Restoration of Hearing in the VGLUT3 Knockout Mouse Using Virally Mediated Gene Therapy

Abstract

Summary Mice lacking the vesicular glutamate transporter-3 (VGLUT3) are congenitally deaf due to loss of glutamate release at the inner hair cell afferent synapse. Cochlear delivery of VGLUT3 using adeno-associated virus-1 (AAV1) leads to transgene expression in only inner hair cells (IHC), despite broader viral uptake. Within two weeks of AAV1-VGLUT3 delivery, acoustic brainstem response (ABR) thresholds normalize, along with partial rescue of the startle response. Lastly, we demonstrate partial reversal of the morphologic changes seen within the afferent IHC ribbon synapse. These findings represent the first successful restoration of hearing by gene replacement in mice, which is an important step towards gene therapy of human deafness.

Rescue of Hearing by Gene Delivery to Inner-Ear Hair Cells Using Exosome-Associated AAV

“…Prior studies had shown some transduction of IHCs with conventional AAVs, but little transduction of OHCs. 12,23 We found that exo-AAV1 efficiently transduces IHCs and transduces OHCs much more efficiently than does conventional AAV1. AAV1 was our standard of comparison because it has been used in prior studies of hearing gene therapy.…”

“…AAV1 was our standard of comparison because it has been used in prior studies of hearing gene therapy. 12,13 In those studies, it was shown to only transduce inner hair cells relatively efficiently. Interestingly, we found that exo-AAV9-GFP was extremely efficient at transduction of cochlear explant cultures, although, in vivo, this serotype performed similarly to exo-AAV1.…”

“…Adenoassociated virus (AAV) vectors are presently the most promising vectors for cochlear gene delivery, but in vivo transduction is mostly limited to IHCs. In previous studies, 12,13 virtually no OHCs were transduced by AAV vectors after in vivo injection in mice. Similarly, it is difficult to express genes in hair cells in vitro for research, which has slowed characterization of proteins involved in hair cell function.…”

“…12,13 We therefore prepared exo-AAV1 and conventional AAV1. AAV1 and exo-AAV1 were isolated from the lysate and culture medium of vector-producing 293T cells, respectively ( Figure 1A; see Materials and Methods).…”

“…We therefore compared conventional AAV1 and exo-AAV1 in vivo using direct injections into P0/P1 mouse cochleas. We compared two injection routes: through the round window membrane (RWM) into the scala tympani (used in two animal models of hereditary deafness 12,13 ), and through the lateral wall of the cochlea by cochleostomy at the basal turn. In both cases, 250 nL of virus-containing solution was injected over $10 min.…”