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

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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.