Engineering Botulinum Toxins to Improve and Expand Targeting and SNARE Cleavage Activity

Fonfría, Elena; Elliott, Mark A.; Beard, Matthew; Chaddock, John A.; Krupp, Johannes

doi:10.3390/toxins10070278

Cited by 28 publications

(22 citation statements)

References 144 publications

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“…BoNT serotype A and E were manufactured by Ipsen as previously described ( Hooker et al, 2016 ; Fonfria et al, 2018a ; Pons et al, 2019 ). Inactive BoNT/A (BoNT/A (0)) was produced by point mutations E224Q, H227Y in BoNT/A ( Fonfria et al, 2018a ). All BoNTs were purified and activated to more than 91%.…”

Section: Methodsmentioning

confidence: 99%

hiPSC-Derived Neurons Provide a Robust and Physiologically Relevant In Vitro Platform to Test Botulinum Neurotoxins

et al. 2021

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Botulinum neurotoxins (BoNTs) are zinc metalloproteases that block neurotransmitter release at the neuromuscular junction (NMJ). Their high affinity for motor neurons combined with a high potency have made them extremely effective drugs for the treatment of a variety of neurological diseases as well as for aesthetic applications. Current in vitro assays used for testing and developing BoNT therapeutics include primary rodent cells and immortalized cell lines. Both models have limitations concerning accuracy and physiological relevance. In order to improve the translational value of preclinical data there is a clear need to use more accurate models such as human induced Pluripotent Stem Cells (hiPSC)-derived neuronal models. In this study we have assessed the potential of four different human iPSC-derived neuronal models including Motor Neurons for BoNT testing. We have characterized these models in detail and found that all models express all proteins needed for BoNT intoxication and showed that all four hiPSC-derived neuronal models are sensitive to both serotype A and E BoNT with Motor Neurons being the most sensitive. We showed that hiPSC-derived Motor Neurons expressed authentic markers after only 7 days of culture, are functional and able to form active synapses. When cultivated with myotubes, we demonstrated that they can innervate myotubes and induce contraction, generating an in vitro model of NMJ showing dose-responsive sensitivity BoNT intoxication. Together, these data demonstrate the promise of hiPSC-derived neurons, especially Motor Neurons, for pharmaceutical BoNT testing and development.

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Section: Methodsmentioning

confidence: 99%

hiPSC-Derived Neurons Provide a Robust and Physiologically Relevant In Vitro Platform to Test Botulinum Neurotoxins

et al. 2021

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“…Figure 1 shows a phylogeny tree of published BoNT sequences and more distantly related homologs. The various serotypes and subtypes of BoNTs differ in key structural and functional sites in the receptor and ganglioside binding domains as well as the catalytic domains, defining the serotype and subtype-specific cell entry and catalytic characteristics [15,16,43,44]. Most BoNT subtypes have not been isolated and functionally and structurally analyzed, but much has been learned about the subtype 1 of the various serotypes.…”

Section: Introductionmentioning

confidence: 99%

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

Pellett

Tepp

Johnson

2019

Toxins

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Botulinum Neurotoxins (BoNTs) are a large protein family that includes the most potent neurotoxins known to humankind. BoNTs delivered locally in humans at low doses are widely used pharmaceuticals. Reliable and quantitative detection of BoNTs is of paramount importance for the clinical diagnosis of botulism, basic research, drug development, potency determination, and detection in clinical, environmental, and food samples. Ideally, a definitive assay for BoNT should reflect the activity of each of the four steps in nerve intoxication. The in vivo mouse bioassay (MBA) is the 'gold standard' for the detection of BoNTs. The MBA is sensitive, robust, semi-quantitative, and reliable within its sensitivity limits. Potential drawbacks with the MBA include assay-to-assay potency variations, especially between laboratories, and false positives or negatives. These limitations can be largely avoided by careful planning and performance. Another detection method that has gained importance in recent years for research and potency determination of pharmaceutical BoNTs is cell-based assays, as these assays can be highly sensitive, quantitative, human-specific, and detect fully functional holotoxins at physiologically relevant concentrations. A myriad of other in vitro BoNT detection methods exist. This review focuses on critical factors and assay limitations of the mouse bioassay and cell-based assays for BoNT detection. Key Contribution:This manuscript reviews two widely used botulinum neurotoxin detection assays, the mouse bioassay and cell-based assays, and the critical factors involved in their methodology as well as interpretation of results. Despite our ability to now reduce animal use and replace the mouse bioassay for certain aspects of botulinum neurotoxin detection, the mouse bioassay is sensitive and is the only in vivo assay considering all aspects of botulinum neurotoxin intoxication on a physiological level as well as pharmacokinetic aspects of the toxins, and thus remains important assay for botulinum neurotoxin detection.Toxins 2019, 11, 713 2 of 23 cells, with preferential entry into motor-neurons. Inside a neuron, the toxins block neurotransmitter release, leading to the long-lasting descending bilateral paralysis characteristic of botulism [6]. Even if applied intramuscularly for therapeutic purposes, a portion of the injected BoNTs, in particular at high doses, can diffuse away from the local injection site leading to distal neuronal effects and at very high doses systemic distribution [7][8][9][10]. BoNTs can also enter human or vertebrate circulation and cause botulism by different routes, including ingestion of contaminated foods, by wound infection with neurotoxigenic clostridia, and by the colonization of the intestinal tract by neurotoxigenic clostridia, causing infant botulism or adult intestinal botulism [11,12]. The latter is rare, as C. botulinum usually does not colonize a healthy intestine with a mature microbiota. The severe symptoms of botulism last from several days to up to a year, depending ...

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“…A natural version of BT may exist, or one could be produced artificially that would affect neurotransmitter release and/or receptor trafficking to the cell membrane in sensory neurons but not in motor neurons. Engineering of BTs has been made possible by recombinant technologies, and some molecules with analgesic properties that do not target the neuromuscular junction have already been produced [ 61 ].…”

Section: The Futurementioning

confidence: 99%

Botulinum Toxin in the Treatment of Headache

Becker

2020

Toxins

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Botulinum toxin type A has been used in the treatment of chronic migraine for over a decade and has become established as a well-tolerated option for the preventive therapy of chronic migraine. Ongoing research is gradually shedding light on its mechanism of action in migraine prevention. Given that its mechanism of action is quite different from that of the new monoclonal antibodies directed against calcitonin gene-related peptide (CGRP) or its receptor, it is unlikely to be displaced to any major extent by them. Both will likely remain as important tools for patients with chronic migraine and the clinicians assisting them. New types of botulinum toxin selective for sensory pain neurons may well be discovered or produced by recombinant DNA techniques in the coming decade, and this may greatly enhance its therapeutic usefulness. This review summarizes the evolution of botulinum toxin use in headache management over the past several decades and its role in the preventive treatment of chronic migraine and other headache disorders.

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Engineering Botulinum Toxins to Improve and Expand Targeting and SNARE Cleavage Activity

Cited by 28 publications

References 144 publications

hiPSC-Derived Neurons Provide a Robust and Physiologically Relevant In Vitro Platform to Test Botulinum Neurotoxins

hiPSC-Derived Neurons Provide a Robust and Physiologically Relevant In Vitro Platform to Test Botulinum Neurotoxins

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

Botulinum Toxin in the Treatment of Headache

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