Systematic identification of enhancers to target the breadth of excitatory and inhibitory neuronal cell types in the cerebral cortex

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PROJECT SUMMARY In this proposal we aim to identify gene regulatory elements that permit the targeting and manipulation of brain circuit models of human brain function. Gaining genetic access to specific neuron populations in nontransgenic animals and humans would enable targeted circuit modulation for hypothesis testing and provide a means to evaluate the safety and efficacy of circuit modulation for the treatment of epilepsy and psychiatric disorders. Our approach capitalizes on our combined expertise in the development and maturation of brain cell-types and circuits (Gord Fishell and Paola Arlotta), identification of CIS-regulatory elements that function across species (Yating Wang), AAV engineering combined with large-scale screening methods (Ben Deverman) and expertise at combining RNA and chromatin biology (Jason Buenrostro). Our efforts will also benefit from an ongoing collaboration with John Reynolds at the Salk Institute on observation and manipulation of cortical circuits during complex visual perception tasks. This project will build upon success that we and others have had in identifying gene regulatory elements that enable cell type-restricted gene expression when used within recombinant adeno-associated virus (AAV) vectors. In particular, the pipeline for enhancer discovery stemming from our recent UG3 grant provides us with the systematic for identifying additional enhancer sequences that function in the context of the limited carrying capacity of AAV. Here we aim to apply the novel high-throughput screening approach we devised in the course of this UG3 for the rapid identification of a suite of enhancers that enable the study and manipulation of genetically defined cell types and circuits across species. Our preliminary data demonstrates that our enhancer identification strategy can yield novel and highly specific enhancers that restrict expression to target populations. In addition, we have demonstrated that it is possible to use the engineered AAV-PHP.eB capsid to screen enhancers across the brain with a single noninvasive injection. These successes have highlighted the need for more rapid and comprehensive assessment of putative enhancers. In addition, we will examine the tolerance to neuronal activity manipulation within the target neuronal populations in several species. This proposal will be transformative in devising methods to target and manipulate the brain activity of specific neuronal cell populations across species, including human cell-derived organoids.