Generation of rod- vs. cone-dominant patient-derived 3D retinal grafts for the treatment of retinal degenerative blindness

Abstract

Purpose : Induced pluripotent stem cell (iPSC)-derived photoreceptor cells hold great promise for restoring vision to patients with retinal degenerative blindness. Unfortunately, traditional cell isolation and subretinal transplantation approaches result in poor donor cell survival. More sophisticated tissue engineering-based strategies, aimed at reducing post-transplant cell death by providing cells as a contiguous graft, are more promising. The purpose of this study was to combine CRISPR-based genome editing and iPSCs generated from patients with enhanced S-cone syndrome to develop rod- vs. cone-dominant photoreceptor cell grafts for the purpose of evaluating the effects of developmental age on photoreceptor cell axon extension and synapse formation.

Methods : IPSCs were generated using dermal fibroblasts isolated from 2 patients with molecularly confirmed NR2E3-associated enhanced S-cone syndrome. CRISPR-Cas9-based homology-dependent repair strategies were designed to correct NR2E3. Retinal organoids were derived using a stepwise 3D differentiation protocol. Organoids were dissociated and seeded in polycaprolactone (PCL) scaffolds generated using two-photon lithography. Retinal cell grafts were analyzed via immunocytochemistry.

Results : To generate cone-dominant grafts, iPSCs from 2 patients (P1 – IVS1/IVS1, P2 – R73/R311) with NR2E3-associated enhanced S-cone syndrome were used. Throughout early stages of development organoids generated from these patients formed polarized neural epithelium expressing PAX6 followed by OTX2 and RORb. In comparison to normal controls, which at 95-120 days post-differentiation contained <1% S-opsin positive cone photoreceptor precursor cells, 10-15% of the cells contained within NR2E3 patient-derived organoids were S-opsin positive (P<0.01). To restore rod generation potential, and allow us to generate rod- vs. cone-dominant grafts from the same individuals, NR2E3 mutations were corrected via CRISPR-Cas9-based homology-dependent repair. 3D PCL cell delivery scaffolds were seeded with retinal progenitor cells isolated from iPSC-derived retinal organoids. At 1-week post-seeding, grafts were densely populated with OTX2-positive photoreceptor precursor cells.

Conclusions : By combining CRISPR-based genome editing and patient-derived iPSCs, we can generate rod- vs. cone-dominant grafts suitable for analysis of cell type specific regenerative capacity.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.