Botulism is caused by Clostridium botulinum neurotoxin (BoNT), a CDC Category A biodefense threat agent for which no antidote exists. BoNT is the most potent toxin known and can be produced with relative ease. If large numbers of people were exposed to even a small dose of this toxin, they would become paralyzed and require assisted breathing which could easily overwhelm the limited supplies of respirators. We have developed small protein agents that, when expressed within intoxicated neurons, promote rapid degradation of BoNT proteases and improve rates of recovery from intoxication. We call these biomolecule agents "targeted F-boxes" (TFBs) because they consist of a 15 kDa F-box domain fused to a 14 kDa camelid VHH domain with binding specificity for a BoNT protease. Here we propose to develop adenovirus-based botulism antidotes that will specifically target motor neurons and lead to cytosolic expression of a TFB agent within intoxicated neurons. Adenoviruses (Ads) are ideal vehicles for delivery of TFBs to motor neurons for several reasons. First, Ad does not integrate their genome into infected cells and Ad vectors have been produced that are non-replicating and safe for therapeutic use. Second, Ad can be modified to dramatically reduce the normal tropism (liver) while increasing infection of a selected cell population. Thirdly, Ad vectors can be modified to produce a transient burst of transgene expression lasting only a few weeks after which the infected cells revert to normal. Finally, Ads have been found to infect motor neurons leading to transgene expression and, surprisingly, BoNT intoxicated motor neurons are much more efficiently infected than normal neurons. In the R21 phase of this proposed project, we will develop an adapter protein that will block normal Ad tropism while imparting tropism for motor neurons by employing a neuron-specific receptor binding domain (RBD) that derives from BoNT. Recombinant Ad will be engineered to express a TFB agent that targets the protease from BoNT serotype A (BoNT/A) for degradation and will be pre-treated with the neuron-targeting adapter. This modified Ad will then be tested as a botulism antidote by local injection into BoNT/A intoxicated muscles in mice. If successful, in the R33 phase we will make additional modifications to the Ad vector to further improve specificity for motor neurons and obviate the need for an adapter protein. We will also engineer an Ad antidote for another BoNT serotype, BoNT/B. These Ad vectors will be tested for the ability to accelerate recovery from botulism paralysis in mice following a systemic administration of the therapeutic agent. If successful, similar agents can be readily developed for all seven known BoNT serotypes by simply replacing the VHH domain of the TFBs with others having specificity for different BoNT proteases. In addition, the neuron-targeted Ad vehicles we develop may have applications in other important neuronal pathologies.

Public Health Relevance

Botulinum neurotoxin is an extremely dangerous, CDC Category A biodefense threat that is widely available, easily produced, exceedingly toxic and for which no antidote is available. Furthermore, at least seven different toxin types exist, that would each require distinct, challenging, expensive and prolonged antidote development programs using conventional approaches. In contrast, the innovative, virus-based strategy we propose to develop should quickly lead to safe, economical and commercially viable antidotes for all BoNT serotypes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI101403-01
Application #
8366687
Study Section
Special Emphasis Panel (ZAI1-LG-M (M1))
Program Officer
Ranallo, Ryan
Project Start
2012-06-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
1
Fiscal Year
2012
Total Cost
$208,606
Indirect Cost
$55,900
Name
Tufts University
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
039318308
City
Boston
State
MA
Country
United States
Zip Code
02111
Mukherjee, Jean; Dmitriev, Igor; Debatis, Michelle et al. (2014) Prolonged prophylactic protection from botulism with a single adenovirus treatment promoting serum expression of a VHH-based antitoxin protein. PLoS One 9:e106422
Kaliberov, Sergey A; Kaliberova, Lyudmila N; Buggio, Maurizio et al. (2014) Adenoviral targeting using genetically incorporated camelid single variable domains. Lab Invest 94:893-905
Shoemaker, Charles B; Oyler, George A (2013) Persistence of Botulinum neurotoxin inactivation of nerve function. Curr Top Microbiol Immunol 364:179-96
Croft, Michael; Benedict, Chris A; Ware, Carl F (2013) Clinical targeting of the TNF and TNFR superfamilies. Nat Rev Drug Discov 12:147-68