ARTICLE https://doi.org/10.1038/s41467-018-08264-w OPEN Improved TMC1 gene therapy restores hearing and balance in mice with genetic inner ear disorders Carl A. Nist-Lund1, Bifeng Pan 1,2, Amy Patterson1, Yukako Asai1,2, Tianwen Chen3, Wu Zhou3, Hong Zhu3, Sandra Romero4, Jennifer Resnik2,4, Daniel B. Polley2,4, Gwenaelle S. Géléoc1,2 & Jeffrey R. Holt1,2,5 Fifty percent of inner ear disorders are caused by genetic mutations. To develop treatments for genetic inner ear disorders, we designed gene replacement therapies using synthetic 1234567890():,; adeno-associated viral vectors to deliver the coding sequence for Transmembrane Channel- Like (Tmc) 1 or 2 into sensory hair cells of mice with hearing and balance deficits due to mutations in Tmc1 and closely related Tmc2. Here we report restoration of function in inner and outer hair cells, enhanced hair cell survival, restoration of cochlear and vestibular function, restoration of neural responses in auditory cortex and recovery of behavioral responses to auditory and vestibular stimulation. Secondarily, we find that inner ear Tmc gene therapy restores breeding efficiency, litter survival and normal growth rates in mouse models of genetic inner ear dysfunction. Although challenges remain, the data suggest that Tmc gene therapy may be well suited for further development and perhaps translation to clinical application. 1 Department of Otolaryngology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, USA. 2 Department of Otolaryngology, Harvard Medical School, Boston, MA 02139, USA. 3 Department of Otolaryngology and Communicative Sciences, University of Mississippi Medical Center, Jackson, MS 39216, USA. 4 Eaton-Peabody Laboratories, Massachusetts Eye and Ear Infirmary, Boston, MA 02139, USA. 5 Department of Neurology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA. These authors contributed equally: Carl A. Nist-Lund, Bifeng Pan. Correspondence and requests for materials should be addressed to J.R.H. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:236 | https://doi.org/10.1038/s41467-018-08264-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-018-08264-w earing loss is the most common neurological disorder and For the current study we used a mouse model of DFNB7/11 Haffects an estimated 466 million people worldwide, 35 recessive deafness to investigate whether sAAVs encoding Tmc1 million of which are children (World Health Organiza- can enhance auditory recovery, beyond that previously reported tion). In addition to direct loss of auditory function, a number of for Tmc1 packaged in conventional AAV1 capsids12. In addition, secondary consequences can lead to diminished quality of life1, we ask whether restoration of function in the auditory periphery including impaired cognitive development in children, social can drive neuronal function at the level of auditory cortex, which isolation, depression, and dementia2,3. Despite the devastating would be required for perception of complex sounds and speech. consequences of hearing loss, there are no biological treatments We also investigate the ability of exogenous Tmc1 and Tmc2 to for auditory dysfunction. The current standards of care provide restore vestibular and balance function in mouse models of only partial restoration for a limited patient population and genetic vestibular dysfunction. Since both normal hearing and include hearing aids, which boost sound levels and require some balance contribute to quality of life in humans, we investigate residual function, and cochlear implants, which bypass the sen- whether sAAVs encoding TMC proteins improve secondary sory organ and directly stimulate the eighth cranial nerve. measures of recovery in mouse models of inner ear dysfunction. In addition to the auditory organ, the inner ear includes five Although challenges remain15,16, we conclude that efficient vestibular organs which mediate sensitivity to linear head delivery of wild-type Tmc1 restores hearing, balance and several moment and gravity, as well as rotational head movements secondary outcomes, suggesting inner ear gene therapy for TMC1 around three orthogonal axes. Together, the vestibular end organs mutations may be well-suited for further development and per- help maintain stable posture and gaze and contribute to the sense haps translation to the clinic. of balance. Loss of vestibular function can lead to vertigo (i.e., the illusion of motion), imbalance, blurred vision and orthostatic intolerance. Vestibular dysfunction is a risk factor for falling, Results which is one of the most common reasons elderly patients seek Tmc1 gene therapy restores sensory transduction. In prior medical attention4. Unfortunately, there are no biological treat- work, we assessed Tmc1 gene therapy using an AAV2/1 serotype ments for genetic vestibular dysfunction. with a chicken beta actin (Cba) promoter driving expression of In a continuing effort to develop biological treatments for inner wild-type Tmc1 in a mouse model of DFNB7/1112. We reported ear dysfunction, here we focus on development of improved gene recovery of sensory transduction in inner hair cells (IHCs), but therapy strategies to restore function in mouse models of genetic little or no recovery in outer hair cells (OHCs), most likely due to inner ear disorders. In particular, we focus on transmembrane lack of expression or targeting in OHCs of AAV2/1-injected channel-like 1 (TMC1) which encodes a protein required for the cochleas12. As a result, recovery of auditory function was modest. proper function and survival of inner ear hair cells. Recent evi- More recently, we demonstrated that synthetic AAV2/Anc80L65, dence demonstrates that TMC1, and probably TMC2, are pore- together with a cytomegalovirus (Cmv) promoter, efficiently forming components of mechanosensory transduction channels expressed eGFP in both IHCs and OHCs when injected via the in auditory and vestibular hair cells5,6. Mutations in Tmc1/TMC1 round window membrane (RWM) at early postnatal stages17. For cause both dominant and recessive forms of deafness in mice and the present study, we combined the two strategies and generated humans7,8. Estimates suggest three to eight percent of genetic gene therapy vectors that encoded a Cmv promoter, an identical hearing loss may be due to TMC1 mutations9,10. Some recessive therapeutic gene, Tmc1ex1, followed by a WPRE sequence, TMC1 mutations cause moderate to severe hearing loss with post- packaged into synthetic AAV2/Anc80L65 capsids: AAV2/ lingual onset10,11. These patients may retain viable hair cells Anc80L65-Cmv-Tmc1ex1-WPRE (for brevity: sAAV-Tmc1). We amenable to gene therapy intervention. injected 1 μL of sAAV-Tmc1 (1.4 × 1014 gc/mL) between post- Prior gene therapy strategies that targeted TMC1 focused on natal day (P) 0 and P2 via RWM of Tmc1Δ/Δ;Tmc2Δ/Δ mice. gene replacement using conventional adeno-associated viral To determine the efficacy of viral targeting, expression of (AAV) vector serotypes to restore function in mouse models of exogenous Tmc1, and restoration of sensory transduction in recessive TMC1 deafness, DFNB7/1112, gene silencing using auditory hair cells, we visualized uptake of FM1-43, a styryl dye either AAV delivery of microRNAs13 or ribonucleotide protein which permeates hair cell sensory transduction channels20,21. complexes to deliver CRISPR/Cas914 to target dominant TMC1 Since both Tmc1 and Tmc2 are expressed at early postnatal stages mutations. While these therapeutic strategies revealed no adverse (<P10)22, we injected inner ears of Tmc1Δ/Δ;Tmc2Δ/Δ mice which effects, they yielded only partial recovery of auditory function. lack expression of endogenous TMC1 and TMC2 proteins, and The recovery was limited due to inefficient delivery of the ther- thus do not take up FM1-4312,22. Likewise, age-matched, sham apeutic DNA constructs to the targeted cell types, inner and outer injected Tmc1Δ/Δ;Tmc2Δ/Δ mice also lacked FM1-43 uptake. hair cells of the cochlea. Although these approaches demonstrated However, in Tmc1Δ/Δ;Tmc2Δ/Δ mice seven days post-injection successful targeting of IHCs, efficient targeting of OHCs has been with sAAV-Tmc1, we found robust FM1-43 uptake in most IHCs problematic for these and other studies15,16. Outer hair cells (91 ± 8%, n = 4 cochleas) and half of the OHCs (50 ± 17%), (OHCs) amplify and tune auditory information, which is trans- suggesting expression of exogenous TMC1 and the presence of duced and transmitted by inner hair cells (IHCs). Both cell types functional sensory transduction (Fig. 1a). are required for the fully functional cochlea, which enables To assess sensory transduction currents we recorded from exquisite sensitivity to faint sounds and fine pitch perception. IHCs and OHCs of Tmc1Δ/Δ;Tmc2Δ/Δ mice injected on P1 with To address the limited recovery of auditory function with sAAV-Tmc1. Cochleas were harvested on P5-P7 and maintained conventional AAV vectors, we previously screened a number of in organotypic culture. On the equivalent of P7, Tmc1Δ/Δ;Tmc2Δ/ viral capsids and identified a synthetic AAV (sAAV), known as Δ hair cells which did not take up FM1-43, lacked sensory Anc80L065, which transduced IHCs and OHCs with high effi- transduction (Fig. 1b, left). FM1-43-positive OHCs within the ciency17. In prior work, the Anc80L065 capsid was shown to same culture displayed robust sensory transduction (Fig. 1b, efficiently transduce a variety of cell