Replacing the mouse bioassay for diagnostics and potency testing of botulinum neurotoxins – progress and challenges

Abstract

Botulinum neurotoxins (BoNTs) are the most potent toxins known and the causative agents of the rare but potentially life-threatening disease botulism. The elaborate mode of action of BoNTs at the molecular level, their exquisite specificity for peripheral motor neurons, and their ability to effectively inhibit neurotransmitter release led to the development of BoNTs into highly valued pharmaceutical products. Both diagnostics of botulism and potency testing of pharmaceutical BoNT preparations still employ the mouse bioassay as “gold standard assay”. This animal experiment can pose a heavy burden on the animal, including a fatal outcome of testing. Additionally, several analytical disadvantages have been described. Consequently, the development of animal replacement methods is a long pursued goal which has been focused mainly on replacement methods for pharmaceutical potency testing so far. However, fundamentally different requirements and challenges apply for diagnostics of botulism and potency testing of BoNT pharmaceuticals, which necessitates the development of different assays tailored for each purpose. Here we review the underlying causes for this intricacy which are rooted in both the biological characteristics of the BoNTs as well as assay specific requirements. We review different functional assays that have been developed to replace the mouse bioassay. Despite significant progress in recent years, further substantial work is needed to pave the way for a fully validated replacement for the mouse bioassay for botulism diagnostics.