Back pain increases with age, affects 70–85% of people during their lives [1], and causes disability with estimated healthcare costs up to $100 billion/year in the US [2]. The cause of back pain is multifactorial; however, intervertebral disc degeneration has been revealed to be one of the independent risk factors [3]. Furthermore, disc degeneration can present not only back pain but also radicular pain, numbness, muscle weakness, and then paralysis in the worst scenario [4]. Intervertebral disc herniation, an injury with degenerative wear and tear, occurs even in the youth, limits daily and sports activities, and requires surgery in 10% [5]. Lumbar spinal stenosis, a clinical condition narrowing the spinal canal and/or intervertebral foramina, arises in conjunction with age-associated degeneration of the lumbar discs and facet joints, resulting in the most frequent indication of spinal surgery in elderly patients [6].

The intervertebral disc has a complex structure with the nucleus pulposus (NP) encapsulated by the annulus fibrosus (AF) and endplates [4]. The collagenous, laminar AF surrounds the central, gelatinous NP, maintaining pressurization of the NP for support of compressive loading and facilitation of multiaxial spinal movement [4]. While the AF comes from the mesenchyme [7,8], the origin of the NP is the notochord [9]. Notochordal cells only exist during the first decade of human life and are subsequently replaced by non-notochordal, chondrocyte-like cells of unknown provenance [7,8]. Disc cells have a chondrocytic phenotype to produce extracellular matrix components comprising proteoglycans (principally aggrecan) and collagens (primarily types I in the AF and II in the NP) [10]. Matrix metabolism is maintained by catabolic enzymes, matrix metalloproteinases (MMPs) and disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs), and anticatabolic inhibitors, such as tissue inhibitors of metalloproteinases (TIMPs) [11]. Increased expression of MMPs and ADADAMTSs relative to TIMP has been observed during clinical human [12-14] and experimental rodent disc degeneration [15-17]. The disc is an immune-privileged, the largest avascular organ in the body [18,19]. Disc cells thus live under an extremely harsh environment—low glucose, oxygen, and pH and high osmolality and load fluctuation [20]. The nutrient supply of central disc NP cells depends on the diffusion from blood vessels at the disc margins compared to peripheral disc AF cells [19]. Therefore, decreased blood supplies, subchondral bone sclerosis, and endplate calcification, caused by mechanical stress, injury, smoking, and aging, can be harmful for the transport of nutrients to discs and are suspected to contribute to the development of disc degeneration [19].

In humans, age-related disc changes characterized by the loss of notochordal cells start from early childhood as observed in people aged 11–16 years [21,22], ~40% of people aged under 30 years and 90% of those aged over 55 years present structural lumbar disc degeneration [23]. Cell decrease is another major characteristic of disc degeneration and primarily results from programmed cell death, apoptosis [24]. Apoptotic cells increase substantially between the ages of 11–16 years, associated with the loss of notochordal cells and chondrocyte proliferation [22], suggesting a relationship between apoptosis and notochordal cell disappearance during the pathogenesis of disc degeneration [7]. Furthermore, a notably high incidence of apoptosis has been observed during human [25] and rodent disc aging and degeneration [17,26]. The incidence of irreversible cell growth arrest due to aging, senescence [27], also increases during human disc degeneration [28,29]. In addition, we have more recently focused on autophagy as a potent treatment target for human degenerative disc disease [30,31]. Autophagy, the intracellular self-digesting and recycling process, is an important cell survival mechanism that sustains metabolism and prevents the accumulation of toxic proteins and organelles under stress conditions, especially nutrient deprivation [29,32-34]. Age-dependent and degeneration severity-dependent changes in the involvement of autophagy-related genes and proteins have been found in human disc specimens surgically collected [30,31]. Understanding of the unique environment, cell fate, and matrix metabolism in the disc is required to design new biological therapies for degenerative disease.

It is difficult to reverse, stop, or even delay the degenerative process by using the currently available treatment options, because of the limited regenerative potential of intervertebral disc tissues [35,36]. Despite successful conservative treatment outcomes with medication and physiotherapy in disc disease [37], nonresponders reluctantly require surgery [5]. Surgical interventions predominantly include the excision of pathological discs, resulting in the function loss, immobilization, and potential additional complications due to the altered biomechanics [38]. Therefore, the development of biological approaches for disc regeneration is an urgent issue.

Biological therapies for intervertebral disc degeneration can be divided into 3 major groups: growth factor injection with or without a carrier [39], cell-based therapy with or without a scaffold [40], and gene therapy modifying endogenous gene expression and function [41]. This review article focuses on backgrounds and applications of gene therapy in the treatment of spinal disorders, especially associated with degenerative disc disease (Table 1).

Gene therapy is defined as the transfer of either RNA or DNA to treat or prevent a disease. Targeted diseases have been mainly classic and fatal genetic disorders, however, the application of gene therapy for such diseases as acquired chronic disorders has been allowed owing to technical advances. A major advantage of gene therapy is its relatively long-term efficacy. Once a therapeutic gene is successfully transferred into the target cells, these genetically modified cells continue to produce the desired gene products (RNAs or proteins). Disc degeneration and related disorders are chronic conditions and therefore good candidates for gene therapy.

In vitro gene therapy for disc degeneration was first reported in 1997, which transferred genes to chondrocytic cells of bovine endplates using retroviral vectors [42]. In vivo gene therapy for disc degeneration was first reported in 1998, which achieved sustained expression of β-galactosidase (lacZ) with adenoviral vectors in a rabbit model at 1 year [43]. Thereafter, a variety of therapeutic genes have been studied and delivered to disc cells via viral and nonviral vectors [41]. These vectors are required to transfer the gene of interest into the target cells, because genetic materials are not well transmitted into cells. Viral vectors have been preferred since they can enter into cells and subsequently express their genes as a part of the life cycle, resulting in greater long-term transgene expression.

Another essential factor in gene therapy is how to deliver the genetic materials to the target organ. Because the intervertebral disc is avascular and encapsulated, and the intradiscal environment is harsh for cells to survive, local and direct gene therapy is the best way for the disc, which gene of interest is administered locally and delivered directly to the cells of the target organ in vivo [41].

From a safety viewpoint, the problem with retroviral vectors was the risk of insertional mutagenesis while the problem with adenoviral vectors was the immunogenicity of transduced cells [49,50]. In clinical trials, several patients died after viral vector administration [51]. In addition, virus-mediated gene therapy requires exclusive facilities and techniques in manufacturing, specialized patient care units in clinical application, and high costs in all these steps. On the other hand, non–virus-mediated gene therapy has increased attention due to its safety and simplicity [52]. Although various types of non–virus-mediated gene transfer techniques have been investigated, a major limitation has been the lower transfection efficacy compared to viral vector methods.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology enabled precise and efficient genome editing in living eukaryotic cells. The CRISPR is an adaptive immune system used by microbes to defend themselves against invading viruses by recording and targeting their DNA sequences [75]. Jansen et al. [76] named the repetitive DNA sequences in the prokaryotes CRISPR, which shared common set of features, and identified 4 CRISPR-associated (cas) genes in 2002. Barrangou et al. [77] demonstrated in 2007 that CRISPR and associated cas genes provided resistance against phages, and resistance specificity was determined by spacer-phage sequence similarity. Thereafter, Jinek et al. [78] found that Cas9, the hallmark protein of type II CRISPR-Cas systems, was a DNA endonuclease guided by dual RNAs and showed site-specific double-stranded breaks in target DNA by a Cas9 endonuclease in 2012. Finally, Cong et al. [79] successfully conducted mammalian genome editing with CRISPR-Cas9 system in 2013. In addition, this system has been shown to have minimal off-target effects [80]. Today, CRISPR-Cas9 techniques can be used not only to knockout but also to repair and/or regulate gene expression.

In the field of the intervertebral disc, Farhang et al. [81] reported the receptor-specific inhibition of tumor necrosis factor-α (TNF-α) and IL-1β signaling in human disc cells in vitro. In this study, lentiviral CRISPR epigenome editing systems were introduced into human degenerative disc cells to downregulate TNF receptor 1 (TNFR1)/IL-1 receptor 1 (IL1R1) expression. The transduction of epigenome editing systems was efficient. Although TNFR1 expression was significantly downregulated, IL1R1 down-regulation was not maintained in the majority of the patient samples. Aggrecan increased and MMP-3 decreased in expression significantly by TGFR1 epigenome editing, while aggrecan and MMP-3 did not significantly change in IL1R1 epigenome-edited cells. These results demonstrated the efficacy and feasibility of CRISPR-Cas9 system in pathological disc cells and also revealed a limitation in epigenome targeting of IL1R1, which indicated that a tailored approach may be required for successful regulation of each gene.