Efficient gene transfer to the mouse inner ear is achieved with a synthetic adeno-associated viral vector. Efforts to develop gene therapies for hearing loss have been hampered by the lack of safe, efficient, and clinically relevant delivery modalities1,2. Here we demonstrate the safety and efficiency of Anc80L65, a rationally designed synthetic vector3, for transgene delivery to the mouse cochlea. Ex vivo transduction of mouse organotypic explants identified Anc80L65 from a set of other adeno-associated virus (AAV) vectors as a potent vector for the cochlear cell targets. Round window membrane injection resulted in highly efficient transduction of inner and outer hair cells in mice, a substantial improvement over conventional AAV vectors. Anc80L65 round window injection was well tolerated, as indicated by sensory cell function, hearing and vestibular function, and immunologic parameters. The ability of Anc80L65 to target outer hair cells at high rates, a requirement for restoration of complex auditory function, may enable future gene therapies for hearing and balance disorders.

Adeno-associated virus (AAV) vectors have emerged as a gene-delivery platform with demonstrated safety and efficacy in a handful of clinical trials for monogenic disorders. However, limitations of the current generation vectors often prevent broader application of AAV gene therapy. Efforts to engineer AAV vectors have been hampered by a limited understanding of the structure-function relationship of the complex multimeric icosahedral architecture of the particle. To develop additional reagents pertinent to further our insight into AAVs, we inferred evolutionary intermediates of the viral capsid using ancestral sequence reconstruction. In-silico-derived sequences were synthesized de novo and characterized for biological properties relevant to clinical applications. This effort led to the generation of nine functional putative ancestral AAVs and the identification of Anc80, the predicted ancestor of the widely studied AAV serotypes 1, 2, 8, and 9, as a highly potent in vivo gene therapy vector for targeting liver, muscle, and retina.

Gene therapy in the inner ear achieves unprecedented recovery of hearing and balance behavior in Usher syndrome mice. Because there are currently no biological treatments for hearing loss, we sought to advance gene therapy approaches to treat genetic deafness. We focused on Usher syndrome, a devastating genetic disorder that causes blindness, balance disorders and profound deafness, and studied a knock-in mouse model, Ush1c c.216G>A, for Usher syndrome type IC (USH1C). As restoration of complex auditory and balance function is likely to require gene delivery systems that target auditory and vestibular sensory cells with high efficiency, we delivered wild-type Ush1c into the inner ear of Ush1c c.216G>A mice using a synthetic adeno-associated viral vector, Anc80L65, shown to transduce 80–90% of sensory hair cells. We demonstrate recovery of gene and protein expression, restoration of sensory cell function, rescue of complex auditory function and recovery of hearing and balance behavior to near wild-type levels. The data represent unprecedented recovery of inner ear function and suggest that biological therapies to treat deafness may be suitable for translation to humans with genetic inner ear disorders.

Adeno-associated virus (AAV) is a highly promising gene transfer vector, yet major cellular requirements for AAV entry are poorly understood. Using a genome-wide CRISPR screen for entry of evolutionarily divergent serotype AAVrh32.33, we identified GPR108, a member of the G protein-coupled receptor superfamily, as an AAV entry factor. Of greater than 20 divergent AAVs across all AAV clades tested in human cell lines, only AAV5 transduction was unaffected in the GPR108 knockout (KO). GPR108 dependency was further shown in murine and primary cells in vitro. These findings are further validated in vivo, as the Gpr108 KO mouse demonstrates 10- to 100-fold reduced expression for AAV8 and rh32.33 but not AAV5. Mechanistically, both GPR108 N- and C-terminal domains are required for transduction, and on the capsid, a VP1 unique domain that is not conserved on AAV5 can be transferred to confer GPR108 independence onto AAV2 chimeras. In vitro binding and fractionation studies indicate reduced nuclear import and cytosolic accumulation in the absence of GPR108. We thus have identified the second of two AAV entry factors that is conserved between mice and humans relevant both in vitro and in vivo, further providing a mechanistic understanding to the tropism of AAV gene therapy vectors.