Present Molecular Limitations of ON-Bipolar Cell Targeted Gene Therapy

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.

Optogenetic Gene Therapy for the Degenerate Retina: Recent Advances

“…This was the first demonstration of robust bipolar cell transduction from an AAV vector but transduction in a wild type mouse does not necessarily translate to degenerate retina or indeed non-human primate and human retina. Further confirmation of human bipolar cell transduction was provided in a later study using human retinal explants that also showed targeting of cone ON-bipolar cells by a reporter AAV8(BP2) vector (van Wyk et al, 2017). This study showed comparable human bipolar cell expression patterns were achieved from both AAV8(BP2) and AAV2(7m8) vectors and also performed similarly in the degenerate 11-week-old rd10 mice.…”

“…A comparison of multiple copies of the Grm6 enhancer identified that including four copies prior to the SV40 promoter improved reporter gene expression (Cronin et al, 2014). Whilst this promoter has been shown to primarily enable transgene expression in ON-bipolar cells, expression has been identified in other cell types (van Wyk et al, 2015(van Wyk et al, , 2017. The expression profile of an optogenetic transgene with fluorescent reporter revealed predominant expression in amacrine cells and a lack of ON-bipolar cell targeting in rd1 mice.…”

Role for Wnt Signaling in Retinal Neuropil Development: Analysis via RNA-Seq and In Vivo Somatic CRISPR Mutagenesis

“…In the outer retina, we found that Cbh and Ubiquitin (Matsuda and Cepko, 2004) regulatory elements drive expression of fluorescent reporters in Rods, Bipolar Cells and Muller Glia. We used a 2.3 kb fragment of the Rhodopsin promoter to drive Cas9 expression in rods (Matsuda and Cepko, 2004; 2007), and a 1082 bp fragment containing a tandem repeat of an mGluR6-SV40 promoter (4XGrm6-Sv40) from the pAAV-4xGRM6-CatCh-EGFP vector (gift from Botand Roska) to drive Cas9 expression in ON BCs (van Wyk et al, 2017). Promoters were cloned between the KpnI and AgeI restriction sites in px458.…”

Innovative Optogenetic Strategies for Vision Restoration

“…AAV never achieves complete infection and is normally “patchy,” so complete restoration of a receptive field looks unlikely. Furthermore, the mGluR6 promoters used to restrict expression to the ON-bipolar cells may not work in many retinal dystrophies due to dis- and downregulation of mGluR6 (van Wyk et al, 2017 ). mGluR6 is tonically activated by the continuous release of glutamate by photoreceptors in the dark.…”