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.

中文翻译：

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符合 CGMP 的微流体转染诱导多能干细胞，用于 CRISPR 介导的基因组编辑。

遗传性视网膜变性是一个术语，用于描述由于感光感光细胞死亡而导致的遗传性疾病。尽管我们和其他人相信可以使用基因疗法在病程早期阻止疾病进展，但对于已经失明的患者可能需要更换感光细胞。虽然自体感光细胞制造方面的进展令人鼓舞，但仍需要开发能够利用现行良好生产规范 (cGMP) 有效地将基因组编辑试剂递送至干细胞的技术。使用 Zephyr 微流体转染平台 (CellFE) 将基因编辑试剂递送至诱导多能干细胞 (iPSC)。使用核酸内切酶测定对 CRISPR 介导的切割进行定量。通过数字 PCR 和桑格测序证实了 CRISPR 校正。所得校正细胞也进行了核型分析并分化为视网膜类器官。我们描述了使用新型微流体转染平台，通过 CRISPR 介导的同源依赖性修复 (HDR)，使用 cGMP 兼容的试剂和方法来纠正患者来源的 iPSC 中致病的 NR2E3 突变。我们表明，所得的细胞系具有校正的基因型，没有表现出脱靶切割，保留多能性和正常的核型，并且可以分化成适合移植的视网膜组织。在不使用专有转染试剂的情况下将 CRISPR/Cas9 和 HDR 模板编码传递至患者来源的 iPSC 的能力将简化生产方案，提高细胞疗法的安全性，并大大减轻临床试验的监管负担。