Botulinum neurotoxin type A (BoNT/A) is a biopharmaceutic widely used for the treatment of neurological diseases and in aesthetic medicine to achieve months-long paralysis of target muscles and glands. Large numbers of mice are used in the mouse bioassay (MBA) for various botulinum-related applications including batch release potency testing, antitoxin testing, countermeasure development, and basic research. BoNT/A intoxication causes severe suffering to the mice used for testing, and application-specific, non-animal alternatives are urgently needed. Cell-based assays (CBA) need to replicate all the physiological steps of botulinum intoxication, comprising neuronal binding, internalization, endosomal escape, and cleavage of synaptosomal-associated protein of 25 kDa (SNAP25). However, the CBA currently in use have limitations. In this study we show that LAN5 cells, a human neuroblastoma-derived cell line, are sensitive to BoNT/A and can be engineered to express a recombinant NanoLuciferase (NanoLuc)-tagged SNAP25 reporter molecule. On exposure, the reporter molecule is cleaved and releases a NanoLuc-SNAP25 fragment that can be captured on a 96-well plate for quantitative luminometry using a cleavage-specific SNAP25 antibody. We demonstrate, using purified BoNT/A and a commercial BoNT/A product, that the sensitivity of this new cell-based assay is in the fM range, comparable to that of the MBA. The new assay could replace the MBA in research and commercial testing of BoNT/A, benefiting both scientific progress and animal welfare.
Keywords: LAN5; NanoLuc; SNAP-25; botulinum neurotoxin; cell assay.
Botulinum neurotoxin type A (BoNT/A) is extensively used in the treatment of neurological disorders and in aesthetic medicine. The toxin targets a protein called SNAP25 in nerve cells and blocks signaling. Traditionally, the potency and safety of BoNT/A has been tested in mice, which causes significant distress to the animals. Our study introduces a new method for detecting BoNT/A activity based on LAN5 cells, which are a self-replicating, human cell line derived from a tumor. We have engineered the cells to express a version of SNAP25 that allows the potency of BoNT/A to be measured. This new assay is as sensitive as the mouse bioassay. This development could lead to fewer animals being used in research and commercial testing of BoNT/A, benefiting both scientific progress and animal welfare.