During this program, Electronic BioSciences, Inc. (EBS) will develop and validate its in-vitro, chip-based, picowellbilayer integrated assay (BP-IA) for characterizing the complete activity of botulinum toxin serotype A (BoNT/A) transmembrane toxin. A transmembrane toxin is a molecule that recognizes/targets a specific cell type viareceptor-mediated targeting/endocytosis, forms a pore in the cell membrane, and transports itself or anothermolecule into the cell to disrupt cellular function, e.g., botulinum neurotoxin, tetanus, diphtheria, shiga, cholera,pertussis, etc. While these toxins are innately hazardous to human health, their inherent cell targeting andenzymatic capabilities can also be harnessed for therapeutic benefit. Today, the emerging therapeutic uses oftoxins include but are not limited to the treatment of muscle spasms, wrinkles, excessive sweating, depression,anxiety, anorexia, neurodegenerative disorders (e.g., Parkinson's disease), and targeted cancer therapy.However, the limitations of current toxin activity assessment methods have constrained the field. There is a needfor a low cost, easy-to-use, rapid, highly sensitive, highly reproducible assay that is capable of individuallyquantifying the separate steps of the intoxication mechanism (i.e., the cell targeting/endocytosis and theintracellular enzymatic activity) to fully understand and utilize toxin functionality. The present standard for toxinactivity assessments is the mouse intraperitoneal injection assay, which has numerous limitations, includingprice, variability, time, lack of sample quantification, and the utilization of live animals, in addition relying on asingle endpoint determination that precludes assessment of the toxin's mechanism. Unknown or poorlyunderstood differences in the potency (or mode of action) of toxin-containing therapeutics can confound clinicaldose findings, result in over or under dosing patients, and delay (or prohibit) the development and/or availabilityof new/novel therapeutics. EBS' BP-IA technology will be capable of unprecedented toxin characterization in alow cost, easy-to-use, rapid, highly sensitive, highly reproducible, in vitro, chip-based platform. Furthermore, themethodology of the BP-IA is customizable such that the complete activity of any transmembrane toxin could bequantified. The development of the BP-IA under this Phase I program will be accomplished by developing andbuilding an alpha prototype BP-IA device, and demonstrating the capability of the BP-IA device to quantitativelyassess the potency of commercial BoNT/A therapeutic toxin formulations. Development of the BP-IA, atechnology for which there is no equivalent commercially available and the future gold standard in toxin,biotherapeutics, cell-targeting, uptake/internal activity, and causation mechanism quantification, will directlyenable the research and development of BoNT/A-based therapeutics, novel toxin agents/samples, thedevelopment of antitoxin agents, the detailed study of toxin and antitoxin mechanisms, the evaluation of thecausative effects of experimental variables on each specific intoxication modality, and the assessment of toxinpotency in general.
Public Health Relevance Statement: Project Narrative
The development of the proposed picowell bilayer integrated assay (PB-IA) will enable more detailed,
quantitative, and customizable characterization of transmembrane toxin activity/potency with a methodology that
is low cost, easy-to-use, rapid, highly sensitive, and highly reproducible, all on a single, in vitro, chip-based
platform. Such technology is presently needed to improve and standardize toxin testing and potency
assessments. The PB-IA would greatly assist with improving the scientific understanding of intoxication
mechanisms, in addition to aiding with the development, screening, and approval of toxin and antitoxin
therapeutics, ultimately resulting in improved human health and patient care, while also aligning with NIH and
FDA guidelines for reducing the use of vertebrate animals for assays of biological activity.
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