AAV dose-dependent transduction efficiency in RGCs and functional efficacy of CoChR3M-mediated vision restoration

Abstract

Purpose : The loss of photoreceptors in retinal degenerative diseases leads to vision impairment
or complete blindness. Optogenetics is a promising approach to restore vision after the death of
photoreceptors. We previously reported that AAV-mediated expression of a highly light-
sensitive ChR variant, CoChR3M, in retinal ganglion cells (RGCs) enables restoring functional
vision in a triple knock-out (TKO; Gnat1 -/- Cnga3 -/- Opn4 -/- ) blind mouse model under ambient
light conditions. In this study, we investigated the AAV dose-dependent transduction efficiency
in RGCs and its correlation with the functional efficacy of CoChR3M-mediated vision
restoration.

Methods : The transgene of CoChR3M-GFP driven by CAG promoter were packaged into
AAV2.7m8-Y444F capsid variant. Viral vectors were injected intravitreally in TKO mice at the
tiers ranging from 1.5 x 10 ⁷ to 1.5 x 10 ¹⁰ vg/eye. Optomotor response (OMR) assays were
performed to assess the restored visual functions with the light sensitivity examined by a home-
designed optomotor system and visual acuity examined by OptoDrum (Stria.Tech). The
transduction efficiency of CoChR3M-GFP in RGCs was assessed by co-labeling antibodies
against GFP and RBPMS.

Results : The viral transduction efficiency in RGCs is viral titer dependent. The percentage of
transduced RGCs rises rapidly from ~12% at the viral titer of 1.5 x 10 ⁷ vg/eye to ~56% at the
titer of 1.5 x 10 ⁸ vg/eye. The transduction efficiency rises more slowly at the higher viral titers
and reaches a plateau of ~87% at the tier of 1.5 x 10 ¹⁰ vg/eye. With a similar but an inverse
relationship, the threshold light intensity required to evoke OMR decreases with the increase of
viral tier. Visual acuity can only be measured in mice with the injection of viral tiers of ≥1.5 x
10 ⁸ vg/eye in which the threshold light intensities required to evoked OMR fell below the light
intensity of OptoDrum (~30 µW/cm 2 ). The values of visual acuity were not significantly
different among the mice injected with the viral tiers ranging from1.5 x 10 ⁸ to 1.5 x 10 ¹⁰ vg/eye.

Conclusions : The light sensitivity of CoChR3M-mediated OMR is correlated with the density of
AAV transduced RGCs. Visual acuity can be measured with OptoDrum in mice injected with the
viral tiers of > 1.5 x 10 ⁸ vg/eye or the transduction of > 50% of RGCs. The injection of higher
viral tiers up to 1.5 x 10 ¹⁰ vg/eye does not significantly improve visual acuity.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.