Although botulinum neurotoxin serotype A (BoNT/A) products are common treatments for various disorders, there is only one commercial BoNT/B product, whose low potency, likely stemming from low affinity toward its human receptor synaptotagmin 2 (hSyt2), has limited its therapeutic usefulness. We express and characterize two full-length recombinant BoNT/B1 proteins containing designed mutations E1191M/S1199Y (rBoNT/B1_MY) and E1191Q/S1199W (rBoNT/B1_QW) that enhance binding to hSyt2. In preclinical models including human-induced pluripotent stem cell neurons and a humanized transgenic mouse, this increased hSyt2 affinity results in high potency, comparable to that of BoNT/A. Last, we solve the cocrystal structure of rBoNT/B1_MY in complex with peptides of hSyt2 and its homolog hSyt1. We demonstrate that neuronal surface receptor binding limits the clinical efficacy of unmodified BoNT/B and that modified BoNT/B proteins have promising clinical potential.

Clinically used botulinum neurotoxins (BoNTs) are natural products of Clostridium botulinum. A novel, recombinant BoNT type A1 (rBoNT/A1; IPN10260) has been synthesized using the native amino acid sequence expressed in Escherichia coli and has previously been characterized in vitro and ex vivo. Here, we aimed to characterize rBoNT/A1 in vivo and evaluate its effects on skeletal muscle. The properties of rBoNT/A1 following single, intramuscular administration were evaluated in the mouse and rat digit abduction score (DAS) assays and compared with those of natural BoNT/A1 (nBoNT/A1). rBoNT/A1‐injected tibialis anterior was assessed in the in situ muscle force test in rats. rBoNT/A1‐injected gastrocnemius lateralis (GL) muscle was assessed in the compound muscle action potential (CMAP) test in rats. The rBoNT/A1‐injected GL muscle was evaluated for muscle weight, volume, myofiber composition and immunohistochemical detection of cleaved SNAP25 (c‐SNAP25). Results showed that rBoNT/A1 and nBoNT/A1 were equipotent and had similar onset and duration of action in both mouse and rat DAS assays. rBoNT/A1 caused a dose‐dependent inhibition of muscle force and a rapid long‐lasting reduction in CMAP amplitude that lasted for at least 30 days. Dose‐dependent reductions in GL weight and volume and increases in myofiber atrophy were accompanied by immunohistochemical detection of c‐SNAP25. Overall, rBoNT/A1 and nBoNT/A1 exhibited similar properties following intramuscular administration. rBoNT/A1 inhibited motoneurons neurotransmitter release, which was robust, long‐lasting, and accompanied by cleavage of SNAP25. rBoNT/A1 is a useful tool molecule for comparison with current natural and future modified recombinant neurotoxins products.

Botulinum neurotoxins (BoNTs) are notorious toxins and powerful agents and can be lethal, causing botulism, but they are also widely used as therapeutics, particularly to treat neuromuscular disorders. As of today, the commercial BoNT treatments available are from native A or B serotypes. Serotype F has shown efficacy in a clinical trial but has scarcely been used, most likely due to its medium duration of effect. Previously, the uniqueness of the light chain of the F7 subtype was identified and reported, showing an extended interaction with its substrates, VAMPs 1, 2 and 3, and a superior catalytic activity compared to other BoNT/F subtypes. In order to more extensively study the properties of this neurotoxin, we engineered a modified F7 chimera, mrBoNT/F7-1, in which all the regions of the neurotoxin were identical to BoNT/F7 except the activation loop, which was the activation loop from BoNT/F1. Use of the activation loop from BoNT/F1 allowed easier post-translational proteolytic activation of the recombinant protein without otherwise affecting its properties. mrBoNT/F7-1 was expressed, purified and then tested in a suite of in vitro and in vivo assays. mrBoNT/F7-1 was active and showed enhanced potency in comparison to both native and recombinant BoNT/F1. Additionally, the safety profile remained comparable to BoNT/F1 despite the increased potency. This new modified recombinant toxin F7 could be further exploited to develop unique therapeutics to address unmet medical needs.