Alternative splicing of nucleoredoxin-like 1 (Nxnl1) results in 2 isoforms of the rod-derived cone viability factor. The truncated form (RdCVF) is a thioredoxin-like protein secreted by rods that promotes cone survival, while the full-length isoform (RdCVFL), which contains a thioredoxin fold, is involved in oxidative signaling and protection against hyperoxia. Here, we evaluated the effects of these different isoforms in 2 murine models of rod-cone dystrophy. We used adeno-associated virus (AAV) to express these isoforms in mice and found that both systemic and intravitreal injection of engineered AAV vectors resulted in RdCVF and RdCVFL expression in the eye. Systemic delivery of AAV92YF vectors in neonates resulted in earlier onset of RdCVF and RdCVFL expression compared with that observed with intraocular injection using the same vectors at P14. We also evaluated the efficacy of intravitreal injection using a recently developed photoreceptor-transducing AAV variant (7m8) at P14. Systemic administration of AAV92YF-RdCVF improved cone function and delayed cone loss, while AAV92YF-RdCVFL increased rhodopsin mRNA and reduced oxidative stress by-products. Intravitreal 7m8-RdCVF slowed the rate of cone cell death and increased the amplitude of the photopic electroretinogram. Together, these results indicate different functions for Nxnl1 isoforms in the retina and suggest that RdCVF gene therapy has potential for treating retinal degenerative disease.
Authors
Leah C. Byrne, Deniz Dalkara, Gabriel Luna, Steven K. Fisher, Emmanuelle Clérin, Jose-Alain Sahel, Thierry Léveillard, John G. Flannery
(A) Photopic ERG amplitudes in rd10 animals injected at P15 with 7m8-GFP, PBS, 7m8-RdCVFL, 7m8-RdCVF, or a mix of 7m8-RdCVF plus 7m8-RdCVFL. Injection of 7m8-scCAG-RdCVFL resulted in a small but statistically insignificant increase in the amplitude of the photopic ERG b-wave compared with that measured in PBS- or GFP-injected eyes. Injection of 7m8-scCAG-RdCVF significantly increased the amplitude of the photopic ERG wave compared with that that of control eyes. Coinjection of 7m8-scCAG-RdCVF and 7m8-scCAG-RdCVFL resulted in greater rescue of the photopic ERG. n = 5 for all groups. Significance was determined using 1-way ANOVA with Tukey’s post-hoc multiple comparisons test. (B) Representative ERG traces from eyes injected with 7m8-GFP, 7m8-RdCVFL, 7m8-RdCVF, or a mixture of 7m8-RdCVF plus 7m8-RdCVFL. Arrows indicate the onset of stimulus. (C) Representative flicker ERGs recorded at 30 Hz of eyes injected with 7m8-RdCVF or a mix of 7m8-RdCVF plus 7m8-RdCVFL indicated improvement over ERGs of control 7m8-GFP–injected eyes. Marks on scale bar indicate 10-μV increments. (D) Flat-mounted retinae labeled with anti–S-opsin and –M/L-opsin antibodies revealed higher density of cones in RdCVF-treated eyes compared with those in GFP-expressing contralateral eyes. Lower images illustrate improved densities of surviving cones in the central retina near the optic nerve head (asterisk) in 7m8-RdCVF–injected eyes. (E) Quantification of cone densities revealed greater numbers of anti–S-opsin– and anti–M/L-opsin–labeled cones in treated eyes compared with those detected in contralateral GFP-expressing eyes (n = 4). *P < 0.05 and **P < 0.01 by 2-tailed, paired Student’s t test.