- Email: [email protected]
Neuronal targeting, internalization and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A
Abstracts Toxins 2011 / Toxicon 68 (2013) 60–123 55
E. Cleavage between 54L and 55E of synaptobrevin-2 has never been reported for any botulinum neurotoxin. Conclusions: BoNT/F5 cleaves synaptobrevin-2 in a location which is unique from all other BoNT/F subtypes. This unique property of BoNT/F5 may have significant implications for medical countermeasure development. http://dx.doi.org/10.1016/j.toxicon.2012.07.059
Neuronal targeting, internalization and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A E. Vazquez-Cintron a, S. Pellett b, W.H. Tepp b, L.H. Stanker c, P.A. Band a, d, E.A. Johnson b, K. Ichtchenko a a
Department of Pharmacology, New York University School of Medicine, New York, NY, USA Department of Bacteriology, University of Wisconsin–Madison, Madison, WI, USA c Agricultural Research Service, U.S. Department of Agriculture, Albany, CA, USA d Department of Orthopaedic Surgery, New York University Hospital for Joint Diseases, New York, NY, USA E-mail address: [email protected] (K. Ichtchenko). b
Purpose of study: Botulinum neurotoxins (BoNTs) have the unique capacity to cross epithelial barriers, target neuromuscular junctions, and translocate an active metalloprotease component to the cytosol of motor neurons. We have taken advantage of the molecular carriers responsible for this trafficking to create a family of recombinant, fulllength BoNT derivatives with native structural features and physiologic trafficking profiles. They have been rendered atoxic by point mutations that disable the light chain (LC) metalloprotease. CDC has excluded these BoNT derivatives from Select Agent status. Methods used: The genetic constructs, which were synthesized de novo with a baculovirus expression system implemented in our laboratory, enabled the facile production of recombinant, full-length, atoxic BoNT derivatives. In addition, we have engineered derivatives with an amino acid sequence at the N-terminus of the LC that allows for site-selective enzymatic attachment of cargo intended for delivery to the neuronal cytosol. Summary of results: Unlike wild-type BoNTs, which effectively disable their own uptake, the atoxic derivatives accumulate in neurons to detectable and quantifiable levels. Our data demonstrate targeting of recombinant atoxic BoNT/A to neuromuscular junctions after systemic administration, uptake into neurons, binding to SNAP-25 in the cytosol of neurons and competition with the wild-type toxin for neuronal binding and toxicity. Conclusions: BoNT/A atoxic derivatives are currently being used as a technology platform to design antidotes to BoNT poisoning that have the capacity to be effective for extended periods post-exposure. In addition to their therapeutic potential, these derivatives can be used to dissect details of endocytosis and exocytosis in neurons and to identify new targets for therapeutic modulation of these events. http://dx.doi.org/10.1016/j.toxicon.2012.07.060
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Are standard doses of incobotulinumtoxinA, botulinum neurotoxin A free from complexing proteins, not detected by the human immune system? H. Hefter, C. Hartmann, U. Kahlen, M. Moll Department of Neurology, Heinrich Heine University of Duesseldorf, Duesseldorf, Germany E-mail address: [email protected] (H. Hefter).
Purpose of study: The development of secondary nonresponse is one of the most limiting factors in long-term treatment with botulinum toxin. Repetitive injections at short intervals or in high single doses, or both, are known to be risk factors for the induction of neutralizing antibodies (NABs) leading to progressive non-responsiveness. This study was conducted to test the influence of incobotulinumtoxinA (botulinum neurotoxin type A free from complexing proteins) on neutralizing antibody titers in patients with cervical dystonia and with beginning secondary nonresponse after pretreatment with BoNT/A in hemagglutinin-complexed preparations. Methods used: In 37 patients with cervical dystonia, progressive non-responsiveness was clinically observed under treatment with onabotulinumtoxinA and/or abobotulinumtoxinA. Therefore, neutralizing antibody titers (NABTS) were determined by means of the mouse hemidiaphragm assay (HDA). Treatment was switched to incobotulinumtoxinA. During the first year patients received 200 U according to the injection scheme of pre-treatment. Afterwards, doses could be raised without restrictions. NABTs were measured every 12 months for up to four years and more. Summary of results: In most patients antibody titers were reduced or remained constant during the first year of treatment. The proportion of patients whose antibody titers increased temporarily in comparison to the baseline titers continuously decreased with ongoing incobotulinumtoxinA treatment. After four years of treatment with incobotulinumtoxinA, titers of all patients dropped below their baseline titers. The decay of titers with ongoing incobotulinumtoxinA treatment was similar to the decay of titers in patients with cessation of treatment. Conclusions: We think that the decay in antibody titers despite ongoing incobutlinumtoxinA treatment is the result of the low immunogenicity of this formulation of botulinum neurotoxin A free from complexing or other bacterial proteins. http://dx.doi.org/10.1016/j.toxicon.2012.07.061
The frontalis test versus research and commercial mouse protection antibody assays and exposure history in clinical resistance to botulinum toxin therapy in cervical dystonia D.D. Duane a, b, B.M. DiVito a, b, H.A. Koch a, b a b
Arizona Dystonia Institute, Scottsdale, AZ, USA Arizona State University, Tempe, AZ, USA E-mail address: [email protected] (D.D. Duane).
Purpose of study: Whether the mouse protection antibody (MPA) assay accurately reflects immune-based
E. Cleavage between 54L and 55E of synaptobrevin-2 has never been reported for any botulinum neurotoxin. Conclusions: BoNT/F5 cleaves synaptobrevin-2 in a location which is unique from all other BoNT/F subtypes. This unique property of BoNT/F5 may have significant implications for medical countermeasure development. http://dx.doi.org/10.1016/j.toxicon.2012.07.059
Neuronal targeting, internalization and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A E. Vazquez-Cintron a, S. Pellett b, W.H. Tepp b, L.H. Stanker c, P.A. Band a, d, E.A. Johnson b, K. Ichtchenko a a
Department of Pharmacology, New York University School of Medicine, New York, NY, USA Department of Bacteriology, University of Wisconsin–Madison, Madison, WI, USA c Agricultural Research Service, U.S. Department of Agriculture, Albany, CA, USA d Department of Orthopaedic Surgery, New York University Hospital for Joint Diseases, New York, NY, USA E-mail address: [email protected] (K. Ichtchenko). b
Purpose of study: Botulinum neurotoxins (BoNTs) have the unique capacity to cross epithelial barriers, target neuromuscular junctions, and translocate an active metalloprotease component to the cytosol of motor neurons. We have taken advantage of the molecular carriers responsible for this trafficking to create a family of recombinant, fulllength BoNT derivatives with native structural features and physiologic trafficking profiles. They have been rendered atoxic by point mutations that disable the light chain (LC) metalloprotease. CDC has excluded these BoNT derivatives from Select Agent status. Methods used: The genetic constructs, which were synthesized de novo with a baculovirus expression system implemented in our laboratory, enabled the facile production of recombinant, full-length, atoxic BoNT derivatives. In addition, we have engineered derivatives with an amino acid sequence at the N-terminus of the LC that allows for site-selective enzymatic attachment of cargo intended for delivery to the neuronal cytosol. Summary of results: Unlike wild-type BoNTs, which effectively disable their own uptake, the atoxic derivatives accumulate in neurons to detectable and quantifiable levels. Our data demonstrate targeting of recombinant atoxic BoNT/A to neuromuscular junctions after systemic administration, uptake into neurons, binding to SNAP-25 in the cytosol of neurons and competition with the wild-type toxin for neuronal binding and toxicity. Conclusions: BoNT/A atoxic derivatives are currently being used as a technology platform to design antidotes to BoNT poisoning that have the capacity to be effective for extended periods post-exposure. In addition to their therapeutic potential, these derivatives can be used to dissect details of endocytosis and exocytosis in neurons and to identify new targets for therapeutic modulation of these events. http://dx.doi.org/10.1016/j.toxicon.2012.07.060
77
Are standard doses of incobotulinumtoxinA, botulinum neurotoxin A free from complexing proteins, not detected by the human immune system? H. Hefter, C. Hartmann, U. Kahlen, M. Moll Department of Neurology, Heinrich Heine University of Duesseldorf, Duesseldorf, Germany E-mail address: [email protected] (H. Hefter).
Purpose of study: The development of secondary nonresponse is one of the most limiting factors in long-term treatment with botulinum toxin. Repetitive injections at short intervals or in high single doses, or both, are known to be risk factors for the induction of neutralizing antibodies (NABs) leading to progressive non-responsiveness. This study was conducted to test the influence of incobotulinumtoxinA (botulinum neurotoxin type A free from complexing proteins) on neutralizing antibody titers in patients with cervical dystonia and with beginning secondary nonresponse after pretreatment with BoNT/A in hemagglutinin-complexed preparations. Methods used: In 37 patients with cervical dystonia, progressive non-responsiveness was clinically observed under treatment with onabotulinumtoxinA and/or abobotulinumtoxinA. Therefore, neutralizing antibody titers (NABTS) were determined by means of the mouse hemidiaphragm assay (HDA). Treatment was switched to incobotulinumtoxinA. During the first year patients received 200 U according to the injection scheme of pre-treatment. Afterwards, doses could be raised without restrictions. NABTs were measured every 12 months for up to four years and more. Summary of results: In most patients antibody titers were reduced or remained constant during the first year of treatment. The proportion of patients whose antibody titers increased temporarily in comparison to the baseline titers continuously decreased with ongoing incobotulinumtoxinA treatment. After four years of treatment with incobotulinumtoxinA, titers of all patients dropped below their baseline titers. The decay of titers with ongoing incobotulinumtoxinA treatment was similar to the decay of titers in patients with cessation of treatment. Conclusions: We think that the decay in antibody titers despite ongoing incobutlinumtoxinA treatment is the result of the low immunogenicity of this formulation of botulinum neurotoxin A free from complexing or other bacterial proteins. http://dx.doi.org/10.1016/j.toxicon.2012.07.061
The frontalis test versus research and commercial mouse protection antibody assays and exposure history in clinical resistance to botulinum toxin therapy in cervical dystonia D.D. Duane a, b, B.M. DiVito a, b, H.A. Koch a, b a b
Arizona Dystonia Institute, Scottsdale, AZ, USA Arizona State University, Tempe, AZ, USA E-mail address: [email protected] (D.D. Duane).
Purpose of study: Whether the mouse protection antibody (MPA) assay accurately reflects immune-based