Improved CoChR Variants Restore Visual Acuity and Contrast Sensitivity in a Mouse Model of Blindness under Ambient Light Conditions

Abstract

Severe photoreceptor cell death in retinal degenerative diseases leads to partial or complete blindness. Optogenetics is a promising strategy to treat blindness. The feasibility of this strategy has been demonstrated through the ectopic expression of microbial channelrhodopsins (ChRs) and other genetically encoded light sensors in surviving retinal neurons in animal models. A major drawback for ChR-based visual restoration is low light sensitivity. Here, we report the development of highly operational light-sensitive ChRs by optimizing the kinetics of a recently reported ChR variant, Chloromonas oogama (CoChR). In particular, we identified two CoChR mutants, CoChR-L112C and CoChR-H94E/L112C/K264T, with markedly enhanced light sensitivity. The improved light sensitivity of the CoChR mutants was confirmed by ex vivo electrophysiological recordings in the retina. Furthermore, the CoChR mutants restored the vision of a blind mouse model under ambient light conditions with remarkably good contrast sensitivity and visual acuity, as evidenced by the results of behavioral assays. The ability to restore functional vision under normal light conditions with the improved CoChR variants removed a major obstacle for ChR-based optogenetic vision restoration.

Towards optogenetic vision restoration with high resolution

“…We have measured the receptive fields of retinal ganglion cells of mouse and primate retinas that were made light sensitive using an optogenetic protein. [11,12] and [15] have measured receptive fields in the mouse retina where ganglion cells are transfected by an optogenetic protein. However, this was not performed in the primate retina.…”

“…Previous studies using optogenetic proteins expressed in ganglion cells mostly measured responses to full field flashes [5][6][7][8][9]. Some used fine-grained stimulation patterns that are necessary to measure receptive fields [11,12], but only in the rodent retina, not in the primate retina. A few studies where light sensitivity was restored at the bipolar or photoreceptor stage used spots of increasing sizes to determine size selectivity [13,14].…”

Problems and outlooks of optogenetic technologies in the 21st century

“…For example, in the experiment with «switching-in» halorhodopsin eNpHR in light-insensitive rods, it was possible to restore the natural behavioral responses of laboratory animals to light [51,52]. Additionally, there is a possibility for creating artificial photoreceptors after incorporation of ChR2 into bipolar cells or ganglion cells [53,54].…”

Degenerated Cones in Cultured Human Retinas Can Successfully Be Optogenetically Reactivated

“…Despite this, non-targeted and GC-targeted expression is still able to achieve a considerable degree of restoration. GC firing to light stimuli flows through to the visual cortex and an impressive amount of visual acuity and contrast sensitivity when measured by optomotor responses has been restored in mice as have light avoidance, freezing and object recognition behaviours [26][27][28][29][30][31][32][33][34], but see also [35]. Targeted expression has also photosensitized GCs in macaque and cultured human retina, enabling them to response directly to light stimuli [27,28].…”

“…For instance, the commonly used light-sensitive actuator, Channelrhodopsin-2, requires dangerously high levels of blue light to activate it [27,28] and most light-sensitive actuators lack a signal amplification cascade so in order to induce a meaningful effect their expression levels in a cell's membrane must be high. Approaches addressing these issues, including red-shifting the channelrhodopsin to spectral sensitivities, were the high intensities required for activation that can be safely used [27], reducing the channelrhodopsin deactivation kinetics to increase its operation light sensitivity [29], using actuators with signal amplification mechanisms such as the G-protein coupled rod opsin [23], and engineering vectors with better tissue penetration [36] or more efficient promotor sequences [28] to increase transgene expression. However, the efficacies of optogenetic therapies utilizing improved actuator biophysical-properties and vector-delivery systems may differ between model systems and humans [37].…”