Multiplex epigenome editing of ion channel expression in nociceptive neurons abolished degenerative IVD‐conditioned media‐induced mechanical sensitivity

Abstract

Background Low back pain is a major contributor to disability worldwide and generates a tremendous socioeconomic impact. The degenerative intervertebral disc (IVD) has been hypothesized to contribute to discogenic pain by sensitizing nociceptive neurons innervating the disc to stimuli that is nonpainful in healthy patients. Previously, we demonstrated the ability of degenerative IVDs to sensitize neurons to mechanical stimuli; however, elucidation of degenerative IVDs discogenic pain mechanisms is required to develop therapeutic strategies that directly target these mechanisms. Aims In this study, we utilized CRISPR epigenome editing of nociceptive neurons to identify mechanisms of degenerative IVD‐induced changes to mechanical nociception and demonstrated the ability of multiplex CRISPR epigenome editing of nociceptive neurons to modulate inflammation‐induced mechanical nociception. Methods and Results Utilizing an in vitro model, we demonstrated degenerative IVD‐produced IL‐6‐induced increases in nociceptive neuron activity in response to mechanical stimuli, mediated by TRPA1, ASIC3, and Piezo2 ion channel activity. Once these ion channels were identified as mediators of degenerative IVD‐induced mechanical nociception, we developed singleplex and multiplex CRISPR epigenome editing vectors that modulate endogenous expression of TRPA1, ASIC3, and Piezo2 via targeted gene promoter histone methylation. When delivered to nociceptive neurons, the multiplex CRISPR epigenome editing vectors abolished degenerative IVD‐induced mechanical nociception while preserving nonpathologic neuron activity. Conclusion This work demonstrates the potential of multiplex CRISPR epigenome editing as a highly targeted gene‐based neuromodulation strategy for the treatment of discogenic pain, specifically; and, for the treatment of inflammatory chronic pain conditions, more broadly.

Therapeutic TNF-alpha Delivery After CRISPR Receptor Modulation in the Intervertebral Disc

“…The observed pain behavior modifications may be due to the observed maintenance of IVD structure (Figures 3G, 4G, 6G), the overall reduction of inflammatory cytokine presence (Figures 5, 7), or TNFR1 modification of innervating neurons in the disc (Figures 2C, 3C). All of these are consistent with our previous in vitro model work with human cells and tissue demonstrating the reduction of inflammatorydriven cell apoptosis/degeneration 57 and neuron sensitization 58,70 .…”