Sequence-specific CRISPR mediated inflammatory cytokine receptor modulation for the treatment of inflammatory intervertebral disc pathology

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Our understanding of the underlying links between degenerative changes in the disc and pain is incomplete, which directly limits our ability to develop targeted and effective therapeutic strategies to treat discogenic pain. Additionally, our ability to both target the degenerative changes and the pain simultaneously and independently is unrealized. IVD degeneration and pain are linked by a pro-inflammatory environment (i.e. TNF-α, Il-1β, Il-6, IFN-γ) that can lead to disc degeneration within the IVD and sensitization of nociceptive neurons that innervate the IVD. Targeting these two distinct outcomes (ECM changes and neuronal sensitization) simultaneously would provide novel treatment strategies that could both treat the pain and prevent disease progression. We will utilize CRISPR epigenome editing to provide simultaneous targeting of the TNF-α, IL-1β, and IL-6 signaling pathways through receptor modulation both in the DRG and directly in the IVD to alter pain signaling and degenerative disc disease progression. In Aim 1, we will investigate regulation of inflammatory receptors in the dorsal root ganglia with CRISPR epigenome editing to modify pain in degenerative disc disease. We will investigate the targeting of TNFR1, IL1R1, IL6ST, and TNFR1/IL1R1/IL6ST simultaneously in nociceptive neurons in vitro and in vivo. Using a translationally relevant human degenerative IVD tissue model of nociceptive neuron sensitization, we investigate thermal (Aim 1A) and mechanical (Aim 1B) sensitization pathways and our ability to modulate them using CRISPR epigenome editing in vitro. (Aim 1C) We will deliver lentiviral epigenome editing vectors to the DRG at two delivery time points (0 and 4 weeks) in a painful rodent lumbar annular puncture model. At both acute and chronic time points, pain related measures of mechanical allodynia and thermal hyperalgesia will be obtained in awake animals, prior to DRG and dorsal horn calcium imaging. These outcomes will provide critical information on redundant inflammatory signaling in the development of pain in degenerative disc disease and our ability to modulate it in the DRG. In Aim 2, we will investigate the regulation of inflammatory receptors at two delivery time points (0 and 4 weeks) in the IVD with CRISPR epigenome editing to slow progression of degenerative disc disease. To investigate our ability to modulate degenerative disease progression and investigate TNF- α, IL-1 β and IL-6 signaling in acute and chronic disease progression, we will target TNFR1, IL1R1, IL6ST, and TNFR1/IL1R1/IL6ST simultaneously by delivering lentiviral epigenome editing vectors to the IVD in a rodent annular puncture model. We will investigate disc height, changes in ECM structure and composition, and inflammation to assess a role for redundant TNF-alpha, IL-1β, and IL-6 in disc degeneration and to understand our ability to prevent degenerative disc disease progression. At both acute and chronic time points, pain related measures of mechanical allodynia/thermal hyperalgesia will be obtained in awake animals, prior to DRG and dorsal horn calcium imaging to determine ability to simultaneously modify IVD degeneration progression and nociception.