RESTORING VISION USING HUMAN OPSINS

Abstract

Restoring vision using human opsinsJasmina Cehajic-Kapetanovic; The University of Manchester, Doctor of Philosophy, 2015Inherited retinal degenerations (IRDs) are progressive degenerative conditions that affect around 1 in 2500 people worldwide and lead to severe visual impairment due to irreversible loss of photoreceptors. These conditions are currently untreatable. However, inner retinal neurons, including bipolar and ganglion cells, can survive representing promising targets for emerging optogenetic therapies that aim to convert them into photoreceptors and recreate the lost photosensitivity. However, efficient targeting of these surviving cells, such as ON-bipolar cells, has not been achieved. In addition, current optogenetic actuators have low sensitivity posing major limitations to vision restoration. The overall aim of this research was to develop an optimised adeno-associated virus (AAV) based gene delivery system for efficient targeting of optogenetic sensors to surviving retinal cells, and to investigate whether this optimised approach can restore visual function in an rd1 mouse model of advanced IRD.Transduction efficiency of AAV serotype 2, AAV2 (carrying enhanced green fluorescent protein, GFP, driven by a non-selective promoter) in conjunction with glycosidic enzymes, was determined by qualitative and quantitative analysis of GFP positive cells in the treated wild-type retinas. In addition, using an optimised AAV2-enzyme combination, GFP expression was analysed in rd1 mice after both untargeted delivery and when GFP was selectively targeted to ON-bipolar cells. Lastly, effects of glycosidic enzymes on the retinal function were determined by flash electroretinograms (ERGs) and pupillometry. The data revealed that a combination of heparinase III and hyaluronan lyase produced the greatest enhancement of gene delivery to the healthy wild-type retinas and that this optimised approach led to a marked improvement in transduction in degenerate rd1 retinas. Retinal function and photosensitivity were unaffected as determined by ERGs and pupillometry at a range of irradiances tested in the acute period and up to at least 12 months post enzymatic treatment.Using the optimised AAV2-enzyme combination, human melanopsin (driven by a non-selective promoter) and human rod opsin (driven by non-selective or ON-bipolar cell-specific promoters) were expressed in rd1 retinas. Mice treated with melanopsin showed enhanced pupil light reflex compared to controls. Analysis of in vivo electrophysiology recordings from the lateral geniculate nucleus (LGN) in the thalamus of melanopsin treated revealed that light-evoked excitatory neuronal responses were more numerous, larger, and of higher sensitivity and shorter latency than those derived from control eyes. Importantly, restored responses were orders of magnitude more sensitive than current microbial or chemical-based optogenetic strategies. Electrophysiological recordings from retinal explants and the LGN in rod opsin treated rd1mice (driven by either promoter) revealed light-evoked responses (excitatory and inhibitory) at light intensities similar to melanopsin-driven responses, but with significantly shorter latencies and these could be induced by simple light pulses, luminance increases, and naturalistic movies. Mice with rod opsin expression driven by the ON-bipolar promoter displayed behavioural responses to increases in luminance, flicker, coarse spatial patterns, and elements of a natural movie at levels of contrast and illuminance typical of natural indoor environments.Collectively, these data reveal that enhanced AAV-mediated ectopic expression of both human melanopsin and rod opsin can drive responses at moderate light intensities, but that rod opsin has superior response qualities. The inherent advantages in employing a human protein, the simplicity of this intervention, and the quality of vision restored, all suggest that rod opsin has the potential to restore vision in patien

Date of Award	1 Aug 2016
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Paul Bishop (Supervisor) & Robert Lucas (Supervisor)