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

早期和晚期视网膜前体细胞(RPC)选择性地在 视网膜发育过程中的离散时间窗。Müler glia(MG)和晚期RPC 相似的基因表达谱并表达许多共同的转录因子(TF)以抑制增殖 和神经性能力。在视网膜损伤时，许多这样的TF在MG中下调，而TF 驱动反应性胶质细胞增生症上调。这一过程对于激活鱼类的神经源性能力是必要的 两栖动物。然而，哺乳动物的MG缺乏神经源性能力，而保持静止的TF是 在损伤后迅速重新表达。我们已经确定了增殖性和神经源性的关键调节因子 通过多组学分析，在RPC和MG方面都有能力。我现在正在探索这样一种可能性：单曲 基于AAV的试剂可用于将MG重新编程为早期RPC样细胞，从而产生早期出生 视网膜细胞类型，包括视锥感光细胞，原位观察。我假设MG可以被重新编程以获得 哺乳动物的增殖和神经发生能力，通过破坏促进晚期的转录因子的功能 RPC同源性，在成人MG中也有表达。此外，我预计TF的过度表达 促进MG祖细胞早期RPC特性可能促进早期出生的视网膜细胞的生成 类型(图1B)。最后，通过将这些过度表达和函数丢失方法与 过量表达的TF可以促进光感受器的规范，我希望能够产生大量的 早期出生的视锥感光细胞的数量。我提出了两个目标来解决这一假设。目标一：改变视网膜 利用全长和显性负性过表达对成年MG的发育轨迹和重编程序 候选TF的构造。多路复用单细胞(Sc)rna-seq分析将用于识别构建体。 促进电穿孔晚期RPC和转导成人的增殖和神经源性能力 Mg.免疫组织化学将用于验证scRNA-seq数据的发现。这一目标将允许 调控卵巢癌早期和晚期过渡的转录因子的功能特征 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的产生的结构，包括锥体 新生视网膜外植体和成熟MG中的光感受器。目的II：Prdm1在BL21细胞中的过表达 对成年MG重新编程以驱动视锥感光细胞的形成。Prdm1选择性强表达于 光感受器前体和刺激光感受器分化。Prdm1的过表达可能诱导 重新编程MG以产生早期RPC样细胞，这些细胞是神经源性的，并特异性地生成成熟细胞 视锥感光器。该项目对新基因的开发具有很大的潜力 光感受器营养不良的治疗，包括老年性黄斑变性、Stargardt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.