Cell therapy is one of the most promising approaches for restoration of lost vision and human induced pluripotent stem cells (hiPSCs) emerged as an attractive source of donor cells. Despite recent improvements in generating, banking and isolating photoreceptor precursors from hiPSCs, a remaining obstacle is that such transplanted cells have to develop into functional light-sensitive photoreceptors, requiring outer segment formation and interaction with the underlying retinal pigmented epithelium (RPE). We therefore proposed to combine stem cell-based therapy and optogenetics insuring light sensitivity to the donor cells thanks to the expression of a microbial opsin and independently from outer segment formation and RPE interaction.

We engineered via a knock-in strategy by CRISPR/Cas9 optogenetically-modified human iPSCs using a hyperpolarizing chloride pump Jaws, a red-shifted halorhodopsin. To ensure stable and homogenous expression of the microbial opsin in photoreceptors, we successfully insert Jaws in fusion of EGFP, driven by the CRX promoter, at the safe AAVS1 locus in iPSCs. Using our previously described retinal differentiation protocol, we produced retinal organoids with Jaws-expressing photoreceptors, identified by GFP co-staining with specific rod and cone photoreceptor markers. Two-photon guided patch clamp revealed robust response to orange light pulse in GFP-Jaws photoreceptors from retinal organoids after 100 days of differentiation.

We tested the ability of these Jaws-expressing photoreceptor precursors to restore visual function through transplantation in an immunodeficient rat model of advanced retinal degeneration (SD-Foxn1Tg(S334ter)3Lav rats). We were able to purify Jaws-expressing photoreceptors, by CD73-targeted magnetic-activated cell sorting (MACS), resulting in a homogenous donor cell population. We demonstrated that transplanted photoreceptors, can survive for up to 6 months in close contact to the host inner retina and respond to light, thanks to Jaws expression, as demonstrated by ex vivo patch-clamp recording. Examination of potential host-graft synaptic interaction on retinal cryosections, showed multiple CtBP2-positive ribbon synapses in the vicinity of PKC-positive bipolar cells, suggesting putative connections between grafted photoreceptors and host bipolar cells.

Overall, these results highlight the potential of our optogenetic strategy for the development of improved therapies in visual restoration.