Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6

Abstract

Botulinum neurotoxins (BoNTs) have proved to be an effective treatment for a large number of neuropathic conditions. BoNTs comprise a large family of zinc metalloproteases, but BoNT/A1 is used nearly exclusively for pharmaceutical purposes. The genetic inactivation of a second BoNT gene in the native strain enabled expression and isolation of a single BoNT/A6 from cultures. Its characterization indicated that BoNT/A subtype A6 has a long duration of action comparable to A1, while it enters neurons faster and more efficiently and remains more localized after intramuscular injection. These characteristics of BoNT/A6 are of interest for potential use of BoNT/A6 as a novel BoNT-based therapeutic that is effective and has a fast onset, an improved safety profile, and a long duration of action. Use of BoNT/A6 as a pharmaceutical also has the potential to reveal novel treatment motifs compared to currently used treatments.

Critical Analysis of Neuronal Cell and the Mouse Bioassay for Detection of Botulinum Neurotoxins

“…In efforts to avoid animal suffering, a multitude of BoNT detection methods has been developed with minimal use of animals. These include in vivo simulation assays, such as the hemidiaphragm assay and local injection assays [57,59,62,[78][79][80][81][82], numerous in vitro assays using immunological detection methods, endopeptidase assays, or a combination of the two such as the endopeptidase-mass spectrometry assay [83][84][85][86][87][88], and cell-based assays [89].…”

“…Potency determinations of different BoNTs by NCB assay and MBA do not necessarily correlate. Several BoNT types (A2, A6, FA) have been shown to enter cultured neurons more efficiently, but BoNT/FA was significantly less potent in mice [55,57,61,126,127]. Thus, greater potency of a BoNT in NCB assays doesn't necessarily mean greater toxicity in vivo.…”

“…For these applications, an assay that can detect the biologic activity of multiple BoNT seroand sub-types is required. While cell-based assays can provide human-specific and highly sensitive BoNT detection platforms fulfilling this requirement, research in the past few years has indicated that the activity of BoNTs in cultured neurons and in vivo in mice does not equally correlate for all BoNTs [55,57,61,126,128]. Thus, the mouse bioassay remains an important detection method for BoNTs, in particular, for investigations of novel BoNTs and for detection of BoNTs in complex matrices such as stool samples and food matrices.…”

“…Intramuscular and subcutaneous injections, usually of sub-lethal doses, combined with assays assessing the resulting local paralysis, are usually used to study pathology, onset, and duration of action of sub-lethal doses of BoNTs. Significant differences between BoNT types have been demonstrated using such studies [57][58][59]62,126,133].…”

“…BoNT/B and/C bind gangliosides with greater affinity than BoNT/A1 [52], while BoNT/A2 appears to interact with polysialogangliosides with greater affinity than BoNT/A1 [53,54]. Cell entry kinetics also differ within the BoNT types, with BoNT/E,/A2, and/A6 entering neuronal cells faster than BoNT/A1 [55][56][57]. BoNT sero-and subtypes also differ in catalytic activity and intracellular persistence, with BoNT/A1 and/C1 having the longest reported intracellular duration of action, followed by BoNT/B and/D, then BoNT/A3, BoNT/E and/F [56,[58][59][60][61][62].…”