A major challenge in development of therapies for neurodegenerative diseases, including botulism, is the low efficiency and low specificity of existing treatments for targeting the therapeutic agents selectively to nerve cells without involvement of secondary sites of action. We have already demonstrated that we can specifically deliver a prototype cargo GFP into neuronal cells in vitro, ex vivo and in vivo, and w are now poised to translate this prototype cargo-delivery vehicle into a medically relevant inhibitory cargo-delivery vehicle(s) against botulism. We propose herein two antitoxin approaches toward combating paralysis from botulism, both based on our BoNT/A-HC platform. To demonstrate feasibility and versatility of our BoNT/A-HC-based platform for therapeutic biomolecule cargos targeted for neuronal-specific delivery, we propose to exchange the GFP cargo in our prototype GFP-BoNT/A-HC construct with two different types of cargo moieties, which will comprise the two aims of this proposal:
Aim 1. Peptide/protein-based inhibitory cargo-delivery vehicles, and Aim 2. Biotin-streptavidin-linked inhibitory cargos-delivery vehicles. The proposed studies will be conducted in 3 phases for each construct of each aim: Phase I (R21 component, years 1-2) - Construction, purification, characterization and optimization of the BoNT/A-HC-based cargo-delivery vehicles and preliminary testing of the cargo-delivery vehicles for functionality in neuronal-specific cellular uptake in cultured neuronal cells. Phase II (R33 component, years 3-4) - Further optimization of cargo-delivery vehicle for stability, expression yield and intracellular cargo release. Testing the cargo-BoNT/A-HC delivery vehicle for functionality (delivery of cargo, inhibition of BoNT/A-mediated SNAP25 cleavage) in neuronal-specific cellular uptake in cultured neuronal cells;ex vivo in peroneal nerve-EDL muscle preparations;and in vivo after injection in mice. Phase III (R33 component, years 4-5) - Testing the cargo-BoNT/A-HC delivery vehicle for functionality and effectiveness in neuroprotective activity, such as protection from or recovery of neurotransmitter release after BoNT/A challenge, detection of increased expression of toxin-neutralizing scFv or camelid antibodies or SNAPI proteins encoded by DNA vectors.

Public Health Relevance

Antidotes are urgently needed that can reverse the detrimental paralysis caused by botulinum neurotoxin, particularly once the toxin has been internalized into nerve cells. Not only is targeting the therapeutic agent to the correct cells critical for successful treatment, but it is also important that the therapy is not delivered to th wrong cells. The proposed neuron-specific delivery vehicle will provide a means for neuronal-specific delivery of a variety of therapeutic biomolecule cargos, including antibody or peptide-based inhibitors that neutralize the paralyzing activity of the neurotoxins.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI101504-01
Application #
8367174
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
$211,123
Indirect Cost
$75,259
Name
University of Illinois Urbana-Champaign
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Haywood, Elizabeth E; Ho, Mengfei; Wilson, Brenda A (2018) Modular domain swapping among the bacterial cytotoxic necrotizing factor (CNF) family for efficient cargo delivery into mammalian cells. J Biol Chem 293:3860-3870
Fan, Yongfeng; Dong, Jianbo; Lou, Jianlong et al. (2015) Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B. Toxins (Basel) 7:3405-23
Fan, Yongfeng; Geren, Isin N; Dong, Jianbo et al. (2015) Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity. PLoS One 10:e0135306
Wilson, Brenda A; Ho, Mengfei (2014) Cargo-delivery platforms for targeted delivery of inhibitor cargos against botulism. Curr Top Med Chem 14:2081-93
Ho, Mengfei; Goh, Cheong-Hian; Brothers, Michael C et al. (2012) Glycine insertion at protease cleavage site of SNAP25 resists cleavage but enhances affinity for botulinum neurotoxin serotype A. Protein Sci 21:318-26