PROJECT SUMMARY/ABSTRACT: Botulinum neurotoxin (BoNT) is a highly potent, neuroparalytic poison that cleaves presynaptic SNARE proteins required for neurotransmitter release. The primary toxic effect of BoNT poisoning is blockade of neuromuscular transmission, leading to flaccid paralysis and death by respiratory arrest. There are no specific countermeasures capable of reversing symptomatic botulism. Current treatment strategies for lethal exposures are limited to post-exposure prophylaxis via passive immunoglobulin therapy, which cannot prevent respiratory collapse once botulism symptoms emerge, and supportive care. In the event of a large-scale botulism outbreak or deliberate attack, there is limited ability to maintain large numbers of symptomatic victims on artificial ventilation for weeks or longer. Consequently, development of effective therapeutics for respiratory paralysis is a high priority for public health and national defense. A major limitation in creating an effective therapeutic for botulinum poisoning has been the inability to identify post-exposure treatments that block or terminate the molecular toxicity of the BoNT light chain (LC) within the nerve terminal. This project aims to develop a new class of botulism antidotes that exploit the natural neuronal receptors and trafficking pathways normally employed by the targeted toxin itself. Unlike available antibody-based therapeutics and small molecule inhibitors of the light chain metalloprotease, the botulinum neurotoxin (BoNT)‐based therapeutics that we have developed can reach an intracellular target in a post‐exposure setting. Unlike conventional small molecule therapeutics, which would equally affect all cells in the body with potentially deleterious side effects, the proposed method will specifically deliver a therapeutic moiety only to the cytosol of the target cells (neurons). This is a unique approach toward treating not only botulism, as embodied in this proposal, but also infectious and neurodegenerative diseases in which the causative agents reside and exert their action intra-neuronally. The team of scientists assembled for this project includes researchers with unique expertise who are well-qualified for the task at hand, including a long and successful history in generating BoNT-neutralizing nanobodies, extensive expertise in expressing neuron-targeted physiologically active fusion proteins that use metalloprotease-inactivated BoNT/C1 as a molecular vehicle for delivery of nanobodies, expertise in test systems for development of compounds promoting accelerated degradation of intracellular targets through the endogenous proteasomal system, and extensive experience in developing in vitro and in vivo models that can be used to test novel botulism antidotes.