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• W2902016625 abstract "Report3 December 2018Open Access Source DataTransparent process A dual-AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock-out mice Hanan Al-Moyed Hanan Al-Moyed Molecular Biology of Cochlear Neurotransmission Group, Department of Otorhinolaryngology, University Medical Center Göttingen, and Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences, University of Göttingen, Göttingen, Germany Search for more papers by this author Andreia P Cepeda Andreia P Cepeda orcid.org/0000-0002-7197-1168 Molecular Biology of Cochlear Neurotransmission Group, Department of Otorhinolaryngology, University Medical Center Göttingen, and Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences, University of Göttingen, Göttingen, Germany Search for more papers by this author SangYong Jung SangYong Jung Institute for Auditory Neurosciences and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Tobias Moser Tobias Moser orcid.org/0000-0001-7145-0533 Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences, University of Göttingen, Göttingen, Germany Institute for Auditory Neurosciences and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Sebastian Kügler Corresponding Author Sebastian Kügler [email protected] orcid.org/0000-0002-5130-2012 Center Nanoscale Microscopy and Physiology of the Brain (CNMPB), Department of Neurology, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Ellen Reisinger Corresponding Author Ellen Reisinger [email protected] orcid.org/0000-0003-3739-7569 Molecular Biology of Cochlear Neurotransmission Group, Department of Otorhinolaryngology, University Medical Center Göttingen, and Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Search for more papers by this author Hanan Al-Moyed Hanan Al-Moyed Molecular Biology of Cochlear Neurotransmission Group, Department of Otorhinolaryngology, University Medical Center Göttingen, and Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences, University of Göttingen, Göttingen, Germany Search for more papers by this author Andreia P Cepeda Andreia P Cepeda orcid.org/0000-0002-7197-1168 Molecular Biology of Cochlear Neurotransmission Group, Department of Otorhinolaryngology, University Medical Center Göttingen, and Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences, University of Göttingen, Göttingen, Germany Search for more papers by this author SangYong Jung SangYong Jung Institute for Auditory Neurosciences and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Tobias Moser Tobias Moser orcid.org/0000-0001-7145-0533 Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences, University of Göttingen, Göttingen, Germany Institute for Auditory Neurosciences and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Sebastian Kügler Corresponding Author Sebastian Kügler [email protected] orcid.org/0000-0002-5130-2012 Center Nanoscale Microscopy and Physiology of the Brain (CNMPB), Department of Neurology, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Ellen Reisinger Corresponding Author Ellen Reisinger [email protected] orcid.org/0000-0003-3739-7569 Molecular Biology of Cochlear Neurotransmission Group, Department of Otorhinolaryngology, University Medical Center Göttingen, and Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Search for more papers by this author Author Information Hanan Al-Moyed1,2, Andreia P Cepeda1,2, SangYong Jung3,4, Tobias Moser2,3,4, Sebastian Kügler *,5 and Ellen Reisinger *,1 1Molecular Biology of Cochlear Neurotransmission Group, Department of Otorhinolaryngology, University Medical Center Göttingen, and Collaborative Research Center 889, University of Göttingen, Göttingen, Germany 2Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences, University of Göttingen, Göttingen, Germany 3Institute for Auditory Neurosciences and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany 4Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany 5Center Nanoscale Microscopy and Physiology of the Brain (CNMPB), Department of Neurology, University Medical Center Göttingen, Göttingen, Germany *Corresponding author. Tel: +49 551 39 8351; E-mail: [email protected] *Corresponding author. Tel: +49 551 39 9688; E-mail: [email protected] EMBO Mol Med (2019)11:e9396https://doi.org/10.15252/emmm.201809396 See also: JR Holt & GSG Geleoc (January 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Normal hearing and synaptic transmission at afferent auditory inner hair cell (IHC) synapses require otoferlin. Deafness DFNB9, caused by mutations in the OTOF gene encoding otoferlin, might be treated by transferring wild-type otoferlin cDNA into IHCs, which is difficult due to the large size of this transgene. In this study, we generated two adeno-associated viruses (AAVs), each containing half of the otoferlin cDNA. Co-injecting these dual-AAV2/6 half-vectors into the cochleae of 6- to 7-day-old otoferlin knock-out (Otof−/−) mice led to the expression of full-length otoferlin in up to 50% of IHCs. In the cochlea, otoferlin was selectively expressed in auditory hair cells. Dual-AAV transduction of Otof−/− IHCs fully restored fast exocytosis, while otoferlin-dependent vesicle replenishment reached 35–50% of wild-type levels. The loss of 40% of synaptic ribbons in these IHCs could not be prevented, indicating a role of otoferlin in early synapse maturation. Acoustic clicks evoked auditory brainstem responses with thresholds of 40–60 dB. Therefore, we propose that gene delivery mediated by dual-AAV vectors might be suitable to treat deafness forms caused by mutations in large genes such as OTOF. Synopsis Gene delivery of large genes exceeding the packing capacity of a single adeno-associated virus (AAV) is challenging. Split-AAV vectors can bypass this problem. This work offers the first application of split-AAV gene therapy to the inner ear to restore hearing in a genetically deaf mouse model. The 6 kb-long otoferlin coding sequence was split into two fragments and packaged into two separate AAV2/6 viruses which were co-injected into cochleae of otoferlin knock-out mice. Both the dual-AAV trans-splicing and the hybrid strategy led to re-assembly of the two otoferlin cDNA fragments and expression of full-length otoferlin. Otoferlin expression from dual-AAVs was restricted to hair cells and reached ˜30% of wild type otoferlin protein levels in inner hair cells. In inner hair cells, fast exocytosis of the readily releasable pool of vesicles was fully recovered, and vesicle replenishment was restored to 35–50% of wild-type controls. Auditory brainstem responses were present albeit with reduced wave amplitudes in dual-AAV transduced otoferlin knock-out mice and indicated hearing thresholds of 40–60 dB. Introduction Mutations in the OTOF gene cause profound congenital non-syndromic autosomal recessive hearing loss (DFNB9, Yasunaga et al, 1999). Otof−/− mice or animals with deleterious point mutations in this gene are profoundly deaf (Roux et al, 2006; Longo-Guess et al, 2007; Pangrsic et al, 2010; Reisinger et al, 2011). The OTOF gene encodes otoferlin, a large multi-C2-domain protein predominantly expressed in inner hair cells (IHCs), the genuine sensory cells in the cochlea. IHCs transform mechanical acoustic vibrations into a neural code via synaptic transmission to auditory neurons. Studies using Otof−/− and Otof knock-in mouse models revealed that this protein plays an essential role in IHC exocytosis and vesicle replenishment, and is involved in vesicle reformation and endocytosis (Roux et al, 2006; Pangrsic et al, 2010; Duncker et al, 2013; Jung et al, 2015; Strenzke et al, 2016). To date, over one thousand pathogenic mutations have been identified within this gene, affecting 2.3–10% of patients from various ethnicities suffering from hereditary non-syndromic hearing loss (Rodríguez-Ballesteros et al, 2008; Choi et al, 2009; Iwasa et al, 2013; Van Camp & Smith). In contrast to many other deafness or Usher genes, otoferlin seems dispensable for auditory hair cell (HC) development (Roux et al, 2006). Given the normal inner ear morphology in these patients, a postnatal transfer of otoferlin cDNA into the inner ear is predicted to ameliorate this hearing loss. Gene therapy might outperform the otherwise necessary cochlear implantation, which transmits only part of the acoustic information. Cochlear implant users often report difficulties in speech understanding during noise and in perceiving vocal emotions, and typically cannot experience music as a person without hearing impairment (Fu et al, 1998; Nelson et al, 2003; McDermott, 2004; Luo et al, 2007; Most & Aviner, 2009; Oxenham & Kreft, 2014; Chatterjee et al, 2015; Paquette et al, 2018). Yet, no delivery method for large genes, like electroporation or viral transduction via adenoviruses, lentiviruses, or semliki forest viruses, transferred cDNA into HCs in the postnatal inner ear with high efficiency. Recombinant adeno-associated viruses (AAVs) are a safe and promising gene therapy tool to treat hearing impairment (reviewed in Ref. Géléoc & Holt, 2014). Prenatal injections of AAVs into the developing otocyst or postnatal cochlear injections have been proven to efficiently transduce IHCs in animal models (Liu et al, 2005; Bedrosian et al, 2006). However, the limited AAV cargo capacity of approximately 4.7–5 kb presents an obstacle for the transfer of large coding sequences (CDS) such as the 6 kb-long otoferlin cDNA. Split-AAV vectors, each containing a fragment of the large transgene expression cassette, have been developed to circumvent this problem. This technique takes advantage of the intrinsic ability of the AAV genome to form tail-to-head concatemers by end-joining of its inverted terminal repeats (ITRs) (Duan et al, 1998). In the “trans-splicing (TS)” strategy, the ITRs are spliced out after transcription by introducing artificial splice donor (SD) and acceptor (SA) sites before and after the ITRs in the respective half-vectors. The reconstitution of the large transgene can also be mediated through homologous recombination of overlapping sequences placed at the 3′-end of the first AAV half-vector and at the 5′-end of the second in the “overlap” split-AAV strategy. The “hybrid” strategy is a combination of both previous strategies (Ghosh et al, 2008). The correct reassembly of the full-length expression cassette in the nuclei of target cells results in the production of full-length mRNA and protein (Yan et al, 2000; Duan et al, 2001; Chamberlain et al, 2016). To date, dual- and triple-AAV vectors have demonstrated efficacy in photoreceptors and muscle cells (Duan et al, 2001; Ghosh et al, 2008; Trapani et al, 2014; Maddalena et al, 2018), but have not been established for IHCs. Results and Discussion In this study, we investigated whether the delivery of full-length otoferlin cDNA into IHCs via dual-AAV vectors can restore defective IHC and auditory functions in Otof−/− mice. We aimed for a rather late time point of treatment, since the early development of the inner ear does not seem to require otoferlin (Roux et al, 2006), making gene therapy of mature Otof−/− IHCs feasible in theory. AAVs were injected into the cochlea at postnatal day 6–7 (P6–7) because the auditory bulla structure covering the round window membrane (RWM) is still soft enough at this developmental stage to be penetrated well with an injection glass pipette. We chose AAVs with ITRs from serotype 2 and capsid proteins from serotype 6 (AAV2/6) as they can be produced with an excellent transducing-unit to vector-genome ratio (Grieger et al, 2016). This prevents administration of excess protein bolus into the delicate structure of the inner ear. To test whether this AAV serotype transduces IHCs efficiently, we injected single AAV2/6 viruses coding for eGFP through the RWM into the scala tympani of the left cochlea of CD1xC57BL/6N-F1 (CD1B6F1) wild-type mice. eGFP fluorescence was observed in IHCs, outer hair cells (OHCs), supporting cells, and spiral ganglion neurons (SGNs), indicating that the AAV2/6 has no specific IHC tropism and targets a variety of different cell types within the organ of Corti (Fig EV1A). 34–99% of IHCs (average = 77 ± 4%, mean ± standard error of the mean (s.e.m., n = 7 cochleae) exhibited eGFP fluorescence, revealing a high IHC transduction efficiency of the AAV2/6. This finding contrasts recent reports showing that AAV2/6 failed to transduce IHCs if injected at P1–2 (Shu et al, 2016). We assume that vector quality, titer, the injection procedure itself, and the animal age at the time of surgery are all factors influencing IHC transduction efficiency. Click here to expand this figure. Figure EV1. AAV2/6 transduces various cell types in the inner ear A, B. Low magnification views for eGFP immunofluorescence in CD1B6F1 wild-type organs of Corti transduced with AAV2/6 vectors, indicating a broad cell type tropism both for a single eGFP-expressing AAV2/6 (A; P23) and eGFP expressed from otoferlin dual-AAV-TS vectors (B; P27). Images were acquired and displayed with the same settings. Organs of Corti were co-immunolabeled for otoferlin (magenta) to visualize IHCs. (B1, B2) High magnification views of (B) displayed with higher intensity showing eGFP immunofluorescence in IHCs and supporting cells (B1) and in spiral ganglion neurons (B2) in dual-AAV-TS-treated wild-type mice. Individual eGFP immunostainings were depicted as color lookup tables with warmer colors representing higher pixel intensities (max). Maximum intensity projections of optical confocal sections. Vg, vector genomes. Scale bars: 100 μm (A, B), 50 μm (B1, B2). Download figure Download PowerPoint For gene replacement therapy in Otof−/− mice, we used mouse otoferlin transcript variant 4 cDNA (NM_001313767), coding for the 1977 amino acid-long protein and previously confirmed to be expressed endogenously in wild-type cochleae (Strenzke et al, 2016). We designed otoferlin dual-AAV-trans-splicing (dual-AAV-TS) and dual-AAV-hybrid (dual-AAV-Hyb) half-vectors, both containing the N-terminal otoferlin CDS in the 5′-AAV half-vector and the C-terminal CDS in the 3′-AAV vector (Fig EV2). Expression from the 5′AAV is driven by a human β actin promoter/CMV enhancer and additionally codes for a separately translated eGFP-fluorescent reporter to identify transduced cells in acutely isolated organs of Corti. These split-AAV vectors were co-injected through the RWM into the left cochlea of P6–7 CD1B6F1-Otof−/− mice. Organs of Corti from these animals were isolated at P18–30 and immunolabeled with two otoferlin antibodies, one binding within the N-terminal part of otoferlin and the other one binding after the transmembrane domain close to the C-terminus of otoferlin (Figs 1A–C and EV3, Appendix Fig S1–S3). Upon dual-AAV injection into Otof−/− cochleae, we found otoferlin immunofluorescence to be restricted to auditory HCs, with stronger expression in IHCs and much weaker in sparsely transduced OHCs (Appendix Fig S3), resembling otoferlin expression in wild-type animals (Roux et al, 2006; Beurg et al, 2008). eGFP fluorescence, on the contrary, was also found in other cell types (Figs 1A and EV1B, Appendix Fig S3), although the expression of both proteins is driven by the same promoter and they are translated from the same mRNA (Fig EV2). Neither eGFP nor otoferlin expression could be detected in contralateral non-injected ears (Fig 2A, Appendix Fig S1 and S2). We presume that a yet unknown mechanism such as post-transcriptional regulation or targeted protein degradation restricts the expression of otoferlin to auditory HCs. A similar finding was reported for AAV1 postnatal RWM injections (P1–3 and P10–12), where Vglut3 expression was found selectively in IHCs despite the broad cell type tropism of this AAV serotype (Akil et al, 2012). The restricted expression of otoferlin to HCs would be very beneficial for human gene therapy applications to avoid potential off-target effects due to expression of exogenous otoferlin in non-sensory cells in the inner ear. Click here to expand this figure. Figure EV2. Dual-AAV vector strategies for full-length otoferlin gene transfer A, B. Schematic representation of the otoferlin dual-AAV-TS (A) and dual-AAV-Hyb (B) half-vectors used for postnatal cochlear injections. Both otoferlin dual-AAV half-vector systems contain the first half of the otoferlin coding sequence (CDS) in the 5′-AAV and the other half in the 3′-AAV half-vector. The correct reconstitution of the full-length otoferlin mRNA in the dual-AAV-TS strategy is mediated by non-homologous end joining of the inverted terminal repeats (ITRs). In the dual-AAV-Hyb strategy, the reassembly is mediated by non-homologous end joining of the ITRs and/or homologous recombination of the highly recombinogenic AK sequence. Splice donor (SD) and splice acceptor (SA) sites facilitate the excision of the ITRs via trans-splicing. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) stabilizes the mRNA. To ensure the production of two separate proteins, a P2A peptide inducing ribosomal skipping is introduced between the eGFP and the otoferlin CDS. hbA: human beta-actin promoter, CMVe: cytomegalovirus enhancer, pA: polyadenylation signal. Download figure Download PowerPoint Figure 1. Dual-AAV-mediated otoferlin expression is restricted to auditory hair cells in the cochlea A. Low magnification views of a CD1-Otof−/− organ of Corti (P23)-transduced with otoferlin dual-AAV-TS vectors. IHCs: inner hair cells, OHCs: outer hair cells. B, C. High magnification views of CD1B6F1-Otof−/− IHCs transduced with otoferlin dual-AAV-TS (P26) (B) and dual-AAV-Hyb (P26) (C) vectors. Individual eGFP and otoferlin immunostainings are depicted as color lookup tables in (A-C) with warmer colors representing higher pixel intensities. See Fig EV3 for comparison to wild-type IHCs. D. Percentage of N- and C-terminal otoferlin labeled IHCs in dual-AAV-TS (n = 10 mice)- and dual-AAV-Hyb (n = 9 mice)-injected CD1B6F1-Otof−/− mice (P18–30). E. Average N-terminal and C-terminal otoferlin immunofluorescence levels in dual-AAV-transduced Otof−/− and wild-type IHCs (P23–30). Otoferlin levels were normalized to immunofluorescence levels in non-transduced B6 wild-type IHCs for each antibody separately. Data information: In (A–C), maximum intensity projections of confocal optical sections. Scale bars: 100 μm (A), 10 μm (B, C). In (D), individual animals are depicted with open symbols. In (E), the number of quantified IHCs is indicated inside the bars. In (D, E), data are displayed as mean ± s.e.m., ns P > 0.05; *P ≤ 0.05; **P ≤ 0.01;***P ≤ 0.001, [Wilcoxon matched-pair signed rank test (D), unpaired t-test with Welch's correction (D), and Kruskal–Wallis test followed by Dunn's multiple comparison test (E)]. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Cellular localization of eGFP and otoferlin in dual-AAV-TS and dual-AAV-Hyb-transduced Otof−/− IHCs compared to wild-type IHCs A–D. High magnification views of dual-AAV-TS (C; P26) and dual-AAV-Hyb (D; P26)-transduced CD1B6F1-Otof−/− IHCs depicted in Fig 1B and C and compared to AAV2/6.eGFP transduced CD1B6F1 wild-type (B; P28) and non-injected B6 wild-type (A; P27) IHCs. Successful virus transduction is monitored via eGFP immunofluorescence. Organs of Corti were immunolabeled against the N-terminal (magenta) and C-terminal (white) part of otoferlin. HCs were immunolabeled with calbindin. Individual eGFP, otoferlin, and calbindin immunostainings are depicted as color lookup tables with warmer colors representing higher pixel intensities. Non-transduced IHCs are labeled with white asterisks, and one transduced IHC displaying only eGFP and N-terminal otoferlin fluorescence, but hardly any C-terminal otoferlin fluorescence with a yellow asterisk. All samples were processed in parallel and acquired and displayed with the same settings. Maximum intensity projections of optical confocal sections. Scale bars: 10 μm. Download figure Download PowerPoint Figure 2. Otoferlin dual-AAV injection at P6–7 partially restores synaptic function in Otof−/− IHCs A. High magnification views of IHCs immunolabeled for otoferlin and synaptic ribbons (CtBP2) from wild-type (B6: P27, CD1B6F1: P29), dual-AAV-injected CD1B6F1-Otof−/− (dualAAV-TS: P26, dualAAV-Hyb: P28), and their contralateral non-injected ears. (*) Transduced cells. Maximum intensity projections of optical confocal sections. Scale bars: 5 μm. B. Synaptic ribbon numbers quantified from IHCs in apical cochlear turns of wild-type (B6: n = 48 IHCs, CD1B6F1: n = 108 IHCs), transduced Otof−/− (dualAAV-TS: n = 59 IHCs, dualAAV-Hyb: n = 37 IHCs), and non-transduced Otof−/− IHCs from injected (-AAV-injected ear, n = 65 IHCs) and contralateral non-injected (-AAV non-injected ear, n = 46 IHCs) ears (P25–29). C. IHC synapses labeled with CtBP2 and the postsynaptic marker Shank1a in B6 wild-type and Otof−/− P6 and P14 organs of Corti. Maximum intensity projections of optical confocal sections. Scale bars: 5 μm. D. Synapse numbers quantified from IHCs in apical cochlear turns (C) of B6 wild-type (P6: n = 53 IHCs; P14: n = 73 IHCs) and B6-Otof−/− (P6: n = 62 IHCs; P14: n = 65 IHCs) mice at two different developmental stages (P6 and P14). E. Ca2+-current–voltage relationship of control CD1B6F1 wild-type (n = 6 IHCs), dual-AAV-TS-transduced (n = 8 IHCs), and non-transduced CD1B6F1-Otof−/− (n = 10 IHCs) IHCs (P14–18). F. Representative Ca2+-currents (Ica) and IHC plasma membrane capacitance increments (ΔCm) of a wild-type control, transduced, and non-transduced Otof−/− IHC in response to a 20 ms depolarization pulse at maximum Ca2+-current potentials (typically −14 mV). G, H. Average exocytosis level measured as ΔCm (G) and corresponding Ca2+-current integrals (QCa2+) (H) in wild-type [CD1B6F1: n = 6 IHCs; B6: n = 11 IHCs (B6 data replotted from Strenzke et al, 2016)], dual-AAV-TS-transduced Otof−/− (n = 8 IHCs), and non-transduced (n = 11 IHCs) Otof−/− IHCs. Individual dual-AAV-TS transduced Otof−/− IHCs expressing eGFP, that had exocytosis (thinner red lines) and almost no exocytosis (broken red lines; not included into the average), are depicted. Data information: In (B, D), individual animals are depicted with open symbols. In (G), individual transduced Otof−/− IHCs are displayed with thinner or broken red lines. In (B, D, E, G, H), data are displayed as mean ± s.e.m., ns P > 0.05; **P ≤ 0.01; ***P ≤ 0.001 (Kruskal–Wallis test followed by Dunn's multiple comparison test). Download figure Download PowerPoint The number of IHCs immunolabeled with both N-terminal and C-terminal otoferlin antibodies along the entire cochlea ranged from 12 to 51% in dual-AAV-TS (average: 30 ± 4%, n = 10 animals) and from 5 to 34% in dual-AAV-Hyb (average: 19 ± 3%, n = 9 animals) injected Otof−/− mice (Fig 1D). Approximately 10% of IHCs were solely labeled by the N-terminal otoferlin antibody, likely indicating no correct reassembly of the two virus half-vectors in on average one out of four transduced cells (Figs 1D and EV3). We did not find any IHC showing only a C-terminal otoferlin immunofluorescence signal, which was expected since the 3′-AAV half-vector does not contain a separate promoter. A previous study indicated that 70% of intact IHCs suffice for proper auditory function (Wang et al, 1997). Further studies will reveal if optimizing the virus administration procedure into the cochlea (e.g., as in ref. Yoshimura et al, 2018) might increase the otoferlin IHC transduction rate. In order to examine whether the split full-length otoferlin expression cassette reassembled to produce the correct full-length mRNA in the target cells, we isolated mRNA from transduced Otof−/− organs of Corti (P26–29) and amplified otoferlin cDNA fragments spanning the dual-AAV split-site (Fig EV4). From dual-AAV-transduced Otof−/− organs of Corti and wild-type control samples, we amplified a PCR product with the size expected for full-length otoferlin cDNA (1,753 bp) that was absent in non-injected Otof−/− organ of Corti samples (Fig EV4A and B). Amplicons from wild-type, control Otof−/−, and dual-AAV-TS-transduced Otof−/− samples were subcloned and representative clones were subjected to Sanger sequencing (n = 2 clones/4 clones/5 clones, respectively; Fig EV4B and C). This confirmed the correct reconstitution of the full-length transgene from the two otoferlin AAV half-vectors and the presence of an artificially introduced AccIII restriction site found only in the dual-AAV-transduced Otof−/− samples (Fig EV4C). In cDNA samples of dual-AAV-injected and non-treated Otof−/− organs of Corti, we amplified three otoferlin cDNA fragments of 1,379, 1,480, and 1,679 bp, all lacking exons 14 and 15 (Fig EV4B and C). The larger amplicons originate from incomplete splicing of the mutant mRNA (Fig EV4C). These splice variants might be translated into shorter fragments, the presence of which we assessed by Western blot (Fig EV4D). In wild-type organs of Corti, we detected two specific bands of ~210–230 kDa, likely corresponding to full-length otoferlin, which were absent in Otof−/− controls. However, due to a strong unspecific band at ~100 kDa, the presence of smaller otoferlin fragments that might interfere with the function of full-length otoferlin could not be excluded. Click here to expand this figure. Figure EV4. Otoferlin dual-AAV-TS-transduced Otof−/− organs of Corti express full-length otoferlin mRNA A. Schematic representation of otoferlin cDNA from otoferlin dual-AAV-transduced, wild-type, and Otof−/− organs of Corti, displaying binding sites of primers used in PCRs to assess dual-AAV reassembly. B. Otoferlin PCR amplicons from organ of Corti cDNA. A 1,753-bp-long amplicon (*), also present in non-injected wild-type controls (WTB6, WTCD1B6F1), indicates successful reassembly of the split otoferlin expression cassette in otoferlin dual-AAV-TS-transduced CD1B6F1-Otof−/− organs of Corti (injected ear). In Otof−/− samples, three shorter products were amplified (a, b and c). C. Sanger sequencing confirmed correct dual-AAV split-site assembly (dashed line) as well as the presence of an artificial AccIII restriction site introduced in the dual-AAV-TS otoferlin cDNA, which is absent in the wild-type (WT) and Otof−/− cDNA (a–c). Amplicons a-c from Otof−/− organs of Corti all lack exons 14–15, while bands “b” (1,480 bp) and “c” (1,679 bp) still contain intron 20–21 (b) or intron 23–24 (c), respectively. D. Western blotting on cell lysates of WT and Otof−/− CD1B6F1 organs of Corti. Two bands of ˜210–230 kDa, corresponding to full-length otoferlin, were detected in WT but absent in Otof−/− ears. (**) refers to an unspecific band detected in both samples. GAPDH was used as loading control. Data information: CDS: coding sequence, Ex: exon, TS: trans-splicing, Hyb: hybrid, control ear: non-treated ears, non-injected ear: contralateral non-injected Otof−/− ears. Source data are available online for this figure. Download figure Download PowerPoint To quantify full-length otoferlin protein expression levels, we measured the fluorescence intensity of the C-terminal otoferlin antibody in transduced Otof−/− IHCs and normalized it to the C-terminal immunofluorescence in wild-type C57BL/6J (B6) IHCs (Figs 1E and EV3). Dual-AAV-mediated gene transfer into Otof−/− IHCs led to the expression of full-length otoferlin protein with 31–37% of wild-type levels (Fig 1E). We found the intracellular pattern of N- and C-terminal otoferlin immunolabeling in transduced Otof−/− IHCs to be similar to wild-type IHCs (Fig EV3). Non-transduced Otof−/− IHCs from contralateral non-injected ears displayed very weak N-terminal and C-terminal background otoferlin fluorescence signals of 3 ± 0.0% and 6 ± 0.0% of wild-type levels, respectively (Fig 1E, Appendix Fig S1 and S2). In dual-AAV-TS-transduced wild-type IHCs, the eGFP fluorescence signal was weaker than in those cells transduced with the single AAV2/6.eGFP virus (Figs EV1 and EV3). An earlier study reported that Otof−/− IHCs have normal ribbon synapse numbers a" @default.
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• W2902016625 title "A dual‐AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock‐out mice" @default.
• W2902016625 cites W1809106246 @default.
• W2902016625 cites W1965234374 @default.
• W2902016625 cites W1971459128 @default.
• W2902016625 cites W1974980963 @default.
• W2902016625 cites W1987022414 @default.
• W2902016625 cites W1991705397 @default.
• W2902016625 cites W1992961518 @default.
• W2902016625 cites W1997083976 @default.
• W2902016625 cites W2001999470 @default.
• W2902016625 cites W2002473366 @default.
• W2902016625 cites W2014354304 @default.
• W2902016625 cites W2020352076 @default.
• W2902016625 cites W2023545713 @default.
• W2902016625 cites W2033347933 @default.
• W2902016625 cites W2034060287 @default.
• W2902016625 cites W2054096183 @default.
• W2902016625 cites W2054771268 @default.
• W2902016625 cites W2055648999 @default.
• W2902016625 cites W2061600749 @default.
• W2902016625 cites W2068759427 @default.
• W2902016625 cites W2070638917 @default.
• W2902016625 cites W2071245802 @default.
• W2902016625 cites W2077882371 @default.
• W2902016625 cites W2078577861 @default.
• W2902016625 cites W2082328650 @default.
• W2902016625 cites W2084711412 @default.
• W2902016625 cites W2091869150 @default.
• W2902016625 cites W2105057076 @default.
• W2902016625 cites W2106378095 @default.
• W2902016625 cites W2107252915 @default.
• W2902016625 cites W2109302572 @default.
• W2902016625 cites W2111035895 @default.
• W2902016625 cites W2111081671 @default.
• W2902016625 cites W2112722770 @default.
• W2902016625 cites W2128604557 @default.
• W2902016625 cites W2137826201 @default.
• W2902016625 cites W2152759640 @default.
• W2902016625 cites W2155756146 @default.
• W2902016625 cites W2172332629 @default.
• W2902016625 cites W2223143741 @default.
• W2902016625 cites W2280525353 @default.
• W2902016625 cites W2314835883 @default.
• W2902016625 cites W2417093940 @default.
• W2902016625 cites W2502831104 @default.
• W2902016625 cites W2531867258 @default.
• W2902016625 cites W2586762515 @default.
• W2902016625 cites W2605246466 @default.
• W2902016625 cites W2746153430 @default.
• W2902016625 cites W2771095058 @default.
• W2902016625 cites W2807140244 @default.
• W2902016625 cites W2886113798 @default.
• W2902016625 cites W2886288118 @default.
• W2902016625 cites W2886400303 @default.
• W2902016625 cites W2888544544 @default.
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