Generation of viral vectors that use alternative splicing to drive cell type-specific gene expression in the nervous system

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Project Summary New tools are urgently needed to selectively target constructs that monitor and manipulate the activity of individual cell types without having to rely on genetic manipulation. This proposal aims to develop viral tools that use cell type-specific alternative splicing events to drive cell type-specific gene expression in the nervous system independent of genetic manipulation, an approach we term splicing-linked expression design (SLED). SLED-based vectors use evolutionarily conserved, highly cell type-specific exons, identified using the ASCOT database developed by our group, to drive expression of reporter and effector constructs. We have demonstrated feasibility of this approach using constructs that selectively target retinal photoreceptors, muscle cells, and cortical neurons. We propose to extend this by combining cell-specific alternative exon/intron sequences with appropriate promoter sequences to generate a toolbox of AAV and lentiviral SLED vectors that selectively target multiple cell types of interest to the neuroscience research community. We will first generate SLED vectors that target primary sensory and motor neurons, as well as multiple subtypes of cortical neurons and glia, and validate the specificity of these reagents in mice. We will next test the cell specificity of SLED reagents that are validated in mice in rats, ferrets, as well as human cortical organoids and rat-human chimeras. Finally, highly specific SLED fluorescent reporter constructs will be converted to drive expression of calcium indicators, as well as optogenetic and chemogenetic constructs. We anticipate that SLED-based reagents will allow highly cell type-specific expression of a broad range of molecular tools useful for analysis of neural circuitry in multiple mammalian species.