Cholera toxin-mediated targeting of botulinum neurotoxin activity to pain-associated sensory neurons

Botulinum neurotoxin injections are used off-label to treat chronic pain, but their efficacy is limited and paralytic effects restrict clinical utility in these applications. Here, we investigated whether combining the light chain and translocation domains of botulinum neurotoxin A (BoNT/A) with the GM1-binding B subunit of cholera toxin would be beneficial in silencing pain-associated sensory neurons. Chimeric ChoBot was assembled via a coiled-coil linking technology and was shown to retain the enzymatic activity of BoNT/A in vitro and in vivo. In cultured dorsal root ganglion neurons, ChoBot cleaved SNAP25 in a calcitonin gene-related peptide (CGRP)-rich subpopulation of sensory neurons, resulting in marked inhibition of CGRP release. ChoBot had a lesser effect on the compound muscle action potentials of the rat gastrocnemius muscle than BoNT/A following subcutaneous injections. In rat models of pain, including chemotherapy-induced peripheral neuropathy, intraplantar administration of ChoBot significantly attenuated mechanical allodynia. Immunohistochemical analysis confirmed SNAP25 cleavage in NF200- and CGRP-expressing sensory fibres in the epidermis following a single injection. ChoBot also mediated SNAP25 cleavage in human neuroblastoma cells in culture. Together, these findings indicate that ChoBot enables a silencing of pain-associated sensory pathways, providing a new strategy for the development of new long-lasting analgesics for chronic pain.