PROJECT SUMMARY A goal of the BRAIN initiative is to develop and validate novel tools to map and manipulate neural circuits. The definition and control of behaviorally relevant circuits requires both retrograde and anterograde trans-synaptic technologies that perform well in vivo. In this project, we pursue the long-term goal of developing a small protein tag for the anterograde delivery of cargos (an “AnteroTag”). The novel concept of AnteroTag is to leverage the endogenous biology of neurotransmission to target tagged payloads to synaptic vesicles where they undergo vesicular release, postsynaptic entry, and desired action. Our consortium of investigators proposes to utilize the pontocerebellar circuit as a model pipeline to further refine and rigorously validate AnteroTag. As the axons of basal pontine neurons (mossy fibers) make specific and quantitative synapses upon cerebellar granule cells, the pontocerebellar circuit affords an established and conserved model circuit to determine how iterations of AnteroTag alter its performance. The investigators of the consortium will leverage their respective areas of expertise to quantify the specificity and biocompatibility of AnteroTag, benchmarked against a current state-of- the-art anterograde viral vector HSV-129; these analyses in mice will include microscopy, electrophysiology, and in vivo activity imaging in behaving animals. Once the pipeline determines the most specific and safe version of AnteroTag in the pontocerebellar circuit, we will assay AnteroTag performance when delivered to diverse starter populations of neurons. The broad utility of AnteroTag will then be determined by testing its efficacy to deliver a variety of genetically encoded markers and modifiers, and by testing AnteroTag derivatives for their utility in accessing second-order anterograde populations. With safety, specificity, and utility thus rigorously demonstrated in mice, we will assay the performance of AnteroTag in the pontocerebellar circuit of higher organisms including tree shrews, ferrets, and NHPs. The outcome of this proposal will be the development, validation, and implementation of a novel technology for the trans-synaptic anterograde delivery of biological agents across multiple brain regions and multiple species. Thus, if successful, our project will ultimately aid in the development of targeted cell-type and circuit-specific therapeutics to treat brain disorders.

PROJECT NARRATIVE Our goal is to develop a new trans-synaptic anterograde technology, as current strategies rely on replication- competent viruses that suffer from unsatisfactory toxicity, efficacy, and/or specificity. To accomplish our goal, we are refining an engineered protein sequence that can deliver active agents to target cells in vivo. If successful, this project will serve multiple goals of the BRAIN to map, monitor, or manipulate neural circuits in model organisms in the short term, and to potentially deliver therapeutic agents in humans in the long term.