Abstract
Low back pain (LBP) represents an enormous unmet medical need. According to a 2002 survey, more than 26% of Americans experienced LBP within the past three months. 1 LBP represents the leading cause of work absence globally. The total estimated cost of LBP in the United States, including both direct costs and lost productivity, exceeds $100 billion annually. 1 Furthermore, the use of prescription opioids for LBP has undoubtedly contributed to the recent epidemic of opioid deaths.
LBP is multifactorial in nature, but many patients with LBP suffer from degenerative vertebral disc disease (DDD). There are several mechanisms by which DDD can lead to chronic pain, but in many cases, the inflammatory signaling from intervertebral disc (IVD) cells directly stimulates a pain response in nocioceptive neurons (Fig. 1). When progressive DDD fails to respond to nonsteroidal anti-inflammatory drugs and acetaminophen, patients often move on to opioid analgesics, with all the intended consequences from those medications. Alternative therapies, such as physical therapy, acupuncture, and spinal manipulation, are sometimes used. Ultimately, unrelieved LBP often prompts surgical intervention. 1
Inflammation and pain signaling in patients with degenerative vertebral disc disease. Inflammation within the nucleus pulposus and annulus fibrosis of the intervertebral disc (IVD) trigger expression of IL1 and TNF-α. Dorsal root ganglion (DRG) neurons have afferent nerve termini in the outer portion of the disc that are signaled by the pro-inflammatory mediators, leading to calcium transients within the DRG neurons and initiating a pain signal to the brain. Epigenome editing strategies aim to interrupt this pathway at both the IVD and the DRG level.
In the context of this unmet need, researchers are seeking to develop appropriate molecular therapies for LBP due to DDD. The pathways of inflammation and pain signaling are complex and interrelated, presenting a potentially difficult set of targets for precision therapies. In this issue, two papers from the laboratory of Dr. Robert Bowles describe the development of two distinct epigenome editing strategies to control LBP due to DDD. 2,3 One of these targets the inflammatory signaling receptors in IVD cells, and the other targets inflammatory signals within the neurons of the dorsal root ganglia (DRG), the primary neuronal system responsible for the sensation of pain.
In the paper by Farhang et al., 2 the authors cultured primary human IVD cells from the nucleus pulposus from patients undergoing surgery for DDD. The pro-inflammatory gene programming of these cells was successfully inactivated using a single guide RNA (sgRNA)-targeted dCas9–KRAB fusion. The dCas9 portion of this fusion is a version of Streptococcus pyogenes Cas9 that has been rendered “dead” for nuclease function but retains the ability for site-specific DNA targeting, as guided by a sgRNA. A Krüppel Associated Box (KRAB) transcription repression domain has been fused to this dCas9. Thus, any promoter sequence targeted by a sgRNA bound to the dCas9–KRAB will be repressed. This approach was used to repress transcription of both TNF-α receptor 1 (TNFR1) and IL1-β receptor 1 (IL1R1) in IVD cells. They demonstrate blockage of NF-κB activation and of TNF1/IL1-β-induced apoptosis. Ultimately, they were able to demonstrate broad reprogramming of the pro-inflammatory state in IVD cells.
In the paper by Stover et al., 3 a similar strategy is described, delivering a sg-RNA-guided dCas9–KRAB fusion to downregulate transcription of redundant IL-6, TNF-α, and IL-1β signaling within DRG neurons. In this context, potentially suitable for in vivo gene therapy, a lentiviral vector is used to deliver the sgRNA and dCas9–KRAB fusion, along with a 2A element allowing for green fluorescent protein expression as a reporter. In addition to showing vector transduction and singleplex, duplex, and multiplex transcription repression of the three targeted genes, the authors also demonstrate a physiologic outcome directly indicative of pain signaling. While tissue taken from a pathologic annulus fibrosis (AF) of a diseased human disc enhances thermal-induced calcium transients in DRGs (a direct indication of pain transmission), multiplex epigenome editing of IL6/TNF/IL1 significantly reduces these AF-induced thermal calcium transients.
Taken together, these papers demonstrate several important concepts for the treatment of inflammatory conditions. First, effective therapies for complex inflammatory disorders such as these generally require regulation, rather than outright ablation or constitutive overexpression, of a gene of interest. The epigenome editing approach may well prove to be an effective means for resetting these inflammatory programs. Second, signaling pathways in inflammation and pain are redundant and may require a multiplex approach to epigenome editing. Finally, such conditions often represent a complex interplay between a number of different cell types and tissues, such as here where pathologic signaling from cells of the IVD lead to hyper-stimulation of pain transmission in the adjacent DRG neurons.
The ability of epigenome editing to address complex, multifactorial disease processes bodes well for prospects with this modality of therapy. “Toning down” pro-inflammatory signaling would have the potential to make therapeutic headway in a broad array of musculoskeletal and cardiovascular diseases that have so far proven resistant to gene therapy. Future studies of these strategies will ultimately determine whether such approaches are superior to improved small-molecule therapies for inflammation and pain.