LAUSR

Leber congenital amaurosis type 2 (LCA2) is an inherited disease that affects the integrity of the retina and results in severe visual impairment early in life [1]. This disease is caused due to mutations in the retinal pigmental epithelium (RPE) 65 kDa protein encoding gene, that generates an isomerohydrolase enzyme in the visual cycle [2]. Gene therapy is an attractive option for this condition, due to the relatively immune privileged nature of the eye. Adenoassociated virus serotype 2 (AAV2) vectors have been utilized to deliver RPE65 gene into LCA2 patients [3]. The long-term follow-up data [4] demonstrated a peak functional rescue, ~1 year after gene therapy, but subsequently a decline in RPE65 expression and immune response was noted [5]. Thus, AAV2 vectors that can augment visual function at significantly lower doses are needed. In our recent study [6], we have identified and demonstrated the role of ubiquitin-like modifiers such as Neddylation in AAV2 vectors and abolition of these sites, augmented coagulation factor IX expression in hemophilia B mice. The present study was designed to evaluate if these Neddylation-site modified AAV2 vectors are effective, during ocular gene therapy. To assess this, we packaged wild-type (WT) AAV2 vector (ssAAV2-RPE65; scAAV2CB-EGFP) and a Neddylation-mutant vector containing a human RPE65 gene or the enhanced green fluorescent protein (EGFP) gene (ssAAV2-K665Q-RPE65; scAAV2K665Q-CB-EGFP), as described earlier [6]. Vector titers were measured by a quantitative PCR and are expressed as vector genomes (vgs)/ml. All the animal experiments were approved by the IIT-Kanpur animal ethics committee. The AAV vectors thus generated were assessed by in vivo ocular gene transfer by different routes of delivery (intravitreal and subretinal) and strains of mice [C57BL6/J and rd12, Jackson Laboratory (Bar Harbor, USA)]. In the first set of investigations, eyes (n= 8 per group) of C57BL6/J mice were either mock injected or injected with AAV2-WT-EGFP and AAV2-K665Q-EGFP vectors by the intravitreal or subretinal route at a dose of 3 × 10 vgs/eye. Fluorescence imaging of the eyes was performed, 2 and 8 weeks after ocular gene transfer in a Micron IV imaging system (Phoenix Research Lab, Pleasanton, USA). Our data shown in Fig. 1a, demonstrate that the K665Q mutant had a significantly higher EGFP expression (7.87–9.72fold, p < 0.05, Fig. 1b) when compared with eyes that were administered with AAV2-WT vectors, intravitreally. We then examined the transduction potential of the AAV2-K665Q-EGFP vector by subretinal administration. Four weeks later, retinal sections (8 μm) of the murine eyes was prepared, stained with DAPI (Sigma-Aldrich, St. Louis, USA) and mounted with FluorSaveTM (Sigma-Aldrich). Images were acquired in a confocal microscope (A1R HD25 Nikon, Tokyo, Japan). Our analysis of K665Q-EGFP vector administered eyes, revealed a markedly enhanced GFP expression within the RPE layer and the outer segment of the retina in comparison to AAV2-WT vector (Fig. 1c). To further evaluate the therapeutic efficiency of the mutant AAV2 vectors in a murine model of retinal degeneration, we administered either PBS (mock group), AAV2WT, and AAV2-K665Q vectors expressing human RPE65, in rd12 mice. Approximately, 1–2 μl of AAV vectors at dose of 7 × 10 vgs was administered via subretinal route into the murine eyes (n= 6 eyes per group). The phenotypic response was measured by scotopic electroretinography (ERG), 16 weeks after vector administration (Ganzfeld ERG, Phoenix Research lab). The representative ERG waveforms from the treated mice are shown in Fig. 1d. We noted a significant visual correction in eyes that received AAV2-K665Q-RPE65 vectors, with a 2.43-fold (p < 0.001) increase in ‘A-wave’ amplitude and a 1.25-fold (p < 0.01) increase in ‘B-wave’ amplitude in comparison to the AAV2-WT injected animals (Fig. 1d, e), at the very low * Giridhara R. Jayandharan [email protected]