Research ArticleBOTULISM

Delivery of single-domain antibodies into neurons using a chimeric toxin–based platform is therapeutic in mouse models of botulism

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Science Translational Medicine  06 Jan 2021:
Vol. 13, Issue 575, eaaz4197
DOI: 10.1126/scitranslmed.aaz4197

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Nontoxic botulinum for drug delivery

Botulism is a severe and potentially fatal disease characterized by muscle paralysis. The causing agent, botulinum neurotoxins (BoNTs), has the ability to enter motor neurons and to block neurotransmission. In two independent studies, Miyashita et al. and McNutt et al. used nontoxic derivative of BoNT to deliver therapeutic antibodies against BoNTs in neurons. Miyashita et al. targeted BoNT/A and BoNT/B and reported therapeutic effects in mice. Using a similar approach targeting BoNT/A, McNutt et al. increased survival after lethal challenge in mice, guinea pigs, and monkeys. This approach provided a safe and effective treatment against BoNT intoxication and could be exploited for targeting other intracellular proteins in neurons.


Efficient penetration of cell membranes and specific targeting of a cell type represent major challenges for developing therapeutics toward intracellular targets. One example facing these hurdles is to develop post-exposure treatment for botulinum neurotoxins (BoNTs), a group of bacterial toxins (BoNT/A to BoNT/G) that are major potential bioterrorism agents. BoNTs enter motor neurons, block neurotransmitter release, and cause a paralytic disease botulism. Members of BoNTs such as BoNT/A exhibit extremely long half-life within neurons, resulting in persistent paralysis for months, yet there are no therapeutics that can inhibit BoNTs once they enter neurons. Here, we developed a chimeric toxin–based delivery platform by fusing the receptor-binding domain of a BoNT, which targets neurons, with the membrane translocation domain and inactivated protease domain of the recently discovered BoNT-like toxin BoNT/X, which can deliver cargoes across endosomal membranes into the cytosol. A therapeutic protein was then created by fusing a single-domain antibody (nanobody) against BoNT/A with the delivery platform. In vitro characterization demonstrated that nanobodies were delivered into cultured neurons and neutralized BoNT/A in neurons. Administration of this protein in mice shortened duration of local muscle paralysis, restoring muscle function within hours, and rescued mice from systemic toxicity of lethal doses of BoNT/A. Fusion of two nanobodies, one against BoNT/A and the other against BoNT/B, created a multivalent therapeutic protein able to neutralize both BoNT/A and BoNT/B in mice. These studies provide an effective post-exposure treatment for botulism and establish a platform for intracellular delivery of therapeutics targeting cytosolic proteins and processes.

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