Development of Targeted Therapeutic Agents for Botulism
Abstract
Botulinum neurotoxin (BoNT) types A and B selectively block transmitter release by cleavage of SNAP-25 and synaptobrevin, respectively; many months are required for full recovery from the resultant neuromuscular paralysis. To decipher the molecular basis for such prolonged poisoning, intoxication of adreno-chromaffin cells was monitored over 2 months. Exocytosis in BoNT/B-treated cells resumed after 56 days due to the appearance of intact synaptobrevin. However, inhibition continued in BoNT/A-treated cells, over the same interval, due to the persistence of cleaved SNAP-25 (1-197). when recovery of exocytosis was attempted by transfection of poisoned cells with the gene encoding full-length SNAP-25 (1-206), no restoration of exocytosis ensued even 3 weeks post intoxication. To ascertain if this failure was due to the persistence of the toxin's protease activity, the cells were transfected with genes encoding mutated forms of SNAP-25, engineered (via point-mutations at residues Q197 and/or R198) to be highly resistant to BoNT/A protease. Importantly, expression of these mutants yielded complete rescue of exocytosis, even 3 weeks after the initial exposure to BoNT/A. Thus, this unusually long-term persistence of protease activity is a major contributing factor to the extended duration of BoNT/A poisoning. These novel findings establish the proof of principle for fast and complete rescue from BoNT/A intoxication by an innovative and straightforward transfection process. Moreover, such a fundamental advance provides the realistic potential of a new and effective therapy for botulism, particularly, because the technology for gene targeting is available.
Document Details
- Document Type
- Technical Report
- Publication Date
- Sep 01, 1999
- Accession Number
- ADB252915
Entities
People
- Oliver J. Dolly
Organizations
- Imperial College London