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The use of induced pluripotent stem cells to reveal pathogenic gene mutations and explore treatments for retinitis pigmentosa

机译：利用诱导多能干细胞揭示致病基因突变并探索色素性视网膜炎的治疗方法

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Background Retinitis pigmentosa (RP) is an inherited human retinal disorder that causes progressive photoreceptor cell loss, leading to severe vision impairment or blindness. However, no effective therapy has been established to date. Although genetic mutations have been identified, the available clinical data are not always sufficient to elucidate the roles of these mutations in disease pathogenesis, a situation that is partially due to differences in genetic backgrounds. Results We generated induced pluripotent stem cells (iPSCs) from an RP patient carrying a rhodopsin mutation (E181K). Using helper-dependent adenoviral vector (HDAdV) gene transfer, the mutation was corrected in the patient’s iPSCs and also introduced into control iPSCs. The cells were then subjected to retinal differentiation; the resulting rod photoreceptor cells were labeled with an Nrl promoter-driven enhanced green fluorescent protein (EGFP)-carrying adenovirus and purified using flow cytometry after 5 weeks of culture. Using this approach, we found a reduced survival rate in the photoreceptor cells with the E181K mutation, which was correlated with the increased expression of endoplasmic reticulum (ER) stress and apoptotic markers. The screening of therapeutic reagents showed that rapamycin, PP242, AICAR, NQDI-1, and salubrinal promoted the survival of the patient’s iPSC-derived photoreceptor cells, with a concomitant reduction in markers of ER stress and apoptosis. Additionally, autophagy markers were found to be correlated with ER stress, suggesting that autophagy was reduced by suppressing ER stress-induced apoptotic changes. Conclusion The use of RP patient-derived iPSCs combined with genome editing provided a versatile cellular system with which to define the roles of genetic mutations in isogenic iPSCs with or without mutation and also provided a system that can be used to explore candidate therapeutic approaches.
机译：背景技术色素性视网膜炎（RP）是一种人类遗传性视网膜疾病，会导致进行性感光细胞丧失，导致严重的视力障碍或失明。然而，迄今为止尚未建立有效的疗法。尽管已经鉴定出遗传突变，但可用的临床数据并不总是足以阐明这些突变在疾病发病机理中的作用，这种情况部分是由于遗传背景的差异。结果我们从携带视紫红质突变（E181K）的RP患者中产生了诱导性多能干细胞（iPSC）。通过使用依赖辅助基因的腺病毒载体（HDAdV）基因转移，可以在患者的iPSC中纠正该突变，并将其引入到对照iPSC中。然后将细胞进行视网膜分化。所得杆状感光细胞用Nrl启动子驱动的增强型绿色荧光蛋白（EGFP）携带的腺病毒标记，并在培养5周后使用流式细胞仪进行纯化。使用这种方法，我们发现具有E181K突变的感光细胞的存活率降低，这与内质网（ER）应激和凋亡标记物表达增加有关。对治疗剂的筛选表明，雷帕霉素，PP242，AICAR，NQDI-1和salubrinal促进了患者iPSC衍生的感光细胞的存活，同时减少了ER应激和凋亡标记。此外，发现自噬标记物与内质网应激相关，表明通过抑制内质网应激诱导的细胞凋亡改变可减少自噬。结论RP患者来源的iPSC与基因组编辑的结合使用提供了一个通用的细胞系统，可用来定义遗传突变在有或没有突变的同基因iPSC中的作用，还提供了可用于探索候选治疗方法的系统。

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