Restore Vision Covering Visible Light By Using Single Gene, Modified Volvox Channelrhodopsin-1

Abstract

Purpose: : The Channelrodopsin-2 gene transfer to retinal ganglion cells is a useful method for restoring vision. However, the sensitive wavelength is limited under 540nm and their sensitivity to the light is lower than those of photoreceptors. To solve these problems, we investigated about the membrane translocation alignment and modified volvox derived channelrhodopsin-1 (vChR1).

Methods: : The vChR1 was designed by the bioinfomatic approach. In vitro study, modified gene was inserted into the expression cassette directed by CAG promoter. The plasmid contain puromycin resistant gene derived from IRES. The linealized plasmid was electroporated into cultured HEK293 cells and cells were selected by puromicin to obtain the stable transformant. Photocurrents were recorded from the cultured cells under the whole-cell patch clamp to examine their light sensitivities and wavelength properties. In vivo study, we used adeno-associated virus vector for transducing modified vChR1 gene into retinal cells. The vector was intravitreously injected into Royal College of Surgeons (RCS) rats. Visually evoked potentials (VEPs) were recorded 2 months later.

Results: : Modified vChR1-cells responded to a wide range of wavelength of light between 400nm and 600 nm, and higher sensitivity to the light at any wavelength than that of clamydomonus derived channelrhodopsin-2 (cChR2). The VEPs in modified vChR1-transferred RCS rats were also recorded by the stimulus wavelength from 450nm to 600nm.

Conclusions: : The modified vChR1 can utilize as light activated cation channel protein having the sensitivity of wide wavelength of light. Only the single gene expression of vChR1 will enable to restore the vision in patients suffering from blindness by photoreceptor degeneration.

Keywords: gene transfer/gene therapy • ion channels • retina: proximal (bipolar, amacrine, and ganglion cells)