Abstract
Purpose :
Conventional optogenetic molecules (opsins) have had limited success in achieving high-acuity vision restoration due to their expression in retinal ganglion cells that extensively overlap surrounding neurons, which leads to significant cross-activation. We therefore developed novel soma-targeted opsins that enable a higher resolution of optogenetic photoactivation in the retina. By achieving single-cell-resolution, these opsins can support an improved acuity of vision restoration for retinal disease therapeutics.
Methods :
We designed and created novel soma-targeted variants of the popular CoChR opsin through molecular engineering. We subsequently expressed these opsins in primary mice cortical cultures and determined the variants that showed soma restriction. Using AAVs, we validated the expression and photostimulation of these somatic opsins through electrophysiology and determined the spatial resolution of activation in mouse cortical slices. To demonstrate the potential for vision restoration, we also performed ex-vivo testing in mice retinal explants.
Results :
We found three novel soma-targeted opsins exhibiting strong targeted expression even as close as 10µm from the cell body (p<0.01), and more restricted expression than known soma restricting motifs in literature (p<0.05), compared to the conventional opsin (Based on Mann Whitney tests). Using electrophysiology in mouse cortical slices, we found that one of the tested somatic opsins had similar current amplitude, rise and decay times compared to the conventional opsins (Based on student t-tests). Using this somatic opsin, we determined that the photoactivation was restricted to the cortical layer where the somatic opsins were expressed (i.e. expressed in layer 6 and photoactivated in layer 6 only). In comparison, conventional opsins expressing in layer 6 neurons could be photoactivated from layers 6, 5, and 4 (~400-600µm). When transduced in mice retinal explants, we found a restricted expression of these soma-targeted opsins to the cell bodies of retinal ganglion cells.
Conclusions :
We have developed novel soma-targeted opsins with a high spatial resolution of activation. As a result, these opsins can achieve single-cell-resolution activation. With these new opsins, optogenetic therapeutics can finally address the limited acuity associated with this form of vision restoration.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.