A fully humanized functional genomic screen to identify and validate pain therapeutic targets

About This Grant

Abstract: This project will leverage a highly innovative and unique CRISPR-based screening platform built and validated by the Neely lab at University of Sydney in Australia and an unparalleled human dorsal root ganglion (DRG) transcriptomic dataset including extensive data from neuropathic pain patients and an entirely human- based in vitro validation platform created by the Price lab at University of Texas at Dallas. The overarching goal of the project will be to identify and validate high quality targets for chronic pain treatment starting with human disease-based data, followed by screening against receptors found in human DRG, and culminating in functional validation using human DRG neurons. Two paths to target identification and validation will be taken. The 1st Aim will be based on the functional regulation of ion channels or receptors that are at the core of human nociceptor function. Nav1.7, TRPV1 and the type 1 cytokine signal transducer GP130 are required for normal nociceptor function and clearly linked to chronic pain, but mechanisms through which they are regulated in human nociceptors are not known. We will generate human cell lines expressing core pain receptors and we will knockout or turn on every human gene evaluating responses to stimulation of these cells. From these screens we will generate knockout or upregulated lines to confirm functional impact of each target. We will then validate using human DRG neurons using CRISPR-based gene manipulation. The work in the first aim will lead to a new understanding of regulation of critical ion channels and receptors for pain that is not possible in a candidate- based animal model testing paradigm traditionally used in the field. We anticipate that this work, which is supported by ample preliminary data from both laboratories, will set a new standard for target identification and validation in pain. Aim 2 will capitalize on extensive datasets identifying factors associated with neuropathic pain in male and female neuropathic pain patients. CRLF1 is the sole gene that is upregulated in both male and female thoracic vertebrectomy neuropathic pain patients. We will use the genome-wide CRISPR screen to identify how this and similar factors act via receptors expressed in the human genome. We will use this information to validate the target receptors on human DRG neurons from organ donors and then perform further functional characterization to understand signaling mechanisms. In both Aims we will use extensive sequencing datasets to evaluate and prioritize validated targets based on potential side-effect profiles and addiction liabilities. The project brings together two labs with unique resources and expertise to use cutting edge technologies to identify and validate new targets for pain treatment based entirely on human functional genomics, molecular neuroscience, neuro-immunology and pharmacology.