Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, cleaves proteins involved in neurotransmitter release, thereby triggering flaccid paralyses, which are responsible for botulism. BoNT is classified into seven serotypes (BoNT/A-G); BoNT/A and BoNT/B are used as medical therapeutics and anti-wrinkle reagents. In this study, we investigated the efficacy of BoNT/CD, a mosaic toxin of BoNT/C and BoNT/D, to assess its potential as a therapeutic alternative for BoNT/A. In a cultured neuron assay, BoNT/CD cleaved syntaxin and SNAP-25 with higher efficacy than BoNT/C and BoNT/A. Intramuscularly administrated BoNT/CD induced dose-dependent muscle paralysis, and the paralysis lasted ~21 days in a mouse digit abduction score assay (BoNT/A-induced paralysis lasted ~30 days). BoNT/C failed to induce local paralysis without systemic toxicity. Multiple alignment analyses of the amino acid sequences of the receptor binding domain (HC) of eight BoNT/CDs and two BoNT/Ds showed sequence clustering in five groups. Comparing BoNT/CD strain 003-9 (BoNT/CD003-9) and strain 6813 (BoNT/CD6813) showed that both BoNT/CDs displayed similar efficacies in cultured neurons, but BoNT/CD003-9 displayed higher efficacy in a mouse model than BoNT/CD6813. These findings suggest that BoNT/CD may be a potential alternative for patients who do not respond to existing BoNT-based therapeutics.
