Poster AA9 - RNA Sequencing of CRISPRi-TNFR1 Edited Human Nucleus Pulposus Cells Shows Shift of TNF-α from Pro-Inflammatory to Anti-Inflammatory via Potential TNFR2 Signaling

Introduction: Low back pain (LBP) is the leading cause of disability worldwide. Degeneration of the intervertebral disc (IVD), a condition known as degenerative disc disease (DDD), is among the prominent causes of LBP. DDD is characterized by a loss of disc height, a breakdown of the extracellular matrix, inflammation, and pain. Many treatment strategies for DDD and its associated pain have been proposed, but the underlying mechanisms remain largely unknown, making it challenging to develop therapeutics effectively targeting the degeneration and pain. Previously presented work demonstrated that lentiviral-delivered CRISPR epigenome editing of TNFR1 expression and TNF-α dosing in a rodent DDD model leads to protection against degeneration and disc height loss more robustly than TNFR1 editing alone. In this study, we have begun elucidating the underlying mechanisms of this work. Human nucleus pulposus (NP) cells were treated with TNFR1-editing vectors, with and without subsequent TNF-α dosing. RNA sequencing was performed and the top significantly altered metabolic pathways were determined. We demonstrated the myriad of gene expression changes that occur with TNFR1 editing and the changes of gene expression that occur with the addition of TNF-α dosing. The TNFR2 signaling pathway emerged as a top significantly altered metabolic pathway in both comparisons. Together, these results demonstrate that this additional protection against degeneration in the animal model could be due to a shift in signaling from TNFR1 to TNF-α’s other receptor, TNFR2, which has a predominantly anti-inflammatory effect.

Materials and

Methods: Human NP Cell Harvest: NP cells were harvested from healthy human trauma patient tissue extracted by surgical intervention. Promptly after surgery, tissue was placed in washing media. NP tissue was then separated from the remaining IVD tissue, minced, and put in fresh washing media overnight. Tissue was placed in digestion media for two hours or until the tissue was visibly digested. The cell solution was filtered, washed, and centrifuged. The pellet was resuspended, and cells were seeded and allowed to proliferate. NP Cell Transduction and TNF-α Dosing: After growth, cells were passaged and plated in 6-well tissue culture plates at 150,000 cells/well and allowed to adhere overnight. Cells were then dosed with lentiviral media containing either nontarget or TNFR1-targetted CRISPRi vectors for 24 hours. After one week, cells received either a 10ng/mL TNF-α dose or no dose for 24 hours. RNA Sequencing Library Preparation: Cells were lysed, and RNA was extracted and purified using a Purelink RNA micro kit. RNA sequencing libraries were constructed with the KAPA Stranded mRNA-seq kit, and subsequent RNA sequencing was performed. RNA Sequencing Analysis: Data from RNA sequencing were obtained and analyzed for the following group comparisons: TNFR1+TNF-α vs. NT+TNF-α and TNFR1+TNF-α vs. TNFR1 following a published method [1]. For each comparison, genes with a significant adjusted p-value (p < 0.05) were input to Enrichr, and the top significantly altered metabolic pathways were obtained using the BioCarta 2016 database [2–4]. The normalized ratios of TNFR2/TNFR1 were also plotted for each group from RNA sequencing count values.

Results, Conclusions, and Discussions: To begin establishing the mechanism(s) by which TNFR1-editing protected against disc height loss and disc degeneration, which was enhanced by the addition of TNF-α (Figure 1A-C), RNA-sequencing was performed on in vitro human NP cells treated with NT-editing vectors (with and without TNF- dosing) or TNFR1-editing vectors (with and without TNF- dosing). When comparing gene expression changes of TNFR1+TNF-α vs. NT+TNF-α groups, the expression of 4765 genes were significantly altered (Figure 1D), highlighting the substantial changes occurring with TNFR1-editing. In contrast, when comparing gene expression changes of TNFR1+TNF-α vs. TNFR1-edited groups, the expression levels of only 15 genes were significantly altered (Figure 1F). One of these genes was TNFAIP3, which encodes for A20, a regulator of apoptosis and nuclear factor-κB (NF-κB) activation. When these two RNA-seq group comparisons were analyzed further, the TNFR2 signaling pathway emerged as a top significantly altered metabolic pathway for each comparison (Figure 1E, 1G). The normalized ratios of TNFR2/TNFR1 RNA-seq counts were also analyzed and found to be higher in each of these comparisons, pointing to an increased expression of TNFR2 compared to TNFR1 in the TNFR1-edited groups (Figure 1H). These results suggest that combining TNFR1-editing and the addition of TNF- reduces the expression of TNFR1 and seems to potentially drive signaling through TNF-α’s other receptor, TNFR2, leading to greater anti-inflammatory signaling and enhanced protection against DDD pathologies.

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