Rational design of a Lfng-enhancer AAV construct drives specific and efficient gene expression in inner ear supporting cells.

Achieving cell-specific gene expression is crucial in the design of safe and efficacious gene therapies for the treatment of sensorineural hearing loss. Although a variety of adeno-associated virus (AAV) serotypes have been used to deliver genes to inner ear hair cells, few serotypes currently allow specific targeting of supporting cells. We sought to specifically target supporting cells by combining an AAV serotype with high tropism for the inner ear with enhancer sequences from the supporting cell-specific gene Lunatic Fringe (Lfng). We identified three candidate Lfng enhancer sequences using bioinformatic analysis to identify accessible chromatin and histone marks associated with active transcription of the Lfng locus in supporting cells. Candidate Lfng enhancers or the ubiquitous CBh promoter driving an EGFP reporter gene were packaged into the AAV-ie capsid, and the virus was introduced into the inner ear of neonatal mice. AAV-CBh-EGFP transduced multiple sensory and non-sensory inner ear cell types, as well as cells in the brain. One of the three Lfng enhancers gave robust EGFP expression in border cells, inner phalangeal cells, pillar cells, and all three rows of Deiters' cells along the entire cochlear duct, as well as in vestibular organ supporting cells. Significantly, no fluorescently labeled cells were detected in the brains of mice injected with this virus. We further designed an AAV-Lfng-CreERT2 vector that drove strong recombination in Cre reporter mice supporting cells after tamoxifen treatment. Our results provide a tool to specifically target supporting cells of the juvenile and adult inner ear.