Friday, May 23, 2025
5/23/2025
Enhancing Non-Viral Neuron-Specific Molecule Delivery Across Species in the CNS - Abstract Neuron-specific molecular delivery, while pivotal to both basic and translational neuroscience, currently faces significant limitations in efficiency and specificity. Nonviral approaches for the delivery of genes, proteins, and chemicals fall short in specificity and feasibility, particularly for delivering non-genetically encoded chemicals and for model species that are non-amenable to transgenic or viral delivery methods. Moreover, delivering molecules into the human nervous system for therapeutic purposes remains an inefficient process. We discovered a 15- amino-acid peptide, N1, capable of selectively targeting neurons, with significant potential to address current limitations. The N1 peptide exhibits strong neuron tropism, and our preliminary data suggest its capability to deliver a range of agents into neurons from diverse species and multiple brain regions. We anticipate that this novel tool will significantly impact neuroscience, enabling the delivery of varied molecules into neurons across species, and has significant translational potential and value for human therapeutics. This proposal outlines a comprehensive approach focusing on characterizing neuron-specific delivery, investigating neuron-targeting mechanisms, and optimization of its stability and delivery capabilities, including non-invasive brain delivery. First, we will further characterize the range of molecules that can be delivered in terms of their size and hydrophilicity by the N1 peptide, its ability for neuron-specific delivery across multiple model species (mouse, rat, treeshrew, zebra finch), and the function of delivered molecules into neurons (e.g., sensor dyes, mRNA, DNA, and Cre- recombinase protein). Second, we propose mechanistic studies to identify putative binding partners (biotin pull- down assay and proteomics), retroviral screen and gain-of-function approaches to identify genes involved in N1 peptide uptake and candidate N1 receptor molecules. Third, we will optimize the N1 peptide by elucidating the structure-activity relationship (e.g., alanine scanning), improving binding by conformationally constrained peptide analogs, and improving its stability and resistance to degradation by metabolically stable peptide analogs. We will test the neuron-targeting with intrathecal injection and non-invasive ultrasound-assisted delivery paving the way for potential translational applications. Lastly, we will investigate delivery of small molecules for neuron imaging and perturbation. The innovative aspects of this work include the first chemical approach for neuron- specific labeling across diverse brain regions and species and mechanistic insights into neuron-specific delivery.
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