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

AAV介导的穆勒胶质细胞重编程为早期视网膜祖细胞

• 批准号: 10605472
• 负责人: Nicole Pannullo
• 金额: $ 4.77万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605472
• 关键词: ATOH7 gene; Address; Adult; Advanced Development; Age related macular degeneration; Amphibia; Blindness; Cell Differentiation process; Cells; Competence; Data; Development; Disease; Dominant-Negative Mutation; Electroporation; Fishes; Gene Expression; Generations; Genes; Gliosis; Immunohistochemistry; In Situ; Individual; Injury; Length; Mammals; Mediating; Muller' s cell; Mus; N-Methylaspartate; Neonatal; Phenotype; Photoreceptors; Process; Production; Proliferating; Reagent; Repression; Retina; Retinal Cone; Retinitis Pigmentosa; Specific qualifier value; Stargardt' s disease; Technology; Testing; Time; Vision; adeno-associated viral vector; cell type; combinatorial; competence factor; daughter cell; gene repression; gene therapy; innovation; loss of function; multiple omics; novel; novel therapeutics; overexpression; postnatal; prenatal; progenitor; programs; response; retinal damage; retinal neuron; retinal progenitor cell; selective expression; single-cell RNA sequencing; transcription factor; transcription regulatory network

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.

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口（MG）和晚期 RPC 具有 相似的基因表达谱并表达许多抑制增殖的共享转录因子（TF） 视网膜损伤后，许多 TF 会在 MG 中下调，而 MG 中的 TF 会下调。 驱动反应性神经胶质细胞增生是激活鱼类神经源能力所必需的。 然而，两栖动物的 MG 缺乏神经源能力，而维持静止的 TF 则缺乏。 我们已经确定了增殖和神经源性的关键调节因子。 通过多组学分析，我现在正在探索单个人在 RPC 和 MG 方面的能力。 基于 AAV 的试剂可用于将 MG 重新编程为早期 RPC 样细胞，从而产生早期出生的细胞 视网膜细胞类型，包括视锥细胞，我认为 MG 可以通过重新编程来获得。 通过破坏促进晚期阶段的转录因子的功能来提高哺乳动物的增殖和神经发生能力 RPC 特性也在成人 MG 中表达。此外，我预计 TF 的过度表达会导致 促进 MG 衍生祖细胞的早期 RPC 身份可能促进早期视网膜细胞的生成 最后，通过将这些过度表达和功能丧失方法与 促进光感受器规范的转录因子的过度表达，我希望能够产生大量的 我提出了两个目标来解决这个假设：改变视网膜。 使用全长和显性失活的过度表达来确定发育轨迹并重新编程成人 MG 候选 TF 的构建体将用于鉴定构建体。 促进电穿孔晚期 RPC 和转导成体的增殖和神经源能力 MG。免疫组织化学将用于验证 scRNA-seq 数据的结果。 调节早期和晚期阶段之间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产生的构建体，包括锥体细胞。 新生儿视网膜外植体和成熟 MG 中的光感受器 目标 II：Prdm1 在视网膜中的过度表达。 重新编程的成人 MG 驱动视锥光感受器形成的 Prdm1 在体内选择性地强烈表达。 光感受器前体并刺激光感受器分化可能会诱导 Prdm1 过度表达。 重新编程 MG 以产生早期 RPC 样细胞，这些细胞具有神经源性并特异性生成成熟细胞 该项目具有促进新基因开发的巨大潜力。 光感受器营养不良的治疗，包括年龄相关性黄斑变性、斯塔加特氏病和 视网膜色素变性。