AAV-mediated Müller glia reprogramming to early-stage retinal progenitor cells

早期和晚期视网膜祖细胞（RPC）选择性地产生视网膜神经元， 视网膜发育过程中的离散时间窗。Müller胶质细胞（MG）和晚期RPC具有 相似的基因表达谱，并表达许多共同的转录因子（TF）， 和神经能力。在视网膜损伤时，这些TF中的许多在MG中下调，而在MG中下调的TF中， 驱动反应性神经胶质增生被上调。这一过程是激活鱼类神经原性能力所必需的， 两栖动物然而，哺乳动物中的MG缺乏神经原性能力，并且维持静止的TF是 在受伤后迅速重新表达。我们已经确定了增殖和神经原性的关键调节因子， 能力在RPC和MG通过多组学分析。我现在正在研究一个单一的 基于AAV的试剂可用于将MG重编程为早期RPC样细胞， 视网膜细胞类型，包括视锥光感受器。我假设MG可以被重新编程， 在哺乳动物中的增殖和神经原性能力，通过破坏TF的功能， RPC的身份，也表达在成人MG。此外，我预计， 促进MG衍生祖细胞中早期RPC的特性可能促进早期出生的视网膜细胞的产生 类型（图1B）。最后，通过将这些过度表达和功能丧失的方法与 过度表达促进感光细胞特化的TF，我希望能够产生大量的 早期出生的锥状光感受器的数量。我提出两个目标来解决这个假设。目的I：改变视网膜 使用全长和显性负性基因的过表达， 候选TF的构建体。多重单细胞（sc）RNA-seq分析将用于鉴定构建体 其促进电穿孔晚期RPC和转导的成人中的增殖和神经原性能力 MG.免疫组织化学将用于验证scRNA-seq数据的发现。这一目标将使 转录因子的功能特征，调节早期和晚期阶段之间的过渡， RPC的发育能力，以及MG从静止状态到神经源性状态的转变。 这一目标还将确定促进早期出生细胞类型产生的构建体，包括锥细胞。 光感受器，在新生儿视网膜外植体和成熟MG。目的II：Prdm 1在大肠杆菌中的过表达 重编程成年MG以驱动视锥光感受器形成。Prdm 1选择性强表达于 感光细胞前体并刺激感光细胞分化。Prdm 1的过表达可能诱导 重编程MG以产生早期RPC样细胞，这些细胞是神经源性的，并特异性地产生成熟的 视锥光感受器该项目具有重要的潜力，有助于开发新的基因， 感光细胞营养不良的治疗，包括年龄相关性黄斑变性、斯塔加特病和 视网膜色素变性

Early-stage and late-stage retinal progenitor cells (RPCs) selectively generate retinal neurons in discrete temporal windows over the course of retinal development. Müller glia (MG), and late-stage RPCs have similar gene expression profiles and express many shared transcription factors (TFs) that repress proliferative and neurogenic competence. Upon retinal injury, many of these TFs are downregulated in MG, while TFs that drive reactive gliosis are upregulated. This process is necessary to activate neurogenic competence in fish and amphibians. However, MG in mammals lack neurogenic competence, and TFs that maintain quiescence are rapidly re-expressed following injury. We have identified key regulators of proliferative and neurogenic competence in both RPCs and MG through multiomic analyses. I am now exploring the possibility that a single AAV-based reagent can be used to reprogram MG to early-stage RPC-like cells that generate early-born retinal cell types, including cone photoreceptors, in situ. I hypothesize that MG can be reprogrammed to gain proliferative and neurogenic competence in mammals by disrupting the function of TFs that promote late-stage RPC identity and are also expressed in adult MG. Furthermore, I anticipate that overexpression of TFs that promote early-stage RPC identity in MG-derived progenitors may promote generation of early-born retinal cell types (Fig 1B). Finally, by combining these overexpression and loss of function approaches with overexpression of TFs that promote photoreceptor specification, I expect to be able to generate substantial numbers of early-born cone photoreceptors. I propose two aims to address this hypothesis. Aim I: Alter retinal development trajectory and reprogram adult MG using overexpression of full-length and dominant-negative constructs of candidate TFs. Multiplexed single-cell (sc)RNA-seq analysis will be used to identify constructs that promote proliferative and neurogenic competence in electroporated late-stage RPCs and transduced adult MG. Immunohistochemistry will be used to validate findings from the scRNA-seq data. This aim will allow for the functional characterization of TFs that regulate the transition between early and late stages of developmental competence in RPCs, as well as the transition of MG from a quiescent to a neurogenic state. This aim will also identify constructs that promote the production of early-born cell types, including cone photoreceptors, in neonatal retinal explants and mature MG. Aim II: Overexpression of Prdm1 in reprogrammed adult MG to drive cone photoreceptor formation. Prdm1 is selectively and strongly expressed in photoreceptor precursors and stimulates photoreceptor differentiation. Overexpression of Prdm1 may induce reprogrammed MG to produce early-stage RPC-like cells that are neurogenic and specifically generate mature cone photoreceptors. This project has significant potential to contribute to the development of novel gene therapies for photoreceptor dystrophies, including age-related macular degeneration, Stargardt's disease, and retinitis pigmentosa.