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）选择性地产生残留神经元 在视网膜开发过程中，离散的临时窗口。 MüllerGlia（MG）和后期RPC具有 相似的基因表达谱并表达许多反映增殖的共享转录因子（TF） 和神经源能力。视网膜损伤后，其中许多TF在Mg中被下调，而TFS则是 驱动反应性神经病已更新。这个过程对于激活鱼类的神经源能力是必要的 两栖动物。然而，哺乳动物中的MG缺乏神经源能力，保持静止的TF是 受伤后迅速重新表达。我们已经确定了增殖和神经源的关键调节剂 通过多组分分析，RPC和MG的能力。我现在正在探索一个单一的可能性 基于AAV的试剂可用于将MG重新编程至产生早期出生的早期RPC样细胞 现场的视网膜细胞类型，包括锥形感受器。我假设可以重新编程MG以获得 通过破坏促进晚期的TF功能，在哺乳动物中增殖和神经源能力 RPC的身份，也以成年Mg表示。此外，我预计TF的过表达 在MG衍生的祖细胞中促进早期RPC身份可能会促进产生早期出生的永久性细胞 类型（图1B）。最后，通过将这些过表达和功能方法的丧失结合在一起 我希望能够产生大量的TF的TFS过表达 早期出生的锥形感受器的数量。我提出了两个目的，以解决这一假设。目标I：改变视网膜 使用全长和显性阴性的过表达的发展轨迹和重编程成人毫克 候选TFS的构造。多路复用的单细胞（SC）RNA-seq分析将用于识别构造 这促进了电穿孔后期RPC中的增殖和神经源能力，并翻译成人 毫克免疫组织化学将用于从SCRNA-SEQ数据中验证发现。这个目标将允许 TF的功能表征，该特征调节了早期和晚期之间的过渡 RPC中的发育能力以及MG从静止状态到神经源状态的过渡。 该目标还将确定促进早期出生细胞类型的生产的结构，包括锥体 光感受器，在新生儿残留外植体和成熟的Mg中。 AIM II：PRDM1的过表达 重编程的成年毫克驱动锥形光感受器形成。 PRDM1在 感光细胞前体并刺激感光体分化。 PRDM1的过表达可能诱导 重新编程的MG产生神经源的早期RPC样细胞，并特别产生成熟 锥光受体。该项目具有为新基因发展做出贡献的巨大潜力 光感受器营养不良的疗法，包括与年龄相关的黄斑变性，Stargardt病和 色素性视网膜炎。