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) Time course of the amplitude of the full-field scotopic ERG a-wave recorded in dark-reared rd10 mice injected with AAV92YF-RdCVFL (n = 5) or AAV92YF-scCAG-GFP (n = 5) revealed a delay in loss of retinal responses in treated animals. Four weeks after injection, a significant difference in the amplitude of the ERG a-wave was observed in RdCVFL-injected animals compared with that seen in GFP-injected mice. Five weeks after injection, both groups had progressively reduced a-wave amplitudes, and no difference in ERG amplitudes between the 2 groups was observed. Significance was determined using a repeated-measures 2-way ANOVA with Sidak’s multiple comparisons test. (B) A more detailed analysis of ERG traces recorded 4 weeks after injection of AAV92YF-RdCVFL or PBS revealed increased a-wave amplitudes over a range of flash intensities. Significance was determined using a 2-way ANOVA with post-hoc multiple comparisons test. (C) Representative ERG traces from RdCVFL-treated (left traces) or PBS-injected (right traces) animals illustrating increased amplitudes of the scotopic a-wave and b-wave 4 weeks after injection in treated mice. (D) Injection of AAV92YF-scCAG-RdCVFL resulted in an increase in the level of rhodopsin mRNA 4 weeks after injection compared with levels in untreated or GFP- or RdCVF-expressing eyes. Significance was determined by 1-way ANOVA with Tukey’s post-hoc multiple comparisons test. (E) TBARS assay revealed reduced levels of MDA, a marker for oxidative stress and lipid peroxidation, in retinae injected with AAV92YF-RdCVFL and collected 4 weeks after injection compared with MDA levels in AAV92YF-RdCVF, AAV92YF-GFP, or PBS-injected eyes (n = 3 for all groups). Data are presented as the percentage of change in MDA concentrations compared with those in untreated rd10 eyes ± SD. *P < 0.05; **P < 0.01.