Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness

Article22 December 2020Open Access Source DataTransparent process Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness Shahar Taiber Shahar Taiber orcid.org/0000-0002-0787-4216 Department of Human Molecular Genetics & Biochemistry, Sackler Faculty of Medicine & Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Roie Cohen Roie Cohen School of Neurobiology, Biochemistry and Biophysics, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Ofer Yizhar-Barnea Ofer Yizhar-Barnea orcid.org/0000-0002-1953-1982 Department of Human Molecular Genetics & Biochemistry, Sackler Faculty of Medicine & Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author David Sprinzak David Sprinzak orcid.org/0000-0001-6776-6957 School of Neurobiology, Biochemistry and Biophysics, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Jeffrey R Holt Corresponding Author Jeffrey R Holt [email protected] orcid.org/0000-0002-7182-8011 Departments of Otolaryngology & Neurology, Boston Children’s Hospital & Harvard Medical School, Boston, MA, USA Search for more papers by this author Karen B Avraham Corresponding Author Karen B Avraham [email protected] orcid.org/0000-0002-4913-251X Department of Human Molecular Genetics & Biochemistry, Sackler Faculty of Medicine & Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Shahar Taiber Shahar Taiber orcid.org/0000-0002-0787-4216 Department of Human Molecular Genetics & Biochemistry, Sackler Faculty of Medicine & Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Roie Cohen Roie Cohen School of Neurobiology, Biochemistry and Biophysics, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Ofer Yizhar-Barnea Ofer Yizhar-Barnea orcid.org/0000-0002-1953-1982 Department of Human Molecular Genetics & Biochemistry, Sackler Faculty of Medicine & Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author David Sprinzak David Sprinzak orcid.org/0000-0001-6776-6957 School of Neurobiology, Biochemistry and Biophysics, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Jeffrey R Holt Corresponding Author Jeffrey R Holt [email protected] orcid.org/0000-0002-7182-8011 Departments of Otolaryngology & Neurology, Boston Children’s Hospital & Harvard Medical School, Boston, MA, USA Search for more papers by this author Karen B Avraham Corresponding Author Karen B Avraham [email protected] orcid.org/0000-0002-4913-251X Department of Human Molecular Genetics & Biochemistry, Sackler Faculty of Medicine & Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Author Information Shahar Taiber1, Roie Cohen2, Ofer Yizhar-Barnea1, David Sprinzak2, Jeffrey R Holt *,3 and Karen B Avraham *,1 1Department of Human Molecular Genetics & Biochemistry, Sackler Faculty of Medicine & Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel 2School of Neurobiology, Biochemistry and Biophysics, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel 3Departments of Otolaryngology & Neurology, Boston Children’s Hospital & Harvard Medical School, Boston, MA, USA *Corresponding author. Tel: +1 617 919 3574; E-mail: [email protected] *Corresponding author. Tel: +972 3 640 6642; Fax: +972 3 640 9360, E-mail: [email protected] EMBO Mol Med (2021)13:e13259https://doi.org/10.15252/emmm.202013259 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 Genetic variants account for approximately half the cases of congenital and early-onset deafness. Methods and technologies for viral delivery of genes into the inner ear have evolved over the past decade to render gene therapy a viable and attractive approach for treatment. Variants in SYNE4, encoding the protein nesprin-4, a member of the linker of nucleoskeleton and cytoskeleton (LINC), lead to DFNB76 human deafness. Syne4−/− mice have severe-to-profound progressive hearing loss and exhibit mislocalization of hair cell nuclei and hair cell degeneration. We used AAV9-PHP.B, a recently developed synthetic adeno-associated virus, to deliver the coding sequence of Syne4 into the inner ears of neonatal Syne4−/− mice. Here we report rescue of hair cell morphology and survival, nearly complete recovery of auditory function, and restoration of auditory-associated behaviors, without observed adverse effects. Uncertainties remain regarding the durability of the treatment and the time window for intervention in humans, but our results suggest that gene therapy has the potential to prevent hearing loss in humans with SYNE4 mutations. Synopsis Syne4 deficiency leads to hearing loss in humans. In this work, auditory function was rescued in Syne4 knockout mice by a synthetic AAV that enables safe and efficient transduction of hair cells in the cochlea. AAV9-PHP.B transduced inner and outer hair cells with high efficiency. Gene delivery preserved nuclear position in cochlear hair cells. Auditory brainstem responses and otoacoustic emissions showed near complete rescue of auditory function. Local delivery into the inner ear was sufficient to rescue hearing-dependent behavior. The paper explained Problem Gene therapy is a promising strategy to treat genetic deafness. Since the auditory systems of humans and mice are very similar in structure, function, and even gene expression, mice serve as an excellent model for basic and translational auditory research. Genetic variants in SYNE4, encoding the nesprin-4 protein, have been shown to cause deafness in humans, and Syne4-deficient mice show a similar phenotype. In Syne4-knockout mice, the nuclei of outer hair cells (OHCs) lose their basal position and degenerate. Results We used a gene-replacement approach to rescue hearing in a mouse model of SYNE4 deafness. This strategy required delivery of the coding sequence of Syne4 into the inner ears of neonatal Syne4-knockout mice by a synthetic adeno-associated virus, AAV9-PHP.B. The results reveal near-complete rescue of hair cell morphology and survival, with normalization of auditory function and behavioral responses. Impact There are currently over 120 genes associated with inherited deafness. It is of paramount importance to test the feasibility of gene therapy in animal models in order to facilitate the development of future treatments. Our results provide proof of concept for the development of gene therapy for SYNE4 and other forms of deafness. Introduction Hearing loss affects approximately 466 million people worldwide (Olusanya et al, 2019). A genetic cause can be identified in 60% of the cases of hearing loss in multiplex families, and more than 120 genes have been associated with non-syndromic hearing loss in humans (Brownstein et al, 2020; Van Camp & Smith, 2020). Although tremendous progress has been made in the understanding of the physiology the auditory system, there are still no biological treatments for hearing loss in humans. Major efforts are currently being made to develop gene, cell, and pharmacological therapeutics for various types of hearing loss, but current treatment options are still primarily restricted to sound amplification and cochlear implants (Muller & Barr-Gillespie, 2015; Schilder et al, 2018). Variants in SYNE4 (Spectrin Repeat Containing Nuclear Envelope Family Member 4) have been found to cause autosomal recessive progressive, high-tone hearing loss in individuals in Israel, the UK, and Turkey (PanelApp.; Horn et al, 2013; Masterson et al, 2018). SYNE4 codes for the protein nesprin-4, a member of the linker of nucleoskeleton and cytoskeleton (LINC) complex (Roux et al, 2009). Nesprins localize to the outer nuclear membrane, where they interact with inner nuclear membrane SUN proteins, and with cytoplasmatic cytoskeleton elements such as actin and intermediate filaments, as well as motor proteins such as kinesins and dynein (Cartwright & Karakesisoglou, 2014). Mice lacking Syne4 or Sun1 exhibit progressive hearing loss, reminiscent of DFNB76; in Syne4 knockout mice (Syne4−/−), hair cells develop normally, but the outer hair cell (OHC) nuclei gradually lose their basal position, leading to subsequent OHC degeneration (Horn et al, 2013). Preliminary results in animal models identified adeno-associated virus (AAV) as a promising candidate for gene therapy in deafness (Landegger et al, 2017; Akil et al, 2019; Isgrig et al, 2019; Nist-Lund et al, 2019). AAVs appear to elicit little to no immune response, and recombinant AAVs integrate into the host at very low rates, which reduces the risks of genotoxicity (Nakai et al, 2001). Initial characterization of natural AAV serotypes revealed a relatively low transduction rate of inner ear cell types, and in particular of OHC (Kilpatrick et al, 2011). However, recently developed synthetic AAV capsids seem to have overcome this hurdle; AAV9-PHP.B has been shown to transduce both inner and outer hair cells at high rates in mice and non-human primates (Gyorgy et al, 2019; Ivanchenko et al, 2020; Lee et al, 2020). In this study, we used Syne4−/− mice as a model of DFNB76 recessive deafness, in order to develop a genetic therapy for this form of human deafness, based on AAV9-PHP.B as a vector. In addition to morphological recovery of transduced OHC, we observed enhanced OHC survival, improved auditory brainstem responses (ABR), and restored distortion-product otoacoustic emissions (DPOAE). In addition, we demonstrate that functional recovery of the inner ear is sufficient to drive complex behavioral responses that rely on processing of auditory cues in the central nervous system. Finally, we characterize the safety of exogenous Syne4 overexpression in both the auditory and vestibular systems. While the feasibility of translating these results to the clinic is still unclear, we conclude that our results in Syne4−/− mice suggest that gene therapy for DFNB76 is a future possibility that should be developed. Results Syne4−/− outer hair cells degenerate at hearing onset Nesprin-4, which is encoded by the Syne4 gene, has been shown to be important for nuclear positioning and OHC survival in mice (Horn et al, 2013). With the aim of developing gene therapy for Syne4−/− mice, we studied the dynamics of OHC loss in order to determine a therapeutic time window for intervention. A schematic illustration of the ear (Fig 1A), with a focus on the organ of Corti, as well as the timeline of the experiments performed in the study (Fig 1B), are shown. We analyzed hair cell survival in the inner ear at P8, P10, P12, and P14 (Figs 2A and B, and EV1). While at P8, the OHCs appeared intact, by P14, their degeneration was readily apparent (Fig 2A and B). This was also reflected in hair cell counts, with an apex-to-base gradient in the reduction of the number of OHCs by P14 (Fig 2C). FM1-43 is a styryl dye that can enter hair cells through the sensory transduction channels (Gale et al, 2001) and is used as a proxy for functional hair cell sensory transduction. We found that Syne4−/− hair cells could take up FM1-43 at P8, in a similar manner to wild-type (WT) hair cells (Fig 2D). This suggests that Syne4−/− hair cells mature and acquire the properties of functional hair cells prior to the onset of damage. In addition, we examined the expression pattern of Syne4 in published datasets via the gEAR portal (Portal) and found Syne4 expression to be relatively restricted to hair cells, with a higher expression in OHCs than inner hair cells (IHC) (Fig EV2A; Scheffer et al, 2015; Liu et al, 2018). Syne4 RNA is detected as early as P0, although the staining at P0 is weaker than at P12 (Horn et al, 2013, Fig EV2B). Interestingly, while Syne4 is also detected in the vestibular system, Syne4−/− mice exhibited no abnormal balance behavior (Fig EV2B and C). Figure 1. Schematic representation of research strategy and timeline Schematic representation of inner ear anatomy, with a focus on the organ of Corti and the cellular function of nesprin-4. Timeline of experiments performed. Download figure Download PowerPoint Figure 2. Syne4−/− hair cells mature normally and then degenerate between P12 and P14 Whole-mount immunofluorescence of a P8 Syne4−/− organ of Corti showing intact hair cells, as labeled with myosin VIIa. Whole-mount immunofluorescence of WT and Syne4−/− organ of Corti from the 8, 16, and 32 kHz regions at P8 and P14. Inner and outer hair cell counts of Syne4−/− organ of Corti at P8, P10, P12, and P14. FM1-43 uptake performed on P8+1 DIV (days-in-vitro) WT and Syne4−/− organ of Corti. Top shows OHC plane, and bottom shows IHC plane. Data information: Scale bars = 100 μm for (A) and 10 μm for (B and D). Source data are available online for this figure. Source Data for Figure 2 [emmm202013259-sup-0004-SDataFig2.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Syne4−/− hair cells at P10 and P12 Whole-mount immunofluorescence of Syne4−/− organ of Corti from the 8, 16, and 32 kHz regions at P10 and P12, labeled with myosin VIIa. Scale bar = 10 μm. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Expression of Syne4 in the inner ear and vestibular phenotype Syne4 expression in RNA-seq results from pooled P28-35 cells from CBA/J mice, n = 3 (Liu et al, 2018). Syne4 expression in RNA-seq results from 16 samples of E16, P0, P4, and P7 Pou4f3-eGFP mice (Scheffer et al, 2015). Plots show average RPKM. Open-field results of WT and Syne4−/− mice at minutes 5–15 of the test, n = 9 for WT and n = 11 for Syne4−/−. Statistical test was unpaired Student’s t-test. Data information: Plots show mean ± SD. Source data are available online for this figure. Download figure Download PowerPoint AAV9-PHP.B transduces cochlear hair cells in neonatal mice AAV9-PHP.B is a synthetic AAV capsid that has been engineered by directed in-vivo evolution (Deverman et al, 2016) and transduces both IHCs and OHCs at high rates (Gyorgy et al, 2019; Lee et al, 2020). Expression of GFP delivered in AAV9-PHP.B begins rising reliably between days 3 and 5 postinjection (Lee et al, 2020). We therefore chose this capsid as a vector for gene therapy in Syne4−/− mice. We cloned turboGFP into an AAV2 backbone, downstream of a CMV enhancer and promoter and upstream to a bGH poly-A sequence, and packaged the construct into AAV9-PHP.B capsids (termed AAV.GFP). We then cloned the coding sequence (CDS) of Syne4 into an AAV2 backbone, downstream of a CMV enhancer and promoter, added a 3XFLAG epitope sequence at the 5′ end of the Syne4 CDS and a bGH poly-A sequence at the 3′ and packaged the construct into AAV9-PHP.B capsids (termed AAV.Syne4) (Fig 3A). The titers of AAV.Syne4 and AAV.GFP were 7.7E + 12 gc/ml and 8.6E + 12 gc/ml, respectively. Figure 3. AAV9-PHP.B transduction and Syne4 expression Schematic representation of AAV.Syne4 and AAV.GFP constructs. Whole-mount immunofluorescence of a P9 organ of Corti of a mouse injected with AAV.GFP at P1 showing complete transduction of both inner and outer HC. Myosin VIIa was used to label the hair cells. Examples of 8, 16, and 32 kHz regions of an organ of Corti of a mouse injected with AAV.GFP. Top shows OHC plane, bottom shows IHC plane, and right shows YZ orthogonal projection. Black asterisks show bright Deiters cells. Quantification of GFP intensity of inner and outer HC from 3 injected mice, normalized to the average intensity of HC in a control, un-injected mouse. Transduction rates of AAV9-PHP.B at 8, 16, and 32 kHz regions based on GFP fluorescence. A total of 162 IHC and 841 OHC were analyzed from 3 injected mice and 1 control littermate. Staining for FLAG at P14 of the organ of Corti of a mouse injected at P1 with AAV.Syne4. Quantification of FLAG and DAPI fluorescence intensity along a line centered at the nuclear envelope. Eight OHCs were measured. 3D surface projection of two adjacent OHC from a mouse injected with AAV.Syne4. In the left cell, myosin VIIa and DAPI signals were removed to only show FLAG staining. Data information: Scale bars = 100 μm for (B), 10 μm for (C and F), and 5 μm for (H). Plots show mean ± SD. Source data are available online for this figure. Source Data for Figure 3 [emmm202013259-sup-0005-SDataFig3.zip] Download figure Download PowerPoint In order to examine the transduction efficiency of this capsid, we injected WT mice at P0–P1.5 with AAV.GFP, using the previously described posterior-semicircular canal (PSCC) approach for inner ear delivery (Isgrig & Chien, 2018). Inner ears were harvested at P9 for immunofluorescence and quantification of transduction rate (Fig 3B). Comparing the results to an un-injected littermate as control for background fluorescence, we observed GFP expression in all IHCs and OHCs in the 8, 16, and 32 kHz regions of the organ of Corti, as well as strong fluorescence in Deiters cells, pillar cells, and Hensen’s cells (Fig 3C). The normalized GFP intensity was similar between three individual injected mice and typically higher in OHC as compared to IHC (Fig 3D). Cells were regarded as GFP positive if the GFP intensity was higher than 2 standard deviations above the average intensity measured in the control mouse. Despite the transduction rate being 100% in all three regions of the cochlea in all three mice examined (Fig 3E), IHC fluorescence was lower than OHC (Fig 3D). In addition, we injected mice with AAV.Syne4 and stained the ears for FLAG. We observed FLAG staining in a pattern that indicated nesprin-4 was localized to the nuclear envelope (Fig 3F–H), as described previously for endogenous nesprin-4 (Roux et al, 2009; Horn et al, 2013), suggesting that the 3XFLAG-nesprin-4 protein was folded correctly. Viral transduction and Syne4 overexpression not associated with long-term ototoxicity or vestibulotoxicity To test the safety of the AAV9-PHP.B capsid, as well as the overexpression of exogenous Syne4, we injected WT mice with AAV.Syne4 at P0-P1.5, and evaluated ABR and DPOAE at 4, 8, and 12 weeks. Injected mice showed no significant difference from control mice in ABR threshold values (P > 0.58 for all frequencies tested at 4 weeks, P > 0.25 for all frequencies tested at 8 weeks, and P > 0.49 for all frequencies tested at 12 weeks, Fig EV3, EV4). DPOAE thresholds at 4w were also not significantly different (P > 0.89 for all frequencies tested) (Fig EV3D). Open-field tests were performed at 12 weeks to exclude possible toxicity to the vestibular system. Injected mice showed no overt balance defects (P > 0.19 for the three parameters tested) (Fig EV3E). Finally, weight gain, which was used as a measurement of general health, remained unchanged in injected mice (P > 0.7 for WT mice compared to WT mice injected with AAV.Syne4 at all time points, Fig EV3F). Click here to expand this figure. Figure EV3. Safety profile characterization of AAV.Syne4 ABR thresholds at 4w of WT mice injected at P1 with AAV.Syne4 and un-injected controls, n = 7 for WT and n = 10 for WT + AAV.Syne4. ABR thresholds at 8w of WT mice injected at P1 with AAV.Syne4 and un-injected controls, n = 6 for WT and n = 6 for WT + AAV.Syne4. ABR thresholds at 12w of WT mice injected at P1 with AAV.Syne4 and un-injected controls, n = 6 for WT and n = 5 for WT + AAV.Syne4. DPOAE thresholds at 4w of WT mice injected at P1 with AAV.Syne4 and un-injected controls, n = 6 for WT and n = 6 for WT + AAV.Syne4. Open-field results of WT and WT + AAV.Syne4 mice at 12w at minutes 5-15 of the test, n = 9 for WT and n = 7 for WT + AAV.Syne4. Weight gain over time in the different groups tested, n = 5 for WT, n = 5 for Syne4−/−, n = 11 for Syne4−/− + AAV.Syne4, n = 6 for WT + AAV.Syne4, and n = 7 for Syne4−/− + AAV.GFP. Data information: Statistical tests were 2-way ANOVA for ABR and DPOAE with Holm–Sidak correction for multiple comparisons, Student’s t-test for vestibular tests, and mixed-effects model for weight gain. Plots show mean ± SD. ns = not significant. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. ABR thresholds of injected Syne4−/− mice at 8w ABR thresholds at 8w of WT, Syne4−/−, Syne4−/− mice injected with AAV.Syne4, and Syne4−/− mice injected with AAV.GFP, n = 6 for WT, n = 5 for Syne4−/−, n = 11 for Syne4−/− + AAV.Syne4, and n = 7 for Syne4−/− + AAV.GFP. Statistical tests were 2-way ANOVA with Holm–Sidak correction for multiple comparisons. Plots show mean ± SD. ****P < 0.0001, *P < 0.05, ns = not significant. Source data are available online for this figure. Download figure Download PowerPoint AAV.Syne4 prevents nuclear mislocalization in Syne4−/− outer hair cells To test the effect of virally mediated expression of Syne4 on hair cell morphology, we injected Syne4−/− mice with AAV.Syne4 at P0–P1.5 and harvested inner ears at P14 (Fig 4). In Syne4−/− mice, the nuclei of OHCs are mislocalized and are positioned close to the cuticular plate (Fig 4A and B). Hair cell nuclear position was quantified semi-automatically, according to the length of an arc measured between the apical and basal ends of the cell and the position of the nucleus along that arc (Fig 4C). As compared to un-injected Syne4−/− mice, the OHC nuclei in injected mice were situated closer to the base, but were not identical to the situation in WT mice (Fig 4D). In contrast, the nuclear position in the IHCs was not affected by either the mutation or the treatment (Fig 4D). Figure 4. AAV.Syne4 rescues OHC morphology in Syne4−/− mice Whole-mount immunofluorescence of the 8 kHz region from P14 organ of Corti from WT, Syne4−/−, and Syne4−/− mice injected with AAV.Syne4. 3D surface projection of IHC and OHC at P14 from the 8kHz region of WT, Syne4−/− mice and Syne4−/− mice injected with AAV.Syne4. Open arrows denote basal end, and white arrows denote cuticular plate. Image analysis of nucleus position of IHC and OHC. Images show an arc fitted through the apical surface, nucleus, and basal end of the cell in 3D. X, Y, and Z axes show position in μm. Quantification of nuclear distance from the cuticular plate. A total of 45 IHC and 135 OHC from 3 WT mice, 45 IHC and 81 OHC from 3 Syne4−/− mice, and 30 IHC and 90 OHC from 2 Syne4−/− mice injected with AAV.Syne4 were measured. Statistical test was 2-way ANOVA with Holm–Sidak correction for multiple comparisons. Plots show mean ± SD. ns = not significant, ****P < 0.0001. Data information: Scale bars = 10 μm for (A) and 5 μm for (B). Source data are available online for this figure. Source Data for Figure 4 [emmm202013259-sup-0006-SDataFig4.zip] Download figure Download PowerPoint AAV.Syne4 rescues auditory function to near wild-type levels To evaluate the therapeutic effect of Syne4 delivery, we injected Syne4−/− mice at P0-P1.5 with AAV.Syne4 or AAV.GFP as a control, and assessed the ABR and DPOAE at 4, 8, and 12 weeks (Figs 5 and EV4). For two mice, there was no evidence of success of the injection, as evaluated by immunofluorescence analysis of Syne4 expression or ABR/DPOAE recovery, and thus, they were excluded from downstream analyses. The results revealed that mice injected with AAV.Syne4 (n = 20) had fully restored auditory function at 4 weeks that was sometimes indistinguishable from WT controls (P < 0.0001 for injected Syne4−/− mice, as compared to Syne4−/− mice injected with AAV.GFP or un-injected) (Fig 5A and B). At 4 weeks, the amplitudes and latencies of the response to a 0.1 ms click stimulus as a function of stimulus intensity were also highly similar to those of WT controls (Fig 5C and D). DPOAE assessments at 4 weeks showed complete recovery of thresholds (P < 0.0001 for injected Syne4−/− mice compared to un-injected Syne4−/− mice at 12.4–3.5 kHz, P = 0.22 for 6 kHz, P > 0.53 for injected Syne4−/− mice compared to wild-type mice for all frequencies tested). This finding suggests that the OHCs of treated mice were functional (Fig 5E). Figure 5. AAV.Syne4 rescues auditory function in Syne4−/− mice Representative example of ABR traces obtained at 4w from a WT, Syne4−/− mouse, and Syne4−/− mouse injected with AAV.Syne4 in response to 18 kHz stimuli. ABR thresholds at 4w of WT, Syne4−/−, Syne4−/− mice injected with AAV.Syne4, and Syne4−/− mice injected with AAV.GFP, n = 7 for WT, n = 8 for Syne4−/−, n = 20 for Syne4−/− + AAV.Syne4, and n = 6 for Syne4−/− + AAV.GFP. Quantification of P1-N1 amplitude delta from (B), n = 4 for WT, n = 4 for Syne4−/−, and n = 7 for Syne4−/− + AAV.Syne4. Quantification of P1 latency from (B), n = 4 for WT, n = 2 for Syne4−/−, and n = 7 for Syne4−/− + AAV.Syne4. DPOAE thresholds at 4w, n = 6 for WT, n = 5 for Syne4−/−, and n = 6 for Syne4−/− + AAV.Syne4. ABR threshold at 12w, n = 6 for WT, n = 8 for Syne4−/−, n = 16 for Syne4−/− + AAV.Syne4, and n = 5 for Syne4−/− + AAV.GFP. Data information: Statistical test was 2-way ANOVA with Holm–Sidak correction for multiple comparisons. Plots show mean ± SD. ns = not significant, *P < 0.05, **P < 0.01 ****P < 0.0001. Source data are available online for this figure. Source Data for Figure 5 [emmm202013259-sup-0007-SDataFig5.zip] Download figure Download PowerPoint At 12 weeks, the ABR thresholds of treated mice were already significantly higher than those of WT mice, but still significantly lower than Syne4−/− mice injected with AAV.GFP or un-injected (P < 0.05 for injected Syne4−/− mice compared to WT mice for 12–35 kHz, P = 0.055 for 6 kHz, P < 0.0001 for 6–24 kHz, P = 0.001 for 30 kHz, and P = 0.032 for 35 kHz for Syne4−/− mice compared to Syne4−/− mice injected with AAV.Syne4) (Fig 5F). It is worth noting that while the parameters in some mice deteriorated over time, others did not, and that the best performing mouse in the injected group still had low ABR thresholds at 12 weeks (30, 15, 15, 30, 35, and 45 dB-SPL for 6, 12, 18, 24, 30, and 35 kHz, respectively). AAV.Syne4 promotes long-term outer hair cell survival In order to examine the effect of treatment on hair cell survival, we counted myosin VIIa-positive cells along the length of the cochlea. The results indicated survival of virtually all OHC in treated Syne4−/− mice, lasting up to 12 weeks postinjection, but no apparent difference in IHC survival between the three groups (Fig 6A–C). To further validate our hypothesis that Syne4 deafness stems predominantly from OHC loss and not impaired IHC function, we examined whether the variability in ABR threshold levels at 12 weeks could be explained by OHC survival. We found a strong negative correlation between the numbers of OHCs per 100 μm and the ABR threshold in the 12 kHz region of the organ of Corti (r = −0.89, P = 0.0003), which we defined as 1.63–1.69 mm from the apex, based on a place-frequency map of the cochlea (Müller et al, 2005) (Fig 6D). Since there was little change in IHC survival, the number of IHC was not correlated with the ABR threshold (r = −0.29, P = 0.36) (Fig 6D). We tested whether the observed deterioration of auditory function in some mice in the treatment group could be explained by a change in the position of the nuclei that does not lead to OHC death but does impair their function. For this purpose, we quantified the position of OHC nuclei at the 12 kHz region of the organ of Corti at 12 weeks (Fig 6E–G). We could not detect a significant change in their position at 12 weeks, suggesting that this could not explain the deterioration we observed in some of the treated mice. Figure 6. AAV.Syne4 promotes long-term survival of OHC in Syne4−/− mice Whole-mount immunofluorescence at 12w of the organ of Corti of a Syne4−/− mouse injected with AAV.Syne4 mouse injected with AAV.Syne4. Whole-mount immunofluorescence at 12w of the 12 kHz region of WT, Syne4−/− mouse, and Syne4−/− mouse injected with AAV.Syne4. IHC and OHC counts, n = 3 for WT, n = 4 for Mut, and n = 6 for Mut + AAV.Syne4. Correlation between HC count and ABR threshold at 12kHz. 3D surface projection of an OHC at 12w from the 12kHz region of WT and Syne4−/− mice injected with AAV.Syne4. Open arrows denote basal end, and white arrows denote cuticular plate. Image analysis of nucleus position of OHC. Quantification of nuclear distance from the cuticular plate. A total of 30 OHC from 3 WT mice and 38 OHC from 4 Syne4−/− mice injected with AAV.Syne4 were measured. Data information: Scale bars = 100 μm for (A), 10 μm for (B), and 5 μm for (E). Statistical tests were Pearson correlation with two-tailed P values for (D) and unpaired Student’s t-test for (G). Plots show mean ± SD. Source data are available online for this figure. Source Data for Figure 6