CRISPR Epigenome Editing of TNFR1 and IL6ST Reduces Behavioral Pain Sensitivity from Disc Degeneration

Introduction:: Chronic low back pain (LBP) is the leading cause of disability worldwide. Among the prominent causes of LBP is degeneration of the intervertebral disc (IVD), a condition known as degenerative disc disease (DDD) and characterized by a breakdown of the extracellular matrix, loss of disc height, inflammation, and pain. While many treatment strategies for disc degeneration and its associated pain have been proposed, the underlying mechanisms remain largely unknown, making it challenging to develop therapeutics effectively targeting this pain. Our strategy utilizes lentiviral-delivered CRISPR epigenome editing of TNFR1 and IL6ST expression to the IVD in an in vivo model of lumbar DDD. Previously, we have shown the ability of CRISPR epigenome editing of TNFR/IL6ST to modulate neuron sensitization in an in vitro model of human IVD interactions with nociceptive neurons. In this study, we established the safety and efficacy of a novel, non-surgical, image-guided approach to deliver to the lumbar spine, utilizing ex vivo luminescent imaging. We then demonstrated a decrease in mechanical and thermal pain behavior by CRISPR epigenome editing of TNFR1/IL6ST in this model using Von Frey and Hargreaves behavioral assays.

Materials and Methods:: All experiments were performed with the approval of the University of Utah IACUC. Rat Luciferase Injections: Lentiviral-packaged vectors containing the luciferase gene (n=5) or PBS (n=5) were delivered using a novel, non-surgical approach to the rat lumbar spine (Figure 1A). After one week, DRGs (Figure 1B), lumbar spine motion segments (Figure 1C), and major organs were harvested and imaged for luminescence levels. Rat Cadaver Injections: Practice injections of blue dye using our novel approach (Figure 1E) were performed in 9 lumbar IVDs to ensure our ability to properly deliver to the IVDs (Figure 1F). After injections, injected IVDs were immediately harvested and imaged (Figure 1D). Intradiscal CRISPR Lentiviral Injections: Following practice injections, in vivo experiments were performed (Figure 1G). Under anesthesia, rat lumbar IVDs received intradiscal injections (Figure 1E) via 30-gauge needles of PBS (n=7), TNFR1 (n=7), or IL6ST (n=7) CRISPRi epigenome editing systems (Figure 1H). Lumbar IVD Puncture: Two weeks following initial injections, under anesthesia, intervertebral disc injury was induced in previously injected rat lumbar IVDs via image-guided annular puncture with 25-gauge needles. Behavioral Pain Assays: Prior to injections, animals were acclimated to the handler and behavioral assays, and pre-procedure Von Frey and Hargreaves baseline data were collected. Each behavioral session allowed for animals to acclimate to the collection equipment and the experimenter. The experimenter was blinded for the duration of data collection. Data were collected three days post-annular puncture and continued weekly for four weeks (Figure 1I-L). Statistical Analysis: One-factor ANOVAs were performed on the behavioral data, α=0.05.

Results, Conclusions, and Discussions:: The luciferase experiments (Figure 1A) demonstrate our ability to deliver lentiviral-packaged vectors to the lumbar IVD and have expression in both the DRGs (Figure 1B) and IVD (Figure 1C) with no major organ expression (data not shown). To establish our ability to deliver lentivirus vectors and an annular puncture to the lumbar IVD with our novel approach (Figure 1E), practice injections on rat cadavers were performed. In our practice injections, utilizing a C-arm guided injection without surgery, we successfully injected blue dye into the nucleus pulposus at the center of the IVD in 9/9 lumbar IVDs (Figure 1F). After we established the safety, efficacy, and efficiency of our delivery method, intradiscal delivery of CRISPR epigenome editing vectors (Figure 1H) targeting the TNFR1 or IL6ST promoter regions was performed, followed two weeks later by an annular puncture to induce IVD degeneration. Von Frey and Hargreaves behavioral data showed successful initiation of pain behavior in the PBS control group. These data also showed significantly reduced normalized paw withdrawal threshold (PWT) and normalized paw withdrawal latency in both the TNFR1 and IL6ST groups compared to the PBS control (Figure 1I&K), as well as a reduction in thermal and mechanical pain sensitivity (Figure 1J&L).

These results demonstrate the safety and efficacy of our novel delivery approach to the lumbar IVD. They also show our ability to deliver lentivirus to the lumbar spine and have expression in the DRG and IVD, providing the possibility of modifying both IVD degeneration progression and neuron sensitization simultaneously. Most importantly, these results establish CRISPR epigenome editing of TNFR1 and IL6ST as a potential treatment strategy for lessening pain due to degeneration of the IVD and are consistent with our previous in vitro discogenic pain models. Additional studies into the epigenome editing of TNFR1 paired with IL6ST and other targets should be explored to affect redundant or synergistic pain signaling associated with DDD and to explore the specific mechanisms by which these changes in sensitization are seen. Additionally, future work will utilize cell-specific targeting to parse whether the DRG or IVD modifications are driving the change in pain response.


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