Enhancer AAV toolbox for cell classes and subclasses in the brains of mouse, macaque and marmoset.

Project Summary We propose to create, screen, characterize, and distribute (through one or more non-profit partners) a large set of recombinant adeno associated virus (AAV) tools to target the cell classes, subclasses, and select types in the mouse, marmoset and macaque brains. We propose to leverage our new whole mouse brain (WMB) single- nucleus multiome dataset to nominate mouse enhancers for classes and subclasses in the mouse brain. We will employ a second-generation enhancer discovery, screening and characterization pipeline to evaluate at least 2000 enhancers in mouse, 100 in macaque and 30 in marmoset. The characterization of enhancer virus specificity and labeling completeness will be carried out in mouse with a tri-modal pipeline: WMB light sheet imaging, WMB 10xFlex sn-transcriptomics, and spatial transcriptomics on select sections with BARseq. The enhancers with desirable expression patterns will be modified to increase expression strength through enhancer core concatenation and tested for driving different cargos (e.g., Cre recombinase). The characterization of enhancer specificity in macaque and marmoset will rely on BARseq for the injected brain regions at the level of class and subclass, and where feasible, supertypes. In parallel with the brain-wide enhancer AAV discovery, we will conduct a focused enhancer AAV discovery at fine cell-type granularity for midbrain dopaminergic neuron types and subtypes with emphasis on cross-species validation and conservation. These cell types are key players in the brain circuits of reward, motivation, and motor control and the tools discovered here could be applicable to addressing circuit dysfunction in basal ganglia disorders. If successful, this award will establish an unprecedented mammalian brain toolbox showcasing both a broad brain-wide approach and a focused cell-type approach for the neuroscience community with reagents, characterization data and protocols for use available to all. The deliverables from this award could have a transformative effect on cell type characterization in the mammalian central nervous systems, from measuring various properties of cell types to defining their function and the detailed understanding of structure-function relationships in mammalian brains, and potentially, development of cell type-specific therapeutics for brain disorders.