Unilateral Cochlear Delivery of Virally Mediated Gene Therapy Demonstrates Bilateral Expression in VGLUT3 Knockout Mice

Abstract

Objectives: (1) Examine whether unilateral round window membrane injection of adeno-associated virus-1 carrying vesicular glutamate transporter-3 (AAV1-VGLUT3) in congenitally deaf VGLUT3 knockout (KO) mice results in bilateral VGLUT3 expression. (2) Quantify hearing recovery in ipsilateral versus contralateral cochleae following VGLUT3 rescue. Methods: Five wild-type (WT) and 7 VGLUT3 rescued KO mice were used; 2 VGLUT3 KO mice served as deaf controls. Postnatal-day 1 (P1)-P3 VGLUT3 KO mice to be rescued underwent left-ear fixed-volume round window injection of AAV1-VGLUT3. On P21-28, auditory brainstem responses (ABRs) were performed to determine bilateral hearing thresholds. Immunofluorescence was used to count inner hair cells (IHCs) expressing VGLUT3. Results: The 2 VGLUT3 KO mice were completely deaf and showed no IHC expression of VGLUT3. Rescued mice demonstrated VGLUT3 IHC expression in the injected side (85.8 ± 4.6% relative to WT) that was greater than the contralateral side (31.1 ± 22.7% relative to WT; P = .03). ABR thresholds were 33.1 (±2.7) dB ipsilaterally and 46.0 (±14.1) dB contralaterally ( P = .03). Conclusions: The cochlea represents an attractive target for gene therapy due to its bony compartmentalization, but the extent of transfection outside the cochlea has yet to be fully studied. Our study suggests that hearing restoration and IHC transfection occurs bilaterally after unilateral injection, although contralateral changes are significantly less. This supports a model of extracochlear viral spread via cerebrospinal fluid and perilymph, and has important potential implications for future implementation of cochlear gene therapy. This preliminary study provides the basis for a larger, future project examining cochlear and brain expression following VGLUT3 rescue.