Location: Foodborne Toxin Detection and Prevention
Title: Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A Authors
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: October 7, 2011
Publication Date: N/A
Technical Abstract: Botulinum neurotoxins (BoNT) have the unique capacity to cross epithelial barriers, target neuromuscular junctions, and translocate active metalloprotease component to the cytosol of motor neurons. We have taken advantage of the molecular carriers responsible for this trafficking to create a family of recombinant, full-length BoNT derivatives with native structural features and physiologic trafficking profiles. They have been rendered atoxic by point mutations that disable the light chain (LC) metalloprotease. Based on murine toxicity studies demonstrating their LD50 to be 100,000-fold reduced relative to wt BoNT/A, CDC has excluded these atoxic BoNT derivatives from Select Agent status. The genetic constructs and expression systems developed in our laboratory enable the facile production of these recombinant, full-length, atoxic BoNT derivatives that preserve key structural features of the native molecule. In addition, we have engineered derivatives with an amino sequence at the N-terminus of the LC for site-selective attachment of cargo intended for delivery to the neuronal cytosol. Unlike native BoNTs, which effectively disable their own uptake, the atoxic derivatives can accumulate in neurons to detectable and quantifiable levels. Our data demonstrate targeting of recombinant atoxic BoNT/A to neuromuscular junctions after systemic administration, uptake into neurons, binding to SNAP 25 in the cytosol of neurons, and competition with the wild type toxin for neuronal binding and inhibition of wt BoNT/A toxicity. This technology platform is currently being used to design antidotes to BoNT poisoning that have potential to be effective for extended periods post-exposure. In addition to their therapeutic potential, this family of recombinant BoNT derivatives provides molecular tools that can be used to dissect details of endocytosis and exocytosis in neurons and to identify new targets for therapeutic modulation of these events.