MICROFLUIDIC CRISPR CORRECTION WITH RETINAL DIFFERENTIATION FOR PHOTORECEPTOR CELL REPLACEMENT THERAPY

Induced pluripotent stem cell (iPSC)-derived retinal progenitor cells are a promising cell type for restoring vision in patients with inherited retinal degeneration . Development of technologies capable of efficiently delivering genome editing reagents to stem cells using current good manufacturing practices (cGMP) are needed. The purpose of this study was to develop a workflow to generate clinical grade CRISPR corrected photoreceptor precursor cells for autologous retinal cell replacement. 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-directed repair (HDR) strategies were designed to correct a disease causing NR2E3 homozygous intronic mutation. Cas9- sgRNA ribonucleoproteins (RNPs) and HDR plasmid were delivered using CellFE's microfluidic technology, which operates using a reagent-free, convective transport mechanism amenable to the delivery of large molecules under cGMP. Retinal organoids were then derived using a stepwise 3D differentiation protocol. Organoids were deeply characterized for safety and corrected phenotype. Delivery to iPSCs from several donors was optimized through biomechanical assessment of the cells. CRISPR correction was confirmed via digital PCR and Sanger sequencing to occur in both monoallelic and biallelic fashion. We found that the resulting cell lines have a corrected genotype, exhibit no off-target cutting, retain pluripotency and a normal karyotype , and can be differentiated into retinal tissue suitable for transplantation. The ability to co-deliver CRISPR/Cas9 and HDR templates to patient-derived iPSCs without using proprietary transfection reagents will streamline manufacturing protocols, increase the safety of resulting cell therapies, and greatly reduce the regulatory burden of clinical trials .