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) Fundus image of P45 WT mouse illustrating strong GFP expression across the retina after intravitreal injection of 7m8-scCAG-GFP at P15. (B) Flat mount image of a retina expressing GFP, photoreceptor-side-up, showing GFP expression in outer segments of the photoreceptors. (C) Confocal stack of transverse sections showing expression of the reporter gene across all retinal layers in a WT mouse. (D) Confocal image of a P45 rd10 retina showing GFP expression in the GCL and INL and a loss of photoreceptors resulting in loss of the ONL (arrowhead). (E) qRT-PCR indicating near-WT levels of Nxnl1 mRNA expression from eyes injected with 7m8-scCAG-RdCVF and 7m8-scCAG-RdCVFL. GFP-injected eyes had markedly reduced Nxnl1 mRNA levels compared with levels in WT or AAV-injected eyes. Levels of rhodopsin mRNA were similarly low across conditions in rd10 eyes. mRNA was collected at P45 from 5 animals injected intravitreally at P15. Data represent the mean ± SD. n = 5 animals for each condition. Scale bars: 40 μm (B); 50 μm (C and D).