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Abstract Inherited retinal degeneration is a term used to describe heritable disorders that result from the death of light sensing photoreceptor cells. Although we and others believe that it will be possible to use gene therapy to halt disease progression early in its course, photoreceptor cell replacement will likely be required for patients who have already lost their sight. While advances in autologous photoreceptor cell manufacturing have been encouraging, development of technologies capable of efficiently delivering genome editing reagents to stem cells using current good manufacturing practices (cGMP) are needed. Gene editing reagents were delivered to induced pluripotent stem cells (iPSCs) using a Zephyr microfluidic transfection platform (CellFE). CRISPR-mediated cutting was quantified using an endonuclease assay. CRISPR correction was confirmed via digital PCR and Sanger sequencing. The resulting corrected cells were also karyotyped and differentiated into retinal organoids. We describe use of a novel microfluidic transfection platform to correct, via CRISPR-mediated homology-dependent repair (HDR), a disease-causing NR2E3 mutation in patient-derived iPSCs using cGMP compatible reagents and approaches. We show 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 codeliver 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.
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This content will become publicly available on June 12, 2026
Device-free isolation of photoreceptor cells from patient iPSC-derived retinal organoids
Autologous photoreceptor cell replacement is one of the most promising strategies currently being developed for the treatment of patients with inherited retinal degenerative blindness. Induced pluripotent stem cell–derived (iPSC-derived) retinal organoids, which faithfully recapitulate the structure of the neural retina, are an ideal source of transplantable photoreceptors required for these therapies. However, retinal organoids contain other retinal cell types, including bipolar, horizontal, and amacrine cells, which are unneeded and may reduce the potency of the final therapeutic product. Therefore, approaches for isolating fate-committed photoreceptor cells from dissociated retinal organoids are desirable. In this work, we present partial dissociation, a technique that leverages the high level of organization found in retinal organoids to enable selective enrichment of photoreceptor cells without the use of specialized equipment or reagents such as antibody labels. We demonstrate up to 90% photoreceptor cell purity by simply selecting cell fractions liberated from retinal organoids during enzymatic digestion in the absence of mechanical dissociation. Since the presented approach relies on the use of standard plasticware and commercially available current good manufacturing practice–compliant reagents, we believe that it is ideal for use in the preparation of clinical photoreceptor cell replacement therapies.
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- Award ID(s):
- 2225488
- PAR ID:
- 10648069
- Publisher / Repository:
- JCI insight
- Date Published:
- Journal Name:
- JCI Insight
- Volume:
- 10
- Issue:
- 14
- ISSN:
- 2379-3708
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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