cGMP compliant CRISPR correction and retinal differentiation protocol for photoreceptor cell replacement therapy

Abstract

Purpose : Induced pluripotent stem cell (iPSC)-derived retinal progenitor cells are a promising cell type for restoring vision in patients with retinal degeneration. The purpose of this study was to develop a protocol to generate clinical grade CRISPR-corrected photoreceptor precursor cells for autologous retinal cell replacement.

Methods : IPSCs were generated using dermal fibroblasts isolated from an unaffected control and a patient with NR2E3-associated enhanced S-cone syndrome. CRISPR-Cas9-based homology-dependent repair strategies were designed to correct disease causing NR2E3 mutations. CRISPR reagents were delivered using a microfluidic device, which operates using a reagent-free, convective transport mechanism amenable to the delivery of large molecules under cGMP. Retinal organoids were derived using a stepwise 3D differentiation protocol testing different cGMP compliant reagents and oxygen tension (5-20%) in a cGMP compliant Biospherix cell culture isolator. Organoids were characterized via light and confocal microscopy.

Results : CRISPR reagents were delivered to patient iPSCs using a Zephyr microfluidic transfection system (CellFE). Different gap sizes (7, 9, and 10µm) and flow rates (achieved by applying pressure of 50 or 90PSI to each well) were tested and cell survival and cutting efficiency were evaluated. A 9µm gap size and 90 PSI consistently gave the greatest cutting efficiency (~31%) with lowest cell death. While we previously found that iPSC reprogramming efficiency is highest under 5% oxygen tension, retinal organoid production was inefficient at this oxygen level. Increasing oxygen tension to 20% resulted in decreased pluripotency and increased ectoderm gene expression by day 7 of differentiation. By day 30, laminated retinal organoids were abundant in cultures differentiated under 20% oxygen tension, while they were smaller and less consistent when differentiated at 5% oxygen tension. By 150 days of differentiation retinal organoids generated at 20% oxygen tension under cGMP conditions contain abundant rod and cone photoreceptor precursor cells expressing markers such as NRL and cone arrestin respectively.

Conclusions : We have generated a cGMP compliant protocol incorporating microfluidic transfection and varying oxygen tensions to enable efficient generation, CRISPR correction and differentiation of transplantable photoreceptor cells for autologous cell replacement.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.