Selective targeting of CNS cells in vivo for multiple transgene delivery and neuronal reprogramming - Project Summary/Abstract Viral vectors enjoy widespread use for gene transfer to multiple cell types and organ systems in both research studies and, increasingly, in therapeutic strategies. For studies of the CNS, the ability of the viral vector to effectively and selectively infect the desired cell types within the CNS plays a critical role in the subsequent interpretation of study outcomes. However, most viral vectors are somewhat promiscuous in their cell type specificity. Although a particular vector pseudotype may primarily infect one type of cell, the additional “off- target” infection of other cell types may introduce unwanted variance to the study outcomes. In some recent studies, the intended infection of astrocytes also resulted in the unwanted infection of neurons, undermining the studies’ conclusions. Thus, there is a need to improve the current state of cell-type tropism to one of cell- type selectivity to specifically and exclusively target the desired cell type and eliminate off-target effects. A similar unmet need is the lack of a tool to selectively target a previously infected cell for a subsequent “follow- up” gene delivery. In this Exploratory/Developmental R21 mechanism project, we propose to build upon the popular expression of the avian TVA receptor used to enable EnvA-pseudotyped rabies virus delivery of a fluorescent reporter gene for transsynaptic labeling in the CNS. Our aims are in the context of our interest to perform direct in vivo cellular reprogramming of glial progenitor cells into neurons. In aim 1, we will use our current retroviral approach to infect proliferating glial progenitor cells in the adult brain to express the avian TVA receptor. We will then be able to selectively target the TVA-expressing glial progenitor cells for gene transfer using EnvA-pseudotyped lentivirus. In aim 2, we will directly target the glial progenitor cells by pseudotyping lentivirus to exclusively bind to a cell-specific membrane receptor. This will facilitate the demonstration of targeted infection of glial progenitor cells through selective and exclusive gene transfer. Including TVA in the cell specific receptor-pseudotyped lentivirus construct will permit subsequent selective targeting by EnvA-pseudotyped lentivirus for delivery of subsequent reprogramming factors or other gene transfer.
