Gene therapy restores vision to the all-cone Nrl^-/-/Opn1mw^-/-/Opn1sw^-/- mouse model of blue cone monochromacy

Abstract

Purpose : Blue cone monochromacy (BCM) is a rare X-linked cone dysfunction syndrome characterized by the absence or severe reduction of both L- and M- cone function. Studies in patients show that remaining foveal L/M cones retain sufficient inner segment structure to be a target for gene therapy. Our previous gene replacement studies for BCM patients with deletion mutations have been conducted in rod-dominant mouse models. Here we investigate gene replacement in the Nrl^-/-/Opn1mw^-/-/Opn1sw^-/- mouse, an all-cone model deficient in cone opsin, mimicking the foveal structure of human BCM patients with deletion mutations.

Methods : Previously generated Opn1mw^-/-/Opn1sw^-/- mice were bred to a Nrl^-/- background to create the triple knockout (TKO) mouse. TKO mice were injected subretinally at 1-month (1M) and 3M with human L- opsin under cone-specific promoter PR2.1 and packaged in rAAV8-Y733F. Photopic electroretinograms (ERGs) assessed short-term (1M post-injection) and long-term (5M post-injection) visual function, while retinal cross-sections were examined by immunohistochemistry (IHC) with antibodies against L/M opsin, PDE6C, and GNAT2 to assess outer segment structure.

Results : TKO cones exhibit shortened outer segments with no photopic ERG response, structurally and functionally mimicking BCM foveae. At 1M post-injection, the average b-wave maximum amplitudes at a light intensity of 120 cd*s/m² were 205±46μV and 138±41μV (avg.±SD) in 1M and 3M treated eyes, respectively. This is ~50% and ~78% of age-matched Nrl^-/- control eyes and significantly higher than untreated eyes, which have a flat ERG response. Structural rescue was also observed as exogenous L-opsin replenished the outer segment along with GNAT2 and PDE6C. In 1M injected eyes, structural and functional rescue persisted for at least 5M with amplitudes of photopic ERG responses higher than age matched Nrl^-/- control eyes, demonstrating treatment to be protective against degeneration caused by Nrl knockout.

Conclusions : Our results demonstrate that all-cone retinas lacking cone opsin expression can be rescued long-term by gene supplementation. Future directions include characterizing the therapeutic window and evalutating the efficacy of gene therapy delivered by intravitreal injection.

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