PROJECT SUMMARY. Current AAV-based methods to target inhibitory interneurons, and their subpopulations, remain too leaky to use with recombinases such as Cre. Furthermore, the specificity of the widely used CaMKII minipromoter to access excitatory neurons also displays off-target expression. The long-term goal of this project is to develop ultra-specific AAVs that can overcome leakage into non-target cells and therefore be compatible with recombinase enzymes. The overall objective of this application is to develop and test hybrid AAVs that harness alternative splicing, through splicing linked expression design (SLED), in combination with current state- of-the-art minipromoters and enhancers. The central hypothesis is that this hybrid approach will increase target specificity, enabling the delivery of recombinases like Cre. The rationale for the project is to generate and share novel tools to speed up basic science research and reduce the reliance on knockin and transgenic Cre lines to target distinct populations of neurons. Increasing the specificity of AAVs would also benefit future gene therapy interventions, where highly controlled gene expression is essential. The central hypothesis will be tested by pursuing two specific aims: 1) Design and rigorously evaluate the ability of miniaturized enhancer-SLED hybrid constructs to deliver Cre with increased specificity; and 2) Generate sequencing datasets from subcortical brain regions to uncover cell type-specific splicing events. Under the first aim, cell-specific exons for inhibitory interneurons (INs), parvalbumin positive interneurons (PV-INs), and excitatory neurons (ENs) will be identified and utilized to make hybrid SLED AAVs with existing minipromoters or enhancers. These AAVs will be screened in vitro, then leading candidates will be thoroughly tested in vivo in mice to rigorously evaluate their specificity. For the second aim, the RiboTag technology will be used to isolate bulk RNA samples from ENs and PV-INs from the amygdala and thalamus, allowing high depth RNA sequencing to map patterns of cell and region-specific alternative splicing. This will enable integration with published cortical and hippocampal datasets so that region- specific alternative splicing patterns can be identified. This will further a basic scientific understanding of alternative splicing and support the future development of SLED vectors targeting subcortical cell populations relevant to psychiatric disease. The proposed research is innovative because it combines SLED with the leading enhancer and minipromoter technologies to create hybrid AAV constructs predicted to achieve a level of specificity beyond what is possible with each individual approach. This proof of principle experiment could open new horizons for the subpopulations of cells that can be targeted genetically. Furthermore, this research project will generate a valuable dataset tailored to comparing alternative splicing across cell types and brain regi...