Engineering Specific Human iPS Reporter Cell Lines to Generate Optogenetically Modified Photoreceptors.

Abstract

Cell therapy, by transplantation of photoreceptors derived from induced pluripotent stem cells (iPSCs), has been proposed as a promising therapeutic approach for photoreceptor degenerative diseases. A remaining obstacle is that such transplanted cells have to develop into functional light-sensitive photoreceptors, which require outer segment formation and interaction with the underlying retinal pigmented epithelium (RPE). To overcome this limitation, a combination of cell therapy and optogenetics allows to confer light sensitivity to the donor cells thanks to the expression of a microbial opsin and therefore independently of the formation of mature outer segment or RPE contact. To ensure stable and homogenous expression of the microbial opsin in photoreceptors, we inserted the coding sequence of the red-light sensitive chloride pump Jaws under specific photoreceptor promoter into the iPSC genome, using the CRISPR/Cas9 system at the safe AAVS1 locus. We successfully generated a knock-in Jaws-EGFP iPSC line and validated its stemness and pluripotency status. These engineered iPSCs will be used to produce photoreceptors expressing Jaws that will be grafted to assess their ability to restore vision in blind animal models.