Abstract
Optogenetic gene-mediated therapy for restoring vision is thought to be a useful treatment for blind patients. However, light sensitivity achieved using this gene therapy is inferior to that of daylight vision. To increase light sensitivity, we designed three mutants using a bioinformatics approach. Nucleotide sequences encoding two sites in the extracellular loops (ex1, ex3) of mVChR1 close to simulated ion-conducting pathways were replaced by homologous amino acid-encoding sequences of ChR1 or ChR2. The light sensitivity of ex3mV1 was higher than that of mVChR1 at 405–617 nm. Visual responses were restored in Royal College of Surgeons rats with genetically degenerating photoreceptor cells transfected with ex3mV1Co, wherein transmembrane of sixth (TM6) in ex3mV1 was additionally replaced with the corresponding domain of CoChR; these rats responded to light in the order of μW/mm2. Thus, ex3mV1Co might be useful for the restoration of advanced visual function.
Highlights
The light-driven cation channel channelrhodopsin-2 (ChR2)[1,2] plays a central role in optogenetics
We found that the transfection of the modified Volvox channelrhodopsin1 (mVChR1) gene into blind rats restored their visually evoked potentials (VEP) upon stimulation at 450–600 nm and that optomotor responses were elicited with all colour stripes
We reported that the dual expression of optogenetic genes in the retina expands the range of wavelength sensitivities but not that of light sensitivities[18]
Summary
The light-driven cation channel channelrhodopsin-2 (ChR2)[1,2] plays a central role in optogenetics. Optogenetic techniques have been used in a variety of fields. The ChR2 gene has been transfected into neuronal cells for the optical control of neural[3] and behavioural[4] activities and for performing studies on brain function[5] and mechanisms of neuronal diseases[6,7]. In the field of vision research, optogenetic techniques have been used to treat blindness[8]. Bi et al.[9] and our group[10,11,12] reported that the transfection of ChR2 into the retinal ganglion cells (RGCs) of blind mice and rats could recover their light responses. Safety studies have been performed in rats[13] and marmosets;[14] continuous expressions of the ChR2 gene derived from the unicellular green alga Chlamydomonas reinhardtii did not result in any adverse effect caused by immunological responses. A few clinical trials (NCT02556736, NCT0332633615) are underway for investigating the efficacy of gene therapies employing optogenetic genes for restoring vision
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