The Pain in a Dish Assay (PIDA): a high throughput system featuring human stem cell-derived nociceptors and dorsal horn neurons to test compounds for analgesic activity

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Pain causes widespread human suffering and loss of productivity across society. Opioids, widely prescribed to treat pain, are addictive, leading to a national surge in drug abuse and deaths resulting from opioid overdose. This Phase I SBIR proposal, submitted in response to solicitation RFA-NS-23-006 (HEAL INITIATIVE: Development of Therapies and Technologies Directed at Enhanced Pain Management), will initiate development of the “Pain in a Dish Assay” (PIDA) to enable screening of compounds to identify potential analgesics to serve as alternatives to opioids. The project will be a collaboration between Vala Sciences Inc., which specializes in automated digital microscopes and cell-based assays for drug discovery, and Anatomic Incorporated, which manufactures neurons derived from human induced pluripotent stem cell (hiPSCs). Dr. Tony Yaksh, an expert on preclinical pain research, will be a consultant. Anatomic has developed functional hiPSC-nociceptors (dubbed RealDRGs), which represent peripheral neurons specialized to respond to noxious stimuli, whose cell bodies reside in the dorsal root ganglia. Similar to primary DRGs, RealDRGs respond to noxious stimuli (e.g., capsaicin, the active ingredient in chili peppers) with large increases in intracellular calcium. Anatomic has also initiated development of neurons that represent dorsal horn neurons of lamina I and II (dubbed hiPSC-DLNs), which are the next step in neuronal pain transmission (nociceptors connect to DLNs). Vala’s Kinetic Image Cytometer (KIC) is an automated microscope designed to quantify effects of compounds on calcium and voltage transients for neurons cultured in 96- or 384-well dishes. For the PIDA, RealDRGs will be loaded with a calcium indicator, and effects of potential pain modulators on responses to noxious stimuli will be quantified with the KIC. Furthermore, an advanced version of the PIDA will be developed in which RealDRGs and hiPSC-DLNs are cocultured in a device that features two wells connected via micro tunnels which will enable the RealDRGs to establish synaptic connections with the hiPSC-DLNs. This will enable visualization of neuronal transmission between these cell types initiated in response to noxious stimuli applied to the RealDRGs. The PIDA will enable discovery of pain modulators that reduce responses of nociceptors to noxious stimuli, or reduce transmission of signals between nociceptors and DLNs, a mechanism that has been relatively unexplored for pain management and for which there are no in vitro models. The PIDA will feature human neurons, which will likely be more predictive of clinical effects vs. animal models, leading to more efficient preclinical drug discovery, and reduced use of animals. The PIDA, RealDRGs, hiPSC-DLNs, and KIC will be marketed to pharmaceutical companies and used for contract research to develop novel, non-opioid, analgesics.