Project Grant R44TR004811

HUMAN NEUROMUSCULAR JUNCTION PLATFORM TO EVALUATE BOTULINUM TOXIN POTENCY - PROJECT SUMMARY: BOTULINUM NEUROTOXIN (BONT) HAS BEEN PROVEN CLINICALLY EFFECTIVE IN TREATING A WIDE VARIETY OF SYMPTOMS FROM VOCAL MUSCLE SPASTICITY TO CHRONIC MIGRAINE TO NUMEROUS COSMETIC APPLICATIONS. WHILE BONT HAS SUBSTANTIAL THERAPAEUTIC APPLICATIONS, IT CAN ALSO CAUSE THE RARE BUT LIFE-THREATING CONDITION CALLED BOTULISM WHICH IS CHARACTERIZED BY THE CLINICAL SYMPTOMS OF WEAKNESS, BLURRED VISION, SPEECH IMPAIRMENT, MUSCLE CRAMPS, VOMITING, DIARRHEA, AND FEVER. AS THE MAJORITY OF BOTULISM TOXICITY CASES RESULT FROM CONTAMINATED FOOD, THE PRESENCE OF BONT IS ROUTINELY TESTED IN COMMERCIAL FOOD PRODUCTS. THE FATALITY OF BOTULISM HAS DECREASED OVER THE PAST CENTURY HOWEVER, THE ECONOMIC BURDEN OF TESTING AND ON HEALTHCARE COSTS RELATED TO TREATMENT REMAIN STEEP. BONT TESTING INVOLVES THE DETECTION OF DISTINCT SEROTYPES THROUGH ANALYTICAL MEANS FOLLOWED BY THE MOUSE LETHALITY BIOASSAY (MLB) TO CONFIRM THE ACTIVITY OF THE TOXIN. WHILE THE AMOUNT OF MICE USED FOR FOOD AND ENVIRONMENTAL TOXIN TESTING EACH YEAR IS UNKNOWN, THERE ARE OVER 1 MILLION ANIMALS SACRIFICED EACH YEAR TO ESTABLISH THE POTENCY OF CLINICAL DOSAGES OF BONT. THIS PROJECT SEEKS TO EXTEND THE PROGRESS MADE AS PART OF THE SUCCESSFUL PHASE II AWARD TO FURTHER IMPROVE THROUGHPUT AS WELL AS EXTEND THE APPLICATION OF THE SYSTEM BEYOND POTENCY AND RELEASE TESTING FOR BOTOX (ONABOUTLINUMTOXINA) AND BOTOX ALTERNATIVES TO SAFETY TESTING IN FOOD PRODUCTS. THE INTENT IS TO ESTABLISH CHARACTERISTIC FUNCTIONAL AND MOLECULAR PHENOTYPES OF EACH OF THE HUMAN TOXIC SEROTYPES OF BOTULINUM (A, B, E, F, G) AND CREATE AN AI PREDICTIVE MODEL FOR DETERMINING IDENTITY AND POTENCY CHARACTERISTICS OF EACH SEROTYPE. WE WILL UTILIZE THESE DATA TO VERIFY ITS APPLICATION AND UTILITY BY TESTING EACH SEROTYPE, AT DIFFERENT CONCENTRATIONS, IN A VARIETY OF FOOD TYPES WHICH HAVE BEEN KNOWN TO CONFOUND CURRENT MOLECULAR TECHNIQUES SUCH AS HIGH-FAT, HIGH-PROTEIN, AND ACIDIC FOODS. LASTLY, WE WILL BEGIN THE BROAD STROKES OF THE VALIDATION PROCESS UTILIZING THE PLATFORM FOR SAFETY TESTING INCLUDING LOWER LIMITS OF DETECTION AND QUANTIFICATION, VARIABILITY, AND REPRODUCIBILITY. THE ULTIMATE GOAL IS TO PROVIDE A MODEL THAT IS FASTER THAN THE MOUSE LETHALITY BIOASSAY, WITH SIMILAR (OR BETTER) SENSITIVITY AND LIMITS OF DETECTION THAT CAN CHARACTERIZE INDIVIDUAL SEROTYPES AS WOULD BE DONE WITH ANTITOXIN APPLICATION OR THROUGH MOLECULAR/ANALYTICAL BIOLOGY (E.G. PCR, ENDO-PEP). COMMERCIALLY, WE WILL PROVIDE THIS PLATFORM AS A CRO TESTING SERVICE FOR ROUTINE FOOD SAFETY MONITORING. THE RESULTS FROM OUR ASSAY CAN BE ACHEIVED UP TO TWO DAYS SOONER THAN THE MLB AND WHEN POSITIVE, THE IDENTITY OF THE SEROTYPE CAN BE ELUCIDATED TO ALLOW ANTITOXIN APPLICATION AND SEQUESTRATION OF CONTAMINATED FOOD PRODUCTS TO OCCUR MORE RAPIDLY THAN THE CURRENT PARADIGM, REDUCING ECONOMIC AND HEALTH BURDEN. THIS VALIDATION PROCESS, WHICH HAS BEEN DEVELOPED IN CONSULTATION WITH DRS. SHARMA AND FITZPATRICK AT THE FDA AND DRS. TAGLE AND KREPKIY FROM NCATS, WILL ESTABLISH A ROUTE BY WHICH MICROPHYSIOLOGICAL SYSTEMS AVAILABLE THROUGH CROS CAN BE VALIDATED AND QUALIFIED FOR ALL PROVIDERS.