Developing microfluidic devices for cellular modelling of inherited eye diseases

遗传性视网膜营养不良是导致失明和视力丧失的主要原因。在英国，有超过2万人因这些疾病而失明或部分失明，这些疾病是由260多种基因突变引起的。然而，许多遗传变异并不具有特定的功能意义。我们目前无法解释“意义不确定的变异”的功能后果，这限制了遗传信息对许多患者的价值。了解“意义不确定的变异”的影响，可以为患者提供准确的咨询，提高我们对疾病发病机制的认识，并促进新疗法的发展。通过使用微流体装置对细胞进行变异建模，有可能极大地提高功能分析的吞吐量和容量，但目前对视网膜细胞的研究和开发不足。成熟的方案将用于将诱导多能干细胞（iPSCs）分化为视网膜细胞类型。通过使用标准的CRISPR-Cas9敲入方法，从患者身上鉴定出的变异将被基因组编辑成iPSCs。选定的敲除视网膜iPSCs将在新型“器官芯片”微流控装置上转化为生理组织系统，以解决当前遗传变异解释的局限性。该项目的总体目标是开发新的和改进的体外细胞建模方法。“器官芯片”设备是一个明显的机会，通过减少培养量和减少细胞操作来提高吞吐量和可重复性。这将使匹配的功能检测集成为可能，特别侧重于使用这些设备进行新的治疗策略的临床前测试，如等位基因特异性敲除和反义寡核苷酸治疗。

Inherited retinal dystrophies are a leading cause of blindness and visual loss. Over 20,000 individuals in the UK are registered blind or partially sighted because of these conditions, caused by mutations in over 260 genes. However, many genetic variants do not have an ascribed functional significance. Our current inability to interpret the functional consequences of "variants of uncertain significance" limits the value of genetic information for many patients. Understanding the impact of "variants of uncertain significance" allows accurate counselling of patients, improves our knowledge of disease pathogenesis and catalyses the development of novel treatments. Cellular modelling of variants by using microfluidic devices has the potential to hugely increase throughput and capacity of functional assays, but it is an approach that is currently under-studied and under-developed for retinal cells. Well-established protocols will be used to differentiate induced pluripotent stem cells (iPSCs) into retinal cell types. Variants identified from patients will be genome-edited into iPSCs by using standard CRISPR-Cas9 knock-in methods. Selected knock out retinal iPSCs will be turned into physiological tissue systems on novel "organ-on-a-chip" microfluidic devices, to address the current limitations of genetic variant interpretation. The overall aim of the project is to develop novel and improved methods of in vitro cellular modelling. "Organ-on-a-chip" devices are a clear opportunity to improve throughput and reproducibility through minimizing culture volumes and reducing cell manipulation. This will enable matched sets of functional assays, with a particular focus on using these devices for pre-clinical testing of new therapeutic strategies such as allele-specific knockdown and antisense oligonucleotide therapy.