A two-pronged approach to generating novel models of photoreceptor degeneration for regenerative cell therapy

项目总结 遗传性视网膜疾病(IRD)，如视网膜色素变性(RP)、Leber先天性黄斑变性(LCA)，是全球范围内导致视力障碍的常见原因。最近的研究表明，细胞替代疗法对于患有严重视网膜变性的IRD患者是一种有希望的治疗选择，这种变性破坏了光感受器。我们和其他人最近报道了人胚胎干细胞(HESCs)或人诱导多能干细胞(HiPSCs)来源的视网膜在一种新的激光诱导的光感受器退化模型中长期存活和整合。虽然激光消融模型可以作为急性变性模型快速建立，但它不具有进行性病理生理特性(例如，疾病时间进程、视网膜重塑等)。美国国税局。为了解决这个问题，我们开发了一种高效的基因组编辑技术，白金Talen，它可以高效和特异性地在多个生物体中导致基因破坏。通过将白金Talen应用于这一新模型，我们在初步研究中实现了高效率(40%-50%)的一步生成双等位基因修饰。由于在激光损伤模型中已经获得了大量关于移植的光感受器细胞或视网膜片的存活和整合的信息，我们提出了一个双管齐下的策略来产生新的翻译使能模型来评估再生的光感受器在视觉系统中的存活和整合：1)使用激光诱导的光感受器退化模型；2)白金Talen一步产生双等位基因突变体。然后，我们将系统地比较两个模型系统中再生光感受器的存活和整合情况。AIM 1将通过激光消融和白金Talen产生新的光感受器退化模型。目的2将对激光诱导和Talen编辑的感光细胞变性模型进行全面的眼部表型鉴定。目的3将评估人诱导多能干细胞(HiPSC)来源的视网膜在两种光感受器退化模型中的存活和整合情况。

PROJECT SUMMARY Inherited retinal diseases (IRD), such as retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), are a common cause of visual impairment worldwide. Recent studies show that cell replacement therapy is a promising therapeutic option for IRD patients with severe retinal degeneration that destroys photoreceptors. We and others recently reported long-term survival and integration of human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) derived retinas in a novel laser-induced photoreceptor degeneration model. While the laser ablation model can be generated rapidly as an acute degeneration model, it does not have the progressive pathophysiological nature (e.g., disease time course, retinal remodeling, etc.) of IRD. To address this issue, we developed a highly efficient genome-editing technology, Platinum Talen, which leads to gene disruption in multiple organisms with high efficacy and specificity. By applying Platinum Talen to this novel model, we achieved one-step generation of biallelic gene modifications with high efficiency (40-50%) in preliminary studies. Since substantial information regarding the survival and integration of transplanted photoreceptor cells or retinal sheets have been obtained in laser injury models, we propose a two-pronged strategy to generating novel translation-enabling models to evaluate survival and integration of regenerated photoreceptors in the visual system: 1) to use laser-induced photoreceptor degeneration model; 2) one-step generation of biallelic mutants by Platinum Talen. We will then systematically compare the survival and integration of regenerated photoreceptors in the two model systems. Aim 1 will generate novel models of photoreceptor degeneration by laser ablation and Platinum Talen. Aim 2 will perform comprehensive ophthalmic phenotyping of laser-induced and Talen-edited models of photoreceptor degeneration. Aim 3 will evaluate survival and integration of human induced pluripotent stem cell (hiPSC)-derived retina sheets in the two models of photoreceptor degeneration.