Abstract
Purpose: :
It has been reported that the intravitreal injection of the channelrhodopsin-2 (ChR2) gene into genetically blind rats restored visual response. However the sensitive wavelength of ChR2 is limited below 540 nm. In this study, we modified a volvox derived channelrhodopsin-1 (VChR1) gene based on a bioinfomatic approach to improve wavelength sensitivities and expression profiles in mammalian cells.
Methods: :
We made various types of modified VChR1 genes by the replacement of the N-terminal or C-terminal fragment of VChR1 gene with other proteins. The modified VChR1 genes were electroporated into cultured HEK293 cells to investigate the expression profiles. The photocurrents were recorded under the whole-cell patch clamp of a conventional system. We also made an adeno-associated virus vector type 2 including the modified VChR1 gene fused to a fluorescent protein, Venus (AAV-mVChR1V) and intravitreously injected into Royal College of Surgeons (RCS) rats. Visually evoked potentials (VEPs) were recorded 2 months later.
Results: :
The mVChR1 which is chimeric protein with the chlamydomonas derived-channelrhodopsin-1 showed the diffused expression in the cell body and plasma membrane. The photocurrents were evoked by 1-s pulse illumination of wavelength between 400 and 600 nm. The VEPs in mVChR1-transferred RCS rats were also recorded by the stimulus wavelength from 450nm to 600nm. The mVChR1 was more sensitive to the white flash than the chlamydomonas derived channelrhodopsin-2. The expression of mVChR1 in the retina were mainly observed in retinal ganglion cells, which was almost same as those of ChR2.
Conclusions: :
The mVChR1 showed wide range spectrum sensitivities. The gene transfer of ChR2 with mVChR1 into blind rats will increase the visible wavelength.
Keywords: gene transfer/gene therapy • photoreceptors • regeneration