Biology and Engineering of Botulinum Neurotoxins.

Project Summary Botulinum neurotoxins (BoNTs) are a family of bacterial protein toxins. They are one of the six most dangerous potential bioterrorism agents. Members of BoNTs, BoNT/A (Botox) and BoNT/B, are also FDA-approved for treating a growing list of disorders, as well as for reducing wrinkles. Understanding the molecular mechanisms for this family of toxins, engineering toxins to improve their therapeutic uses, and developing effective countermeasures have been the focus of our lab. Our prior funding cycle has led to major discoveries in understanding toxin receptors, identification of novel BoNT-like toxins, and development of new treatment that can neutralize BoNTs inside motor neurons. In this renewal proposal, we will address both side of BoNTs through protein engineering approaches: Aim 1 seeks to develop a novel chimeric toxin that can preferentially bind to the toxin receptor isoform dominantly expressed in autonomic and sensory neurons and evaluate its improvement over the standard Botox for treating overactive bladder conditions in rodent models. Aim 2 seeks to develop and evaluate novel nanobody-based triple-epitopic antibodies (NTAb) for neutralizing and clearing BoNTs in the circulatory system. NTAb is constructed by fusion of three nanobodies targeting distinct regions on BoNTs and then to a Fc, thus generating an artificial antibody that can achieve binding of three NTAb to a single toxin molecule, which promotes toxin clearance. In this proposal, we will develop two sets of NTAb, targeting two most common toxins, BoNT/A and BoNT/B, respectively, and thoroughly evaluate their efficacy, pharmacokinetics, and toxin clearance using rodent and guinea pig models.