Develop delivery systems and therapeutic proteins for reversal of botulinum neurotoxin intoxication

About This Grant

Project Summary Botulinum neurotoxins (BoNTs) are a family of bacterial toxins including seven serotypes (BoNT/A-G). They cause botulism and are classified as one of the most dangerous potential bioterrorism agents. BoNTs enter neurons and remain active in neurons for a long period of time, causing persistent paralysis in humans and animals. There is currently no inhibitor available that can block BoNT activity once they enter neurons, and such a post-symptom inhibitor is urgently needed for treating botulism patients and for biodefense. We previously developed an intraneuronal delivery protein based on a BoNT-like toxin, BoNT/X, which was discovered in our lab, with its receptor-binding domain replaced by the corresponding region of a BoNT. Such a chimeric protein can send anti-toxin nanobodies into motor neurons in vivo and reverse BoNT intoxication inside neurons. Besides BoNT/X, our lab recently uncovered a series of novel BoNT- like toxins, many of which exhibit biochemical properties for recombinant expression superior to those of BoNT/X. In addition, a series of new nanobodies against BoNT/A and BoNT/B have been generated, with higher binding affinity or neutralization efficacy than the previously available nanobodies. Therefore, in the exploratory R61 Phase, we propose to build and evaluate a new generation of delivery proteins based on novel BoNT-like toxins (Aim 1). We will also screen newly available nanobodies to identify those with higher efficacy as a cargo for intraneuronal delivery than the previous nanobodies (Aim 2). We will then fuse them to create at least one improved therapeutic protein that can reverse BoNT intoxication (our milestone for transition from R61 to R33 Phase). Once such a lead protein is finalized, we will then collaborate with the experienced team at the Biological Process Development Facility (BPDF) at the University of Nebraska – Lincoln to develop and establish the scale-up production process, analytical assays, and pharmacokinetic parameters (Aim 3). BPDF will then finish the large-scale (150-L fermentation) engineering production of a toxicity batch (Aim 4). Success of our proposal will generate and produce a first-in-class therapeutic protein that can reverse BoNT intoxication.