The Botulinum Toxin Potency Assay using Tissue Chips program from FDA and NCATS highlights the need for novel engineered systems capable of reliably evaluating botulinum toxin (BoT) potency. Human induced pluripotent stem cell (iPSC)-derived motor neurons and muscle cells have been previously maintained in co- culture and shown to form functional synaptic contacts representing in vivo neuromuscular junctions (NMJs). However, the ability to effectively model NMJ functional responses to BoT using in vitro platforms amenable to high-throughput screening has yet to be achieved due to the complexity of generating mature and functionally competent NMJs in culture. The development of a high-throughput platform capable of promoting NMJ development across a multiplexed assay will have a substantial positive impact on BoT production, as well as advanced therapy development, drug efficacy/toxicity screening, and mechanistic studies of neuronal and NMJ pathophysiology in neurodegenerative diseases. Based on preliminary work and published data, we posit that a culture platform integrating electrode-based stimulation of neuronal firing and magnet-based measurement of engineered muscle contraction will enable real-time analysis of NMJ development and function at baseline and in response to BoT exposure. Using iPSC-derived motor neurons and muscle cells, we will establish organized co-cultures within a culture plate that is compatible with our company's existing muscle contractility assay, MantarrayTM. Microchannels in the walls separating the cells will allow neuronal processes to grow into the muscle compartment, facilitating synaptic contact. Tests with reference batches of BoT, in terms of their ability to alter synaptic communication between our cell populations, will then be used to demonstrate the suitability of this model for assaying NMJ function and BoT potency in vitro (Phase 1). Once validated, our NMJ assay will be further evaluated to determine its accuracy, precision, specificity, and reproducibility in modeling BoT responses in human tissues (Phase 2). Reference batches of BoT will be tested across a wide range of donor cell sources and in comparison to an array of reference compounds with known and predictable effects on NMJ function. The central hypothesis of this work is that differences in NMJ function between engineered skeletal muscle and motor neuron co-cultures treated with different doses of BoT will enable stratification of phenotypes that can be successfully used to plot dose response curves comparable to output data from mouse lethality bioassays (the current gold standard for BoT potency screening). Successful completion of this study will provide a new prototype human-based platform for modeling human peripheral neuropathies as well as a valuable preclinical screening tool for assessing novel therapeutics. The consumable plate will be integrated with Curi's existing hardware/software packages and so can be quickly disseminated to customers upon validation.
Public Health Relevance Statement: PROJECT NARRATIVE Current approaches to measuring the potency of manufactured lots of Botulinum toxin (BoT) involve laborious and ethically dubious mouse lethality assays. Organ on chip technologies based on human induced pluripotent stem cells offer an attractive solution as they can be performed rapidly and in absence of ethical concerns. In this proposal, we will develop an integrated, next-generation platform consisting of instrumentation and stem- cell based models of the neuromuscular junction that can be used for BoT potency assays.
Project Terms: Tissue Chip; Validation; validations; Process; Development; developmental; Output; pre-clinical; preclinical; preclinical study; pre-clinical study; neuromuscular; muscle engineering; computer based prediction; prediction model; predictive modeling; designing; design; next generation; Population; driving force; new drug treatments; new drugs; new pharmacological therapeutic; new therapeutics; new therapy; next generation therapeutics; novel drug treatments; novel drugs; novel pharmaco-therapeutic; novel pharmacological therapeutic; novel therapy; novel therapeutics; iPS; iPSC; iPSCs; induced pluripotent cell; inducible pluripotent stem cell; induced pluripotent stem cell; develop therapy; intervention development; treatment development; therapy development; prototype; commercialization; screenings; screening; Assessment instrument; Assessment tool; Drug Screening; model of human; human model; organ on chip; organ on a chip; stem cell based model; stem cell derived model; stem cell model; iPS neurons; iPSC derived-neurons; induced pluripotent stem cell neurons; neurons derived from induced pluripotent stem cells; Induced pluripotent stem cell derived neurons; multiplex assay; National Center for Advancing Translational Sciences; NCATS; manufacture; Acetylcholine; Adoption; Animals; Axon; Biological Assay; Assay; Bioassay; Biologic Assays; Biomedical Engineering; bio-engineered; bio-engineers; bioengineering; biological engineering; Body Regions; Botulinum Toxins; Bontoxilysin; Botulin; Clostridium botulinum Toxins; botulinum neurotoxin; Cell Culture Techniques; cell culture; cell cultures; Cells; Cell Body; Communication; Cosmetics; cosmetic product; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; Electrodes; Engineering; Ethics; ethical; Human; Modern Man; In Vitro; nerve supply; innervation; instrumentation; Interview; Marketing; Methods; Modernization; Motor Neurons; Motor Cell; motoneuron; Mus; Mice; Mice Mammals; Murine; Muscle Contraction; Muscle Cell Contraction; Muscular Contraction; Muscle; Muscle Tissue; muscular; Nerve; Nerve Degeneration; Neuron Degeneration; neural degeneration; neurodegeneration; neurodegenerative; neurological degeneration; neuronal degeneration; Neuromuscular Junction; Myoneural Junction; Neurons; Nerve Cells; Nerve Unit; Neural Cell; Neurocyte; neuronal; Neurotoxins; neurotoxicant; Patients; Peripheral Nervous System Diseases; PNS Diseases; Peripheral Nerve Diseases; Peripheral Nervous System Disorders; Peripheral Neuropathy; Phenotype; Physiology; Production; Publishing; Safety; Sales; Computer software; Software; Specificity; Synapses; Synaptic; synapse; Technology; Testing; Time; Voice; Work; Writing; Measures; culture plates; Area; Phase; Medical; screening tools; Screening procedure; Muscle function; Measurement; Voluntary Muscle; Skeletal Muscle; Myotubes; Rhabdomyocyte; Skeletal Fiber; Skeletal Muscle Cell; Skeletal Muscle Fiber; Skeletal Myocytes; Muscle Fibers; Dysfunction; Physiopathology; pathophysiology; Functional disorder; Co-culture; Cocultivation; Coculture; Coculture Techniques; Exposure to; programs; Frequencies; human tissue; Protocols documentation; Protocol; Source; cell type; System; Neurodegenerative Disorders; Degenerative Neurologic Diseases; Degenerative Neurologic Disorders; Nervous System Degenerative Diseases; Neural Degenerative Diseases; Neural degenerative Disorders; Neurodegenerative Diseases; Neurologic Degenerative Conditions; degenerative diseases of motor and sensory neurons; degenerative neurological diseases; neurodegenerative illness; Muscle Cells; Myocytes; drug efficacy; neuronal cell body; neural cell body; soma; Toxic effect; Toxicities; skills; novel; Synaptic Cleft; Disease model; disorder model; Devices; Botox; Modeling; response; drug development; high throughput screening; High Throughput Assay; animal facility; preventing; prevent; Dose; Data; Reproducibility; Stratification; in vitro Model; in vivo; Tissue Microarray; Tissue Arrays
