been evaluated (Fig. 4). After transfection of equal copies of the different plasmids containing either the N- or the C-terminal part or both in HEK293T cells, spliced, full- length otoferlin for the two selected split sites were obtained in identical amounts as the full-length otoferlin positive control, confirming near 100% splicing efficiency via Western Blot (WB). The splicing/construct design is by far superior to any of the protein trans-splicing combinations recently described by Tang et al. 2023 (cited above), where the signal of spliced otoferlin is only a fraction of the signal of the split otoferlin halves and a full-length otoferlin reference is lacking.

Additionally, the efficiency and safety of AAV inner ear injection in mice, through the round window membrane (RWM) has already been tested with good inner hair cell transduction levels and no hearing loss, with recently published results of AAV2-GFP injections via the RWM technique with canal fenestration in wild-type adult mice, approaching 100% of inner hair cell transduction levels (Fig. 5 A, B and C) [Du et al. (2023) Mol. Therapy 31, 2796-2810].

To develop an ultra-efficient dual AAV intein-based gene therapy product with superior otoferlin expression, the following approach is followed:

In total four more split sites for otoferlin (8 split-constructs) are used by transfection in HEK293T cells whereby the best performing split otoferlin constructs are identified via SDS-PAGE and WB. In addition, the two best performing split otoferlin constructs described by Tang et al. 2023 (cited above) are additionally be generated (4 splitconstructs) to benchmark the splicing efficiency of the constructs in vitro.

Splicing efficiency is evaluated by SDS-PAGE and WB, optionally with the addition of a tag to also visualize the N-terminal otoferlin fragments. For a more quantitative comparison, mouse otoferlin levels are determined via an enzyme linked immunosorbent assay (ELISA) on HEK293T cell lysates. As WB data does not allow to draw conclusions on the correct folding of spliced otoferlin proteins, their biophysical properties will be compared to full-length unspliced otoferlin. Therefore, the C-terminal fragments of a selection of the most efficient split-otoferlin constructs is equipped with e.g. a His-tag to produce the differently spliced otoferlin proteins and the full-length unspliced otoferlin protein using the ExpiCHO expression system. Subsequently, the proteins are purified using HisPur Ni-NTA agarose prior to sizeexclusion chromatography coupled to multi-angle light scattering (SEC-MALS) analysis to compare the biophysical properties of the spliced otoferlin proteins with the full-length otoferlin protein. Alternatively, the thermostability of the purified, differently spliced otoferlin proteins can be compared with the thermostability of the purified, full-length unspliced otoferlin in a thermal shift assay using the thermofluor dye SYPRO orange in a real-time thermal cycler. Comparing the biophysical properties of the differently spliced otoferlin proteins with full-length otoferlin allows to eliminate potentially misfolded proteins and hence reduce the number of vectors that need to be made and ultimately tested in vivo.

Dual AAV vectors of the two best performing split-otoferlin constructs are produced (mini-scale) and the levels of spliced otoferlin expression after in vitro transduction in HEK293T cells is gauged by SDS-PAGE and WB. Transgene cassette elements and configurations are tested to guarantee optimal expression levels for splicing, considering their potential impact on vector production yield.

In parallel to the transgene cassette optimization for otoferlin delivery, the most suitable natural AAV serotype is identified in a biodistribution study. Therefore, GFP vectors of commonly used natural serotypes (AAV2, AAV6, AAV9) for inner hair cell transduction [Al-Moyed et al. (2019), cited above; Tao et al. (2018) Hum Gene Ther 29, 492-506; Yoshimura et al. (2018) Sci Rep 8, 2980] are produced at large-scale in HEK293T. GFP expression is herein driven by the CMV promoter, which is most commonly used in cochlear gene therapy, yielding the highest transduction efficiency in IHC. Because of unknown production yields associated with engineered AAV vectors such as AAVAnc80L65 [Landegger et al. (2017) Nat Biotechnol 35, 280- 284.], AAV2-4YF [Akil et al. cited above], AAV-PHP.B [Ivanchenko et al. (2020) Hear Res. 394, 107930] and AAV-PHP.eB [Tang et al. cited above], alternative embodiments relate to natural serotype. Herein, the AAV2 serotype has a high IHC co-transduction efficiency obtained in dual-vector configuration in adult murine cochleae. An example of a synthetic AAV serotype to evaluate for its IHC transduction capacity is AAV-TT, an AAV2 variant. Compared to AAV2, AAV-TT demonstrates strong neurotropism in rodent and significantly improved distribution throughout the central nervous system [Tordo et al. (2018) Brain 141, 2014-2031.]

In brief, recombinant AAV (rAAV) is produced in Cell Stacks using the adherent HEK293T cell line in combination with a two-plasmid transient transfection system in which rep and cap genes and Adenoviral helper genes are on the same plasmid. Prior to large-scale productions, the effect of total plasmid DNA (pDNA) amount, DNA:PEI (polyethylenimine, transfection reagent) ratio, and plasmid ratio on viral genome (vg) titer, viral particle (VP) titer, and % full capsids (%fu II) is determined in a set of mini-scale productions to establish the optimal production parameters in terms of yield (vg titer) and %full for the different serotypes. A large-scale produced rAAV vectors is purified by affinity chromatography and polished by anion exchange chromatography to obtain high potency vectors that are subjected to vector analytics (endotoxin levels, ELISA, ddPCR, alkaline gel electrophoresis, SDS-PAGE, WB and Nanopore sequencing) to guarantee the highest possible vector quality.

Preferably, purified vectors with a high (> 1E13 vg/ml ) vg titer and a high (> 50%) %full to minimize the volume (±lpl) are used for intracochlear injections.

For biodistribution analysis in wild-type mice on an FVB background (both female and male mice), four serotypes (AAV2, AAV6, AAV9, AAV-TT) of AAV-GFP vectors are applied to the inner ear at the age of P20-P30 via a round window membrane (RWM) injection with canal fenestration to facilitate longitudinal flow During general anesthesia, a retroauricular incision is made and the auditory bulla is opened to optimally visualize the round window niche (RWM). A slow RWM injection is performed using a microinjector after making a perforation in the posterior semicircular canal. When required, an adhesive is used to control the fluid leaks that result from inner ear perforation. Four weeks after injection, anesthetized mice are tested for hearing by ABR and distortion-product oto-acoustic emissions (DPOAE) by placing an electrode on the vertex, below the pinna and above the tail. Sound is administered via an output channel placed in the external ear canal. Subsequently, the mice are sacrificed and the cochleae are microdissected into the different cochlear turns, enabling immunostaining of the fragments for GFP and cochlear hair cells (MYO7A), followed by confocal microscopy and quantitative analysis. The transduction efficiency in IHCs obtained with the different serotypes of AAV-GFP is compared as well as the transduction spread from apex to base (highest transduction is typically found at the apex, lowest at the base of the cochlea). As both the choice of the vector and the surgical approach play a role in enabling efficient cochlear gene transfer, these experiments are performed in vivo.

Dual AAV vector sets for intein-mediated otoferlin delivery are produced at large- scale and packaged in the AAV serotype of choice. Dual AAV approaches have been used to express otoferlin in IHCs of Otof ^/_ mice albeit to relative low protein levels (~30%). This indicates that the basic rescue of ABR thresholds is quite robust and not strictly dependent on high protein levels of a specific otoferlin isoform, transgene cassette design, AAV serotype or mouse strain.

In the present invention, otoferlin expression is restored to near physiological levels to obtain optimal vesicle replenishment rates and cochlear function. Therefore, to demonstrate that a highly optimized dual AAV approach based on intein-mediated otoferlin splicing is superior to the current prior art hybrid dual AAV approaches in clinical development, benchmarking is performed to demonstrate the difference between dual AAV vector sets of the invention and reference dual AAV vector sets (REFI, REF2) is the strategy for otoferlin reconstitution (Fig. 6).

The ubiquitous CMV promoter from dual AAV vector sets is replaced by a hair celltype specific promoter such as the Myol5a promoter. As selective expression of the transgene improves safety relative to broad promoters, demonstration of the compatibility of the highly efficient intein-mediated otoferlin delivery with the use of a cell-type specific promoter will increase its commercialization potential.

AAV vector sets of the invention and dual AAV vector sets (REFI, REF2) are applied to the inner ear of otoferlin knock-out mice at the age of P20-P30, more specifically via the round window membrane with posterior semicircular canal fenestration. Four weeks after single, unilateral injection, hearing (ABR and DPOAE) is evaluated, compared to the untreated ear as well as to wild-type mice, and is repeated on a monthly basis until 8 months of age. Mice are sacrificed at 8 months of age for microscopic inner ear evaluation, with the addition of immunostaining for synaptic ribbons (CtBP2) and otoferlin. Two different otoferlin antibodies are used, directed against the C-terminal part or the N-terminal part of the protein- which can be used to verify co-transduction efficiency of the dual vectors. In addition, normalized otoferlin immunofluorescence levels is quantified in wild-type, dual AAV vector transduced Otof ^/_, and Otof ^/_ IHCs to evaluate otoferlin expression for AAV vector sets of the present invention and reference dual AAV vectors. Alternatively, mouse otoferlin ELISA is performed on cochlear homogenates of wild-type; Otof ^/_, and treated Otof ^/_ mice to more accurately determine otoferlin expression levels.

The effect of dual-AAV mediated re-expression of otoferlin on fast exocytosis and sustained exocytosis is investigated in Otof ^/_ IHCs via patch-clamp electrophysiological recordings in IHCs of acutely dissected apical turns of organs of Corti.

EXAMPLES

Screening of potential hOtofv5 split sites in a split-luciferase assay

To make the c/s-splicing Firefly luciferase constructs (Fig. 8A), a Swal restriction site replacing the 6 AA 229-234 in Firefly luciferase (pGL4.53 vector, E6681, Promega) was inserted by site directed mutagenesis (Q5 Site directed mutagenesis kit, E0554, NEB [Cheriyan et al. (2013) J Biol Chem. 288, 6202-6211] Subsequently, a bridging approach with two primers containing the extein sequences of each split site was used to assemble (NEBuilder Hifi DNA assembly, E2621, NEB) a PCR product (Q5 polymerase, M0492, NEB) of the entire intein in the Swal linearized, dephosphorylated (Quick CIP, M0525, NEB) backbone. The trans-splicing Firefly luciferase constructs were made by replacing the entire Firefly luciferase via assembly of a PCR product (Q5 polymerase) containing the N-terminal fragment of Firefly including Int^N or Int^c including the C-terminal fragment of Firefly to generate the 5' plasmid or the 3' plasmid of Fig. 8B, respectively. Kozak sequences and stop codons were inserted where required.

To investigate the splicing efficiency at potential hOtofv5 split sites, the effects of the corresponding N-extein and C-extein sequences on splicing were evaluated using a Nano-Gio Dual-Luciferase reporter assay system (Promega N1610). For each split site and intein under investigation, two corresponding split luciferase plasmid constructs were generated. As neither the N- terminal nor the C-terminal experimental plasmids transfected alone produce any luminescence, Firefly luciferase activity can be used as a measure for the trans-splicing efficiency at the extein junctions under investigation (Fig. 9 - Fig. 12).

HEK293T cells were cultured in DMEM high glucose GlutaMAX (61965026, ThermoFisher Scientific) supplemented with 10%FBS (F7524, Sigma) and 1% Penicillin-Streptomycin (P/S) (15140130, ThermoFisher Scientific) at 37 °C with 5% CO2. 24h prior to transfection, cells were seeded at a seeding density of 35K cells per well (V=80 pl) in opaque, white tissue-culture 96 well-plates. In total, 125ng of pDNA was transfected per well (V=10 pl) consisting of lOng of each experimental plasmid (N- and C-terminal corresponding split luciferase constructs), and 105 ng of a filler plasmid containing 10 ppm of a NanoLuc control plasmid constitutively expressing a control reporter, 8.3 pl OptiMEM (11058021, ThermoFisher Scientific), and 0.3 pl Genius transfection reagent (7-1050, Westburg). After combining the DNA in PCR tubes, 8.6 pl OptiMEM-Genius mix was added to each of the tubes, the content was briefly vortexed and incubated for 15 min at room temperature. After complexation, the 10 ul droplets of transfection mix were added to the center of each well of a 96- well plate. All luciferase experiments were carried out in triplicate. Dual assays were used to normalize experimental reporter luminescence to that of a co-transfected control, thereby improving data quality by minimizing or eliminating experimental variability arising from such factors as differences in transfection efficiency, cell number, cell viability, temperature and measurement time. The filler plasmid does not express any luminescent signal and is only added to reach the optimal total pDNA amount required for efficient transfection in 96-well plate format using the Genius transfection reagent. 48h after transfection, plates were equilibrated at room temperature before 90 pl of One-Gio EX Luciferase reagent (Promega) was added to each well. Prior to measurement of the Firefly luminescent signal using a TECAN Spark plate reader (Tecan Life Sciences), the plates were incubated for 3 minutes on an orbital shaker at 600 rpm. Subsequently, 90 pl of NanoDLR Stop & Gio reagent (Promega) was added to each well and incubated for 3 min on an orbital shaker at 600 rpm prior to readout of the Nanoluc luminescent signal. Each Firefly luciferase data set contained a negative control (NCI) that should not allow splicing (KQA/FQL) since the mandatory catalytic residue is lacking as well as a positive control (native) whereby the intein split luciferase constructs were equipped with their native extein junction sequences. All data shown in Fig. 9 - Fig. 12 are normalized to the negative control and include the native extein junction of the intein under investigation as a reference for efficient splicing.

Fig.9 and Fig. 10 show Firefly luciferase activity resulting from splicing at extein junctions from the different hOtofv5 Cysteine and Serine split sites, respectively, in combination with intein 1. Best split sites for intein 1 are at position C798, S785, S901, and S1259, which result in at least 2-fold higher signal than the native control. Intein 1, however, performs best at split site C798, which results in a 4-fold higher signal compared to the native control (Fig. 9).

Fig. 11 shows Firefly luciferase activity resulting from splicing at extein junctions from the different hOtofv5 Cysteine split sites in combination with intein 2. Best split sites are at position C798 and C1162, which result in 4-fold and 3-fold higher signal than the native control, respectively.

Figure 12 shows Firefly luciferase activity resulting from splicing at extein junctions from the different hOtofv5 Serine split sites in combination with intein 2. Best split sites are at position S901, S1220, S1259, and S1263, which result in between 5-fold to 9-fold higher signal than the native control. Intein 2 performs best at Serine split sites.

The purpose of the split-luciferase screen was to identify splits sites that have an extein junction favorable for highly efficient protein splicing. As mentioned earlier, extein junction dependency is only one key parameter affecting intein splicing, which was studied in isolation in the split-luciferase assay as the extein junction of the different split sites were all introduced at the same location (AA 229-235) and thus identical secondary structure in the Firefly luciferase protein. Split sites with a high Firefly luciferase signal have therefore optimal extein junction for protein splicing but the actual otoferlin splicing efficiency could be decreased by a suboptimal location of the split site in the protein and/or stability of the otoferlin fragments. Conversely, a mediocre extein junction in the split-luciferase assay could result in higher than average protein splicing in case the secondary structure and/or the stability of the split fragments would be highly favorable for protein splicing. Therefore, split sites identified in the split-luciferase assay were always confirmed by SDS-PAGE analysis.

Confirmation of identified split sites by SDS-PAGE analysis.

Split otoferlin plasmids were transfected in adherent HEK293T cells. One day before transfection, 4.6M HEK293T cells were seeded in a 10 cm dish. The day after, the HEK293T cells were transfected using a 1 :2 ratio DNA:PEI MAX transfection reagent (24765-1, Avantor). Plasmid DNA was prepared for the different conditions and 500 pL of high glucose DMEM (61965026, ThermoFisher Scientific') 0% FBS was added. For the transfections, a total of 6 pg DNA, comprising of equimolar ratios of N- and C-terminal split otoferlin constructs were used. In cells transfected with only one of the constructs a filler plasmid expressing GFP was added to keep the total amount of plasmid DNA constant. Non-transfected cells were used as negative control, cells transfected with a plasmid with identical regulatory elements expressing double tagged full-length otoferlin were used as positive control. In parallel, transfection reagent was prepared and 500 pL DMEM 0% FBS was added. Next, the plasmid DNA solution and the transfection reagent solution were combined and briefly vortexed, to start the complexation reaction for 15 min. Subsequently, 7 mL of DMEM 0% FBS was added, gently mixed by inversion, and the medium in the 10 cm dish was replaced with this transfection mix. The HEK293T cells were incubated at 37 °C with 5% CO2. After overnight incubation, the transfection medium was switched to expression medium (DMEM, 5% FBS, 1% P/S). Expression was allowed for 36h, after which the cells were collected with ice-cold PBS. The collected cells were centrifuged for 5 min at 300 g. Harvested cells were lysed in 220 pL lx RIPA buffer (20-188, Millipore') containing lx complete Protease Inhibitor Cocktail (11863145001, Roche) and incubated on ice for 15 min followed by 10 min centrifugation at 3500 x g at 4 °C. The protein concentration was determined using a BCA protein assay kit (23225, ThermoFisher Scientific) . Protein samples were denatured for 5 min at 95 °C in a mix of lx loading dye (#1610747, Bio-Rad) and 50mM DTT. Equal amounts of denatured protein were loaded in the wells and separated by SDS-PAGE with a NuPAGE 4-12% Bis-Tris gel (NP0323BOX, Invitrogen) in lx MOPS buffer (NP0001, Invitrogen). Proteins were transferred onto a polyvinylidene fluoride membrane (#10026933, BioRad) using lx transfer buffer (#10026938, Bio-Rad) for 30 min at 25 V/1.0 A. After transfer, the membranes were incubated for lh in blocking solution (PBS, 0.05% Tween20, 5% milk). Subsequently, the membranes were incubated in primary antibody solution (PBS, 0.05% Tween20, 5% milk) containing mouse IgG2a anti- FLAG (1:500 dilution, NBP1-97410, Novus Biologicals), mouse IgGl anti-HA (1:500 dilution, 901513, BioLegend) and IgGl anti-GAPDH (1 : 10000 dilution, ab8245, Abeam) on a tilting table o/n at 4 °C. Next, the membranes were washed 3 times with PBS/0.05%Tween20 for 15 min. The membranes were incubated for 2h at RT in secondary antibody solution (PBS/0.05%Tween20/5% milk) containing goat antimouse antibody (1: 12500 dilution, P0447, Agilent Technologies), followed by 3 times the wash step as described above. Finally, the secondary antibody was visualized by incubating the membranes in Western Blot ECL Blotting Substrate (NEL105001EA, PerkinElmer) for 30 sec and immediately after imaged using a Cytiva AI800 imager.

Splicing efficiency of murine and human otoferlin.

Splicing efficiency was tested for split sites in human otoferlin and the homologous murine otoferlin. Figure 15 shows a highly efficient splicing at 724/725 of murine mOTOF.

Figure 16 shows that split sites in human and murine otoferlin at the corresponding positions in the sequence show a highly comparable. Based upon these data it is highly plausible that the split site 710/711 of human counterpart is as efficient as split site 724/725 of murine otoferlin.

Claims

-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,

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. 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. 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. 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. A set of nucleic acids encoding the fusions proteins according to any one of claims 1 to 5.

7. The set of nucleic acids according to claim 6, wherein the nucleic acids are AAV vectors.

8. The set of nucleic acids according to claim 7, wherein expression of the proteins is under control of a tissue specific promotor.

9. The set of nucleic acids according to claim 8, wherein the tissue specific promoter is a Myol5a promoter.

10. A set of AAV vectors according to any one of claims 7 to 9, for use in the treatment of hearing loss.