Abstract
Recent studies have demonstrated the safety and efficacy of ocular gene therapy based on adeno-associated viral vectors (AAVs). Accordingly, a surge in promising new gene therapies is entering clinical trials, including the first optogenetic therapy for vision restoration. To date, optogenetic therapies for vision restoration target either the retinal ganglion cells (GCs) or presynaptic ON-bipolar cells (OBCs). Initiating light responses at the level of the OBCs has significant advantages over optogenetic activation of GCs. For example, important neural circuitries in the inner retina, which shape the receptive fields of GCs, remain intact when activating the OBCs. Current drawbacks of AAV-mediated gene therapies targeting OBCs include (1) a low transduction efficiency, (2) off-target expression in unwanted cell populations, and (3) a poor performance in human tissue compared to the murine retina. Here, we examined side-by-side the performance of three state-of-the art AAV capsid variants, AAV7m8, AAVBP2, and AAV7m8(Y444F) in combination with the 4xGRM6-SV40 promoter construct in the healthy and degenerated mouse retina and in human post-mortem retinal explants. We find that (1) the 4xGRM6-SV40 promoter is not OBC specific, (2) that all AAV variants possess broad cellular transduction patterns, with differences between the transduction patterns of capsid variants AAVBP2 and AAV7m8 and, most importantly, (3) that all vectors target OBCs in healthy tissue but not in the degenerated rd1 mouse model, potentially limiting the possibilities for an OBC-targeted optogenetic therapy for vision restoration in the blind.
Highlights
Blindness caused by photoreceptor degeneration affects approximately 1 in 3000 people worldwide
In light of the ongoing efforts to develop an ON-bipolar cells (OBCs)-based gene therapy for optogenetic vision restoration, this study aimed to investigate side-by-side the OBC transduction efficacies of three state-of-the-art associated viral vectors (AAVs) capsid variants in healthy and degenerating mouse retinas and in human retinal explants
In AAV7m8 treated retinas, we observed off-target expression of mCitrine in a single homogenous and evenly spaced amacrine cell type (Figures 2A,C). We identified these amacrine cells morphologically as AII amacrine cells based on their lobular processes in the OFF-sublamina of the IPL, their extensive dendritic arbors in the ON-sublamina including multiple close contacts with the axon terminals of PKCα-positive rod bipolar cell (RBC) (Figure 2G) and their strong reactivity to an antibody against Glycine Transporter 1 (GLYT1; Figure 2H)
Summary
Blindness caused by photoreceptor degeneration affects approximately 1 in 3000 people worldwide. When introduced to the retinal ganglion cells (GCs) or ON-bipolar cells (OBCs) of blind rd mice, Channelrhodopsin-2 (ChR2) restored light sensitivity and basic levels of visual processing (Bi et al, 2006; Lagali et al, 2008; Doroudchi et al, 2011). ON-Bipolar Cell Targeted Gene Therapy light-sensitive mammalian proteins (Cehajic-Kapetanovic et al, 2015; van Wyk et al, 2015a) These next-generation optogenetic therapies target the OBCs and re-activate the native OBC signaling cascade. Therapies that target OBCs have the advantage that they can re-activate early-stage processing of visual information within the inner retina It is at the level of the bipolar cells that incoming visual information is divided into ON- and OFF-pathways, which might be imperative for visual perception in higher visual centers (Thyagarajan et al, 2010). Since the RBC signal diverges into cone ON- and OFF-pathways via the AII amacrine cell, optogenetic activation of OBCs restores responses in both, the ON- and OFF-pathways (Macé et al, 2015; van Wyk et al, 2015a)
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