Abstract
Purpose :
Intravitreal multi-characteristic opsin (MCO) gene therapy has demonstrated improvement in patients with severe vision loss due to retinitis pigmentosa and Stargardt disease. Here, we present structure and functioning of individual subunits of MCO that imparts broadband light sensitivity with high dynamic range and fast kinetics. We also present comparison of key characteristics of MCO with other opsins that are being developed for vision restoration.
Methods :
Studies were conducted on HEK293 cells and retinal explants from rd10 mice (animal model of retinal degeneration) which had been transduced with an adeno-associated virus-2 carried MCO (MCO-010). A combination of predictive in-silico protein modeling, Western blot-based protease protection assays, immunostaining as well as electrophysiological techniques were utilized to gather structural and functional data.
Results :
Our multi-disciplinary studies revealed that the first subunit of MCO-010 acts as an ion-channel, while the second subunit is partially embedded in the membrane extending to the third intracellular cytoplasmic subunit. Patch-clamp recordings showed that MCO-010 expressing cells get depolarized upon light activation with large dynamic range across the visible light spectrum. Deletion of transmembrane subunit 1 or intracellular subunit 3 led to either hyperpolarization in response to broadband visible light or significant decrease in photosensitivity. MCO-010 sensitized cells were activated with fast on-off kinetics (time-constants of ~ 2 to 3 ms). Furthermore, multi-electrode array recordings confirmed multi-color light-activation of MCO-010 expressing bipolar neurons in the photoreceptor degenerated mouse retina.
Conclusions :
The unique structure and functioning of MCO-010 represent key characteristics needed to restore vision in subjects with degenerative retinal conditions. Broadband light sensitivity with high dynamic range of MCO has opened new vistas in restoring vision at ambient lighting environments, without the need for a light stimulation device.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.