Attenuation of botulinum toxin mediated blockade of exocytosis at neuromuscular junctions by viral vector mediated gene transfer into spinal motor neurons
Year: 2009
Type: dissertation
Abstract: Botulinum neurotoxin type A (BoNT/A) is implicated in a majority of human cases of botulism and causes long-lasting neuromuscular paralysis due to the persistence in nerve endings of its light chain protease that cleaves synaptosomal-associated protein of 25 kDa (S25).This highly debilitating and, often fatal, neuroparalytic condition cannot be adequately treated by traditional pharmacological methods due to difficulties in efficiently accessing either the pre-synaptic terminals or the cell body of the supplying spinal motor neurons and because of the lack of efficient drugs that can block or reverse the paralysis.A viral vector mediated gene delivery technique was therefore utilised to confer protection against toxin-induced blockade of exocytosis by the expression of cleavage-resistant S25 (S25-R198T) in susceptible cells.Recombinant adeno-associated viral (AAV) vectors expressing a His 6 -tagged S25-R198T (AAV-S25-R198T) were initially used to obtain proof-of-principle of this approach in chromaffin cell cultures, neuroendocrine cells that mimic the exocytotic pathway found in neurons.Stereotaxic injection of AAV-S25-R198T into anterior horns of rat spinal cords resulted in substantial protection against a subsequent challenge by BoNT/A injected into the soleus muscle.Interestingly, intra-muscular injection of this virus also resulted in protection against neuroparalytic action of a subsequently injected toxin.In order to obtain more efficient neuronally-targeted, peripherallydeliverable viral vectors -which would also confer great utility in the treatment of genetic diseases affecting the spinal cord -the more versatile lentiviral vectors (LV) were used.Biotinylated LVs were attached to targeting agents, different core-streptavidin (CS) tagged protease-inactive BoNT/E (BoTIM) mutants, and applied to cerebellar granule neuron cultures.These targeting molecules substantially reduced the viral infection of non-neuronal cultures with only minimal effects on the transfection of neurons.The results obtained in this study demonstrate the applicability of gene therapy to ameliorate the neuroparalytic effects of botulism.In addition, the novel tools and techniques developed, especially in the targeted, peripheral, delivery of viral vectors, could also be adapted to treat genetic disorders of motor neurons.
Author: Arvind Raghunath
Language: English
Subfield: Clinical Neurology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W19348760