I/O Tags: Genetically Encoded Tags for Trans-Neuronal Control Across Specific Synapse Classes - PROJECT SUMMARY The ground-truth definition, experimental control, and therapeutic treatment of neuronal circuits requires technologies that perform well in vivo. Powerful tools that control and report signal transduction, neuronal activity, and gene expression continue to emerge, but the field lacks robust technologies to deliver those tools throughout long-distance neuronal circuits in the mammalian brain. The state-of-the-art relies on viruses that spread from neurons to their retrograde (e.g. rabies) or anterograde (e.g. HSV1) partners. However, these viruses are pathogens that can exhibit neurotoxicity and spread non-specifically, infecting any type of pre- or postsynaptic neurons. This is problematic because the types of synaptic inputs to, and targets of, a given class of neuron are diverse. Consequently, the unmet need for precision trans-neuronal delivery methods severely constrains analyses of how interaction between different cell types influences brain function. While deliberate experimental designs can enable the evaluation of specific types of inputs or outputs in some model systems (e.g., in mice, zebrafish, or fruit fly), these designs rely on genetic modifications to the organism that grant access to unique cell types. There is no such method for robust access from neurons to specific types of their inputs and outputs in “higher” organisms. To address this gap, we propose a transformative project to develop tools that will go from a given neuron starter population to different user-selectable types of its synaptically-connected cells, without the need for replicative neurotropic viruses. Our central goal is to create proteins that achieve targeted trans-neuronal delivery. Our novel strategy is to encode the specificity for distinct types of connected neurons directly into designer proteins, I/O Tags. The I/O Tags will function as fusions: when appended to other proteins, the latter will be ferried into selectable classes of pre- or post-synaptic targets. The Tags will be comprised of combinations of motifs that, in concert, target payloads to synapses, enable release, achieve entry into the preferred type cells, and execute a desired action. We will screen deep libraries of Tag permutations in vivo by utilizing the mouse cerebellar cortex as a model circuit to screen and validate I/O Tags. The investigators will leverage their areas of expertise to assay I/O Tags’ delivery from Purkinje cells into their synaptic partners, ensuring biological compatibility through microscopy, electrophysiology, and behavior. With safety and utility demonstrated in mice, we will validate I/O Tags in ferrets, tree shrews, and macaques. If successful, the outcome of this high-risk proposal will be transformative technologies for trans-synaptic anterograde and retrograde delivery of biological agents. Our Tags will thus seamlessly augment and catalyze existing approaches by enabling the targeted delivery of a diverse array of existing tools, agnostic to the vector or species utilized. If successful, our project will fundamentally change the state-of-the-art to enable targeted cell-type and circuit-specific experimentation and therapy evaluation.
