An in vitro/in vivo system for targeted retinal ganglion cell subtype manipulation

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PROJECT SUMMARY Glaucoma is characterized by the gradual degeneration of retinal ganglion cells (RGCs). RGCs are highly heterogeneous, and greater than 40 RGC subtypes in the mouse retina have been identified. Currently, our understanding of RGC subtypes is hindered by the general inaccessibility of RGCs due to their small number. Therefore, techniques that enrich the population of viable adult RGCs and RGC subtypes for experimentation, such as immunopanning, are of great value. In this new application, we will develop a novel in vitro/in vivo system for targeted RGC subtype manipulation based on modifications of the immunopanning technique. Our overall hypothesis is that Cre-dependent ectopic expression of avian tumor virus receptor A (TVA) in RGCs and RGC subtypes will allow for the isolation and culture of highly purified RGC populations. This will be achieved through TVA-mediated immunopanning (TVAMI), a new technique we will develop which integrates a positive immunopanning step based on protein binding to TVA. Furthermore, through transduction with lentivirus pseudotyped with EnvA, a selective TVA ligand, we will attempt to induce gene expression in the same TVA-expressing RGC populations both in vitro and in vivo. There are two Specific Aims: 1) establish and optimize the TVAMI system for RGC isolation and culture; and 2) manipulate gene expression in targeted RGC subpopulations. Throughout Aim 1, we will study transgenic mice that express TVA on the cell surface of nearly all adult RGCs. We will compare several TVAMI variations against standard immunopanning both after isolation and one week of cell culture with cell staining and immunocytochemistry to develop an optimized protocol. Throughout Aim 2, we will study transgenic animals that express TVA in αRGCs, a population of RGCs which includes four RGC subtypes. First, we will isolate αRGCs with TVAMI and confirm the identity, validate the purity, and measure the relative survival of the four αRGC subtypes after isolation and one week of cell culture with cell staining and immunocytochemistry. We will also perform single cell RNA sequencing on this enriched population to define the transcriptional signature of αRGCs. Second, after αRGC isolation, we will assess the specificity and efficiency of αRGC-specific virus transduction in vitro with EnvA-pseudotyped lentivirus or AAV2 vector containing an RGC-specific GFP expression cassette. Third, we will introduce the same viruses to RGCs via intravitreal injection and compare rates of αRGC transduction in vivo with immunohistochemistry in whole mounted and sectioned retinas and after subsequent TVAMI. If successful, this system has the potential to unlock multiple new approaches to study RGCs and RGC subtypes in normal and disease states, and enable a broad range of novel applications with potential therapeutic value.