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Title: Developing neuronal blockers for long-lasting pain relief
Author: Bresnahan, Rebecca
ISNI:       0000 0004 7425 7327
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2018
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The inadequacy of available pharmacotherapy for managing chronic pain imposes a huge burden on both patients, healthcare services and the economy. The exploitation of biologics, specifically botulinum neurotoxin A (BoNT/A), is important for the development of novel long-acting analgesics. BoNT/A induces analgesia in numerous chronic pain models, via the disruption of SNARE-mediated cellular processes. To aid translation into humans, reengineering BoNT/A can eliminate its paralytic effects and improve its safety profile. BiTox/A, an elongated version of BoNT/A, was generated using SNARE-stapling technology and effectively reduced mechanical hypersensitivity in multiple pain models, presumably via actions at the A-nociceptors. Producing a chimera as efficacious against other clinical features of pain, including thermal hyperalgesia, requires the retargeting of BoNT/A's catalytic activity to different sensory neuron subpopulations. The light chaintranslocation domain of BoNT/A (LcTd/A) was thus conjugated to the receptor binding domain (Rbd) of alternate BoNT serotypes, /C to /E. Staining of cultured sensory neurons revealed that conjugation of LcTd/A to BoNT/D-derived Rbd produced SNAP25 cleavage in a subpopulation of smaller myelinated neurons. Unexpectedly, intraplantar injection of the chimera into an inflammatory pain model potentially prolonged thermal hyperalgesia and failed to alter mechanical hypersensitivity. Conversely, LcTd/A conjugated to cholera toxin-derived binding domain attenuated the development of, and reversed, mechanical hypersensitivity in a post-operative pain model and a chemotherapy-induced peripheral neuropathy model, yet remained ineffective against thermal hyperalgesia. BoNT/A's analgesic effect is hypothesised to be predominantly centrally-mediated. To determine whether this theory was also relevant to BiTox/A, the chimera's in vivo activity was examined. Cleaved SNAP25 was revealed in the ipsilateral ventral horn with sparse labelling in the dorsal horn, as well as in the peripheral afferent terminals, thus implying that a peripheral and central action contribute to the analgesic effect. After demonstrating penetration of the central nervous system, the application of toxin chimeras for drug delivery to the spinal cord was investigated using tetanus binding domain (Tbd). Tbd successfully chaperoned fluorescent tags and LcTd/A to the spinal cord. Attachment of a second Tbd to chimeras significantly increased the detectable fluorescence and amount of cleaved SNAP25 in the ventral horn. In conclusion, the smaller Adnociceptors, targeted by BoNT/D-derived Rbd, likely contribute to the resolution of inflammation and the recovery of normal thermal sensation. Targeting of LcTd/A by cholera toxin resulted in a functional analgesic that displayed less penetration of the ventral horn, indicating a reduced motor effect. This chimera should thus be pursued as a potential long-lasting analgesic. Equally, tetanus proved an efficacious tool for spinal cord delivery. Attaching a second binding domain successfully increased its potency, emphasising the versatility and technical advancement of the SNARE-stapling approach.
Supervisor: Davletov, Bazbek Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available