Bipolar Cell Targeted Optogenetic Gene Therapy Restores Parallel Retinal Signaling And High-Level Vision In The Degenerated Retina

Abstract

Optogenetic gene therapies to restore vision are in clinical trials. Whilst current clinical approaches target the ganglion cells, the output neurons of the retina, new molecular tools enable efficient targeting of the first order retinal interneurons, the bipolar cells, with the potential to restore a higher quality of vision. Here we investigate retinal signaling and behavioral vision in blind mice treated with bipolar cell targeted optogenetic gene therapies. All tested tools, including medium-wave opsin, Opto-mGluR6 and two new melanopsin based chimeras restored visual acuity and contrast sensitivity. The best performing opsin was a novel melanopsin-mGluR6 chimera, which in some cases restored visual acuities and contrast sensitivities that match wild-type animals. Light re- sponses from the ganglion cells were robust with diverse receptive-field types, inferring elaborate inner retinal signaling. Our results highlight the potential of bipolar cell targeted optogenetics to recover high-level vision in human patients with end-stage retinal degenerations.

Functional Optimization of Light-Activatable Opto-GPCRs: Illuminating the Importance of the Proximal C-terminus in G-protein Specificity

“…While the bRhod-b2AR variant was optimally designed by introducing the b2AR TM5-IL3-IL6 and C-terminus after the palmitoylation site in bRhod, a proximal C-terminus replacement of melanopsin by the whole C-terminus of the mGluR6 receptor, including the proximal C-terminus, sufficed to retarget to the Gi/o protein class. Overall, the Mela(palm)-mGluR6(CT) seems to be the favorable chimera for functional applications since it activates G-proteins with high efficacy and shows a strong tropism towards Gi/o (Kralik et al, 2022).…”

“…While the bRhod-b2AR variant was optimally designed by introducing the b2AR TM5-IL3-IL6 and C-terminus after the palmitoylation site in bRhod, a proximal C-terminus replacement of melanopsin by the whole C-terminus of the mGluR6 receptor, including the proximal C-terminus, sufficed to retarget to the Gi/o protein class. Overall, the Mela(palm)-mGluR6(CT) seems to be the favorable chimera for functional applications since it activates G-proteins with high efficacy and shows a strong tropism towards Gi/o (Kralik et al, 2022). Of course, since Cryo-EM studies on mGluR2 showed that the G-protein’s C-terminus is stabilized by a pocket formed by IL2 and the C-terminus (Seven et al, 2021), IL2 may also be involved in Gi/o-selectivity together with the proximal C-terminus of mGluR6, which was, however, not investigated in this study.…”

“…Pioneering studies were performed with bovine rhodopsin (bRhod), where all intracellular domains were swapped by analogues of ligand-gated target receptors and the chimeras were tested functionally for their G-protein binding selectivity (Kim et al, 2005;Yamashita et al, 2001). However, as shown in recent work, engineering all intracellular domains results in poorer expression and functionality in in vivo experiments (Kralik et al, 2022). Thus, the second aim of this study was to determine which intracellular domains were crucial to replace to induce the desired downstream signaling.…”