Intravitreal injection of AAV7m8.hLCA5 restores photoreceptor function in Lca5-/- mice to nearly WT levels.

Abstract

Purpose : Early onset vision loss results from mutations in LCA5, which encodes Lebercilin, a protein critical for photoreceptor health and function. We tested the possibility that gene augmentation therapy restores photoreceptor and retinal function using multi electrode array (MEA) in LCA5 mouse model (Lca5-/-).

Methods : Postnatal day 5 Lca5-/- mice received unilateral intravitreal injection with AAV7m8.CMV/CBA.hLCA5 (~9.87E+10 vg/eye) spiked with 5% (v/v) AAV7m8.CBA.GFP. Contralateral eyes were uninjected. 5 age-matched wildtype (WT) mice were positive controls. Retinas from light-adapted mice were dissected under red light and mounted ganglion cell side down in the MEA chamber. Calibrated full field flashes of 455 nm light (efficiency of pigment excitation ~40%:rhodopsin and M-opsin; ~0.2%:S-opsin) of different intensities were used for light stimulation (2 s flashes at 0.1 Hz or 50 ms flashes at 4 Hz). Data were analyzed using custom code in Matlab; spike sorting was performed in Plexon Offline Sorter.

Results : Presence of GFP confirmed exposure of photoreceptors to AAV. After 3-4 months, 3 out of 4 injected retinas had strong light responses and 1 showed minimal response in MEA testing. Responses became detectable at scotopic intensities (42-112 photons*s^-1*μm^-2, 455 nm) and strong responses were observed through the brightest photopic intensity of 2.00e9 photons*s^-1*μm^-2. Retinas from uninjected eyes showed minimal to abolished responses. As expected for rod/cone driven responses, after exposure to brightest light, scotopic responses disappeared but photopic responses were not significantly affected. Response kinetics were similar to those in WT retinas. Light sensitivity was slightly lower in treated Lca5-/- vs the most sensitive WT retinas where first responses were detected at 8-21 photons*s^-1*μm^-2. All ganglion cell types identifiable in the WT retinas under full field stimulation (ON-, OFF- and ON/OFF-types) were detected in the Lca5-/- treated retinas after spike sorting. WT and treated Lca5-/- retinas were responsive to 4Hz flicker stimulation at 3.53e2-2.00e9 photons*s^-1*μm^-2. All untreated Lca5-/- retinas showed slow melanopsin driven responses at brighter intensities which were absent in the light sensitive treated Lca5-/- and WT retinas.

Conclusions : Given successful transduction, gene therapy can restore degenerating retinal cells to a state close to the WT conditions.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.