Validation of a cell-based assay for BoNT/A detection against the mouse bioassay

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DESCRIPTION (provided by applicant): Botulinum neurotoxins (BoNT) are the most toxic substances known to humans, causing respiratory failure upon intoxication. Despite their lethality, BoNTs have clinical health and cosmetic applications and are currently FDA-approved for treating cervical dystonia, blepharospasm, cranial nerve VII disorders, glabellar lines (wrinkles), and cosmoses. BoNT provides relief of muscle tension by silencing neurons that cause muscle contraction. For many disorders, BoNT-based treatments provide significant and long-lasting reductions in pain. BoNT's exquisite specificity for neurons and long time of action make it a lead candidate for the treatment of neurological and muscle disorders where conventional treatments have failed. Quality control and quantification of BoNT-based therapies are currently achieved using the mouse bioassay which involves injecting samples into mice and counting the number of mice that die one or more days post-injection. Each manufacturer of a BoNT-based pharmaceutical uses a unique protocol for the mouse bioassay; the result is non-standardized units of activity. This non-uniformity has led to confusion about manufacturer-to-manufacturer activity and equivalent dosing, thus putting patients at risk. In addition, the mouse assay is low-throughput, time- and labor-intensive, and sacrifices large numbers of animals. The overall objective of this Phase I proposal is to validate BioSentinel's unique cell-based assay for BoNT/A detection against the mouse bioassay. Because BoNT has three activities-cell receptor binding and uptake, vesicle translocation, and target cleavage-only a cell-based assay can effectively measure BoNT activity with the same fidelity as the mouse bioassay. BioSentinel, in collaboration with Metabiologics, proposes to optimize BioSentinel's cell-based assay for BoNT/A detection, generate a BoNT/A reference standard, determine whether the assay offers improved precision compared to the mouse bioassay, and, using thermally stressed BoNT/A samples, determine whether the cell-based assay accurately measures the activity of BoNT/A samples with the same fidelity as the mouse bioassay. BioSentinel's validated cell-based assay will enable researchers and manufacturers to quantitate BoNT/A preparations, perform high-throughput screens for BoNT/A inhibitors, and develop new BoNT-based therapies using an animal-free, cost-effective system. This assay will also satisfy the requirements of SBIR solicitation PA-09-179, "Development of In-Vitro Assays to Assess the Potency of Botulinum Neurotoxin Type A." PUBLIC HEALTH RELEVANCE: Botulinum neurotoxins are extremely lethal bacterial toxins that are also widely used for cosmetic and pharmaceutical applications providing relief of muscle contraction and pain. Currently, botulinum neurotoxin-based therapies are quantified using the mouse bioassay where animal death is the assay endpoint. BioSentinel proposes to validate its animal-free, cell-based assay for detecting botulinum neurotoxin against the mouse bioassay with the goal of replacing the mouse bioassay for quality control and quantification of botulinum neurotoxin-based products.