A universal protocol for isolating retinal ON bipolar cells across species via fluorescence-activated cell sorting - RNAseq of cell populations

Many inherited retinal dystrophies (IRDs) are characterized by progressive degeneration and loss of light-sensitive photoreceptors. The most promising treatment concept for IRDs is gene supplementation therapy with viral vectors, which relies on the presence of viable photoreceptors at the time of intervention. At later stages, when the pathology is too advanced, photoreceptors are lost and cannot be rescued. For late-stage patients, conferring light sensing abilities to the remaining ON circuit interneurons (i.e., ON bipolar cells) using optogenetic tools poses an alternative treatment approach. However, the development of such therapies is hampered by the lack of efficient gene delivery tools targeting ON bipolar cells, which in turn rely on effective cell isolation for tool development. In this work we have developed a pipeline to isolate ON bipolar cells by relying on the recognition of two intracellular markers and the isolation via fluorescence-activated cell sorting (FACS). As fixation proved to be a necessary prerequisite, RNA sequencing and real-time PCR were used not only to confirm the identity of the isolated ON bipolar cells but also to prove the compatibility of this method with highly sensitive techniques. Moreover, we have shown that this protocol can be applied without any substantial modification to the retinas of wild-type and Rd1 mutant mice as well as retinas of non-human primates (NHP).多种遗传性视网膜营养不良（inherited retinal dystrophies, IRDs）以感光光感受器的进行性变性与缺失为典型特征。目前针对IRDs最具前景的治疗方案为基于病毒载体的基因补充疗法，该疗法依赖于干预时仍存活的感光光感受器。而在疾病晚期，病理进程已十分严重时，感光光感受器已完全丢失，无法被挽救。对于晚期患者，借助光遗传学工具（optogenetic tools）向剩余的ON环路中间神经元（ON circuit interneurons，即ON双极细胞（ON bipolar cells））赋予光感应能力，成为另一可行的治疗思路。然而，此类疗法的开发却因缺乏靶向ON双极细胞的高效基因递送工具而受阻，而工具开发本身又依赖于有效的细胞分离技术。本研究开发了一套分离ON双极细胞的流程：通过识别两种细胞内标志物，并借助荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）完成细胞分离。由于细胞固定被证实为必要的前置步骤，本研究通过RNA测序与实时定量PCR（real-time PCR），不仅验证了所分离ON双极细胞的身份，同时证实了该方法与高灵敏度检测技术的兼容性。此外，本研究证实该实验方案无需大幅修改，即可应用于野生型及Rd1突变型小鼠的视网膜，以及非人灵长类（non-human primates, NHP）的视网膜。