UC Berkeley

There are currently over 300 genetic causes for inherited retinal disease (IRD). Retinitis pigmentosa is part of the many diseases under the IRD umbrella, where rod photoreceptors progressively degenerate, followed by the outer segments of cone photoreceptors. The increasing number of genes found to be responsible for IRDs suggest the need to design a mutation agnostic approach to treat a wider range of IRD patients. Adeno-associated viral (AAV) optogenetic gene therapy is a promising mutation-independent treatment that aims to restore light-sensitivity to the degenerating retina. Studies directing ectopic expression of opsins to add a light-receptive function to surviving retinal neurons have been successful and currently there are four AAV-delivered optogenetic gene therapies in clinical trials. These clinical studies report restored perception of shape, color, contrast, and more. However, many new optogenetic tools have been engineered and have the potential to improve the vision restored after optogenetic gene therapy treatment.

There are many factors to consider when designing AAV optogenetic gene therapies. An ideal optogenetic protein should combine both light sensitivity over a broad range of intensities and response speed to permit vision with motion. To this end, we examined the characteristics of the candidate opsins for optogenetic gene therapy, such as response kinetics, light-sensitivity, and peak excitation wavelength within the visible light spectrum. The current study explores targeting retinal ganglion cells (RGCs) to become photoreceptors using novel channelrhodopsins developed by structure-guided mutagenesis and never previously tested for vision restoration: 1) ChRmine, from the algae Rhodomonas lens, 2) T119A-ChRmine, a more sensitive ChRmine variant, and 3) ChroME2S, a second-generation ChroME-based opsin. We use behavioral tests to show that treated rd1 mice recover high-sensitivity vision and use binocular vision to perceive depth. Using these opsins, we demonstrate the feasibility of using new and improved opsins to enhance vision in patients. We additionally show that AAV constructs designed with opsins tagged with a membrane-targeting sequence are more light-sensitive than opsins without the tag. We thus demonstrate that level of membrane localization may be important for a sensitive treatment. Further, we tested multiple treatment titers and found mice with diluted titers are more light-responsive and have better binocular vision than the highest titer.

A major concern in the degenerating retina is spontaneous activity due to deafferented bipolar cells. RGCs have discrete bursts of activity, which can manifest as visual hallucinations in IRD patients. Most optogenetic therapies choose to target the optical control of RGCs, which may restore vision, but spontaneous activity may affect visual acuity. These oscillations block any remnant light responses from residual photoreceptors. We wanted to understand if functional behavior would recover if we target inhibitory opsin expression in a portion of RGCs. We used a promoter that not only transfects RGCs, but also transfects upstream neurons that are likely cone cell bodies left after outer segment degeneration. We show that expression of inhibitory opsins in cones and RGCs restored an important and complex aspect of vision, binocular depth perception. We may have inadvertently suppressed spontaneous activity by also forcing cone hyperpolarization in response to light, but this study additionally confirms that visual pathways in late-stage retinal disease are still functional. The field of optogenetics has consistently discovered or engineered increasingly potent and fast opsins, some of which we have used in this study. Our most sensitive candidate opsin drove the most light-responsive behaviors. When we used a hyperpolarizing opsin to reduce spontaneous activity in RGCs, additional off-target expression in upstream neurons caused recovery of depth perception in treated animals. This not only showed restoration of binocular vision that suggests correlated brain activity but revealed that the retinal connectome is well preserved and can function even in late-stage degeneration. Together, these results demonstrate that optogenetic proteins must be carefully considered when designing effective AAV optogenetic gene therapies in order to translate the visual scene most accurately.