Human botulism can result from the ingestion of food contaminated with botulinum neurotoxin (BoNT), from the infection of wounds or intestine, or from direct inhalation of the toxin, which can be transcytosed across epithelial barriers as a physiologically active molecule. Intoxication, if untreated, may lead to death due to respiratory muscle failure, while recovery from botulism is a prolonged process (up to several months) and BoNTs are classified as one of the six highest?risk threat agents for bioterrorism by the CDC. Developing may require intensive and expensive hospital care. Due to their extreme toxicity and ease of production, antidotes to botulinum neurotoxins is a priority goal of the Biodefense Research Agenda. During the previous funding period, we developed a molecular engineering platform that takes advantage of structural features and trafficking of BoNTs to enable the delivery within the presynaptic compartment of intoxicated neurons of single domain camelid antibodies (sdAbs) that bind and inactivate the wt light chain (LC) of BoNT metalloproteases. We developed a post-exposure anti-botulinum therapeutic (PEABT) targeting wt BoNT/A1, which is the first compound ever to show reversal of respiratory symptoms in mice at times post-intoxication when standard antibodies are ineffective. Preliminary data also demonstrated the effectiveness of a second lead candidate against wt BoNT/B, and attest to the generalizability of the platform. We succeeded in developing a novel principle for delivering single domain antibodies to the presynaptic compartment of neurons without a viral vector. In addition, we developed a murine model of botulism that is specifically appropriate to test botulism antidotes with an intra-neuronal mode of action. The murine model is currently being translated to a guinea pig, in response to an FDA suggestion that the guinea pig was the species to use for an Animal Rule pivotal study. Our objective is to expand the therapeutic range of this platform to two other known human pathogenic serotypes ? wt BoNT/B and wt BoNT/E, and to further increase the potency and therapeutic window of the wt BoNT/A1 countermeasure. We will identify sdAbs that bind and broadly neutralize BoNT/A, /B, and /E LC subtypes, and we will pursue strategies to increase the potency of PEABTs by increasing the affinity of sdAbs for the wt LC target and by improving therapeutic efficacy. 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 sdAbs, extensive expertise in expressing neuron-targeted physiologically active fusion proteins that use metalloprotease-inactivated BoNT/C1 as a molecular vehicle for delivery of sdAbs, 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.

Public Health Relevance

This project aims to develop novel botulism therapeutics based on the use of natural neuronal receptors and trafficking patterns normally employed by the targeted toxin itself. Unlike available antibody- based on atoxic derivatives of botulinum neurotoxin (BoNT) can reach an intracellular target in a post?exposure based therapeutics and small molecule inhibitors of the light chain metalloprotease, botulism therapeutics setting; unlike conventional, small molecule therapeutics which would equally affect all cells in the body with potentially deleterious cytotoxic side effects, the proposed atoxic BoNT-based molecular vehicle will specifically deliver a therapeutic moiety only to the cytosol of the target cells (motor neurons). This is a unique and innovative approach toward treating not only botulism, as embodied in this proposal, but also toward treating numerous infectious and neurodegenerative diseases in which therapeutic targets reside and exert their action intracellularly.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI093504-07
Application #
9512642
Study Section
Drug Discovery for the Nervous System Study Section (DDNS)
Program Officer
Ranallo, Ryan
Project Start
2011-04-15
Project End
2022-06-30
Budget Start
2018-07-01
Budget End
2019-06-30
Support Year
7
Fiscal Year
2018
Total Cost
Indirect Cost
Name
New York University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
Beske, Phillip H; Hoffman, Katie M; Machamer, James B et al. (2017) Use-dependent potentiation of voltage-gated calcium channels rescues neurotransmission in nerve terminals intoxicated by botulinum neurotoxin serotype A. Sci Rep 7:15862
Pellett, Sabine; Tepp, William H; Johnson, Eric A et al. (2017) Assessment of ELISA as endpoint in neuronal cell-based assay for BoNT detection using hiPSC derived neurons. J Pharmacol Toxicol Methods 88:1-6
Vazquez-Cintron, Edwin J; Beske, Phillip H; Tenezaca, Luis et al. (2017) Engineering Botulinum Neurotoxin C1 as a Molecular Vehicle for Intra-Neuronal Drug Delivery. Sci Rep 7:42923
Beske, Phillip H; Bradford, Aaron B; Grynovicki, Justin O et al. (2016) Botulinum and Tetanus Neurotoxin-Induced Blockade of Synaptic Transmission in Networked Cultures of Human and Rodent Neurons. Toxicol Sci 149:503-15
Vazquez-Cintron, Edwin; Tenezaca, Luis; Angeles, Christopher et al. (2016) Pre-Clinical Study of a Novel Recombinant Botulinum Neurotoxin Derivative Engineered for Improved Safety. Sci Rep 6:30429
Pellett, Sabine; Tepp, William H; Scherf, Jacob M et al. (2015) Botulinum Neurotoxins Can Enter Cultured Neurons Independent of Synaptic Vesicle Recycling. PLoS One 10:e0133737
Lin, Guangyun; Tepp, William H; Bradshaw, Marite et al. (2015) Immunoprecipitation of native botulinum neurotoxin complexes from Clostridium botulinum subtype A strains. Appl Environ Microbiol 81:481-91
Pellett, Sabine; Schwartz, Michael P; Tepp, William H et al. (2015) Human Induced Pluripotent Stem Cell Derived Neuronal Cells Cultured on Chemically-Defined Hydrogels for Sensitive In Vitro Detection of Botulinum Neurotoxin. Sci Rep 5:14566
Pellett, Sabine; Tepp, William H; Scherf, Jacob M et al. (2015) Activity of botulinum neurotoxin type D (strain 1873) in human neurons. Toxicon 101:63-9
Scherf, Jacob M; Hu, Xiaoyang Serene; Tepp, William H et al. (2014) Analysis of gene expression in induced pluripotent stem cell-derived human neurons exposed to botulinum neurotoxin A subtype 1 and a type A atoxic derivative. PLoS One 9:e111238

Showing the most recent 10 out of 16 publications