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 may require intensive and expensive hospital care. Due to their extreme toxicity and ease of production, BoNTs are classified as one of the six highest-risk threat agents for bioterrorism by the US Center for Disease Control and Prevention (CDC). Developing antidotes to botulinum neurotoxins is a priority goal of the Biodefense Research Agenda. Our laboratory has developed genetic constructs and expression systems based on recombinant, full- length, atoxic BoNT derivatives that preserve key structural features of the wt BoNT/A required for physiological trafficking, and enable the development of novel anti-BoNT therapeutics (ABTs) that will be effective in a post-exposure setting by oral or inhalational routes. The construction and expression of these atoxic BoNT derivatives provide molecular delivery vehicles that target motor neurons, and can contain specific sites for enzymatic attachment of a wide variety of cargo and tracker molecules. The genetic constructs are modular by design, and enable the facile production of diverse candidate ABTs for evaluation. In this application we will test two approaches for delivering therapeutic moieties to target neurons: a) For the ABT-1 approach, therapeutic moieties effective against all BoNT serotypes will be incorporated directly into the amino acid sequence of the BoNT atoxic light chain (LC) metalloprotease, which will be expressed in combination with the BoNT/A heavy chain (HC) as full-length ABT-1;b) For the ABT-2 approach, a therapeutic moiety specifically active against wt BoNT/A will be coupled to the full length ABT-2 by site- selective enzymatic attachment to the ABT-2 precursor, carried by the BoNT/A heavy chain. The full-length BoNT derivatives we propose are expected to have unique advantages with respect to their ability to compete with wild type toxin for neuronal binding sites, and to mimic the endosome-to-cytosol channeling required for delivery of ABTs to the neuronal cytosol. Our current methods can produce sufficient quantities for in vitro and in vivo testing. These ABTs can be administered in a post-exposure setting by oral or inhalational routes.

Public Health Relevance

The goal of this project is to create anti-botulinum neurotoxin therapeutics and to test these therapeutics for efficacy and toxicity in model assays and in preclinical in vivo systems. This project provides product-ready alternatives to currently existing treatment modalities, including passive immunotherapy and vaccination, which have well described limitations. It is recognized that a large botulism outbreak would be disastrous due to lack of sufficient numbers of ICUs equipped with mechanical ventilators for prolonged life support, and the lack of an antidote that is effective after the toxin has entered neuronal cells (24-48 hours after intoxication), which emphasizes the need for a therapeutics that can treat botulism by inactivating BoNT after neuronal entry.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI093504-02
Application #
8253685
Study Section
Special Emphasis Panel (ZAI1-LG-M (J1))
Program Officer
Ranallo, Ryan
Project Start
2011-04-15
Project End
2016-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
2
Fiscal Year
2012
Total Cost
$1,238,429
Indirect Cost
$182,501
Name
New York University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
Whitemarsh, Regina Clare Meyer; Tepp, William Howard; Johnson, Eric Arthur et al. (2014) Persistence of botulinum neurotoxin a subtypes 1-5 in primary rat spinal cord cells. PLoS One 9:e90252
Vazquez-Cintron, Edwin J; Vakulenko, Maksim; Band, Philip A et al. (2014) Atoxic derivative of botulinum neurotoxin A as a prototype molecular vehicle for targeted delivery to the neuronal cytoplasm. PLoS One 9:e85517
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
Whitemarsh, Regina C M; Strathman, Monica J; Chase, Lucas G et al. (2012) Novel application of human neurons derived from induced pluripotent stem cells for highly sensitive botulinum neurotoxin detection. Toxicol Sci 126:426-35