Degenerative spinal disorders including low back pain are one of the most common and costly problems for modern society. It has been recognized that these disorders are directly or indirectly associated with intervertebral disc degeneration. The disc is a cartilaginous connective tissue, composed of the nucleus pulposus(NP), annulus fibrosus and endplates, which connects adjacent vertebrae (the functional spinal unit) and plays major roles in promoting the flexibility and stability of the lumbar spine. The NP is comprised of chondrocytic cells (nucleus pulposus cells) in an extensive extra-cellular matrix such as proteoglycan or collagen. Large molecules of proteoglycan exist in collagen networks and the high osmotic pressure produced by proteoglycan can result in inbibing water into the encapsulated NP, leading to high positive pressure inside the disc (Nachemson et al., 1970). The maintainance of this high positive presure inside of the disc is important in ensuring the biomechanical strength of the disc (Figure 1).
In the process of disc degeneration, the loss of an important extra-cellular matrix such as proteoglycan leading to loss of water from the nucleus pulposus, results in loss of the biomechenical stability of the disc. In this process, since the capability for regeneration of the degenerated disc is very limited, clinical treatment of degenerative disc disorders, especially if there is mechanical instability in this segment, often necessitates removal of the degenerated disc and fusion of this segment (spinal fusion) with or without metal instruments (Figure 2). Spinal fusion has been one of the main surgical treatments for degenerative disc disorders with acceptable clinical results. However, there are several problems associated with spinal fusion surgery, such as accerelated degeneration in the disc adjacent to the fused segnment (Lee, 1988), breakage of instruments and damage to the nerve tissue during the instumentation procedure (West et al., 1991). In addition, the procedure is highly invasive, costly, and the risc of infection is comparatively high.
To overcome these problems attention has recently turned to biological treatment methods in an effort to stimulate the regeneration process of the degenerated discs. Due to the relatively well encapsulated and avascular enviroment of the disc, it seems preferable to deliver bioactive materials into the disc to obtain positive biological effects which can maintain or regenearate disc tissue. Biological treatment for the degenerated disc can be divided into three major categories: 1) growth factor injection with or without a carrier, 2) cell transplantation including stem cells, and 3) gene therapy. However, it is clear that the disc has unique anatomical and physiological properties, which challenge many traditional biological approaches. Therefore, we discuss the possiblity of biological treatment approaches especially from the view point of delivery of molecular materials to the disc. One of the most significant advantages of gene therapy may be the sustained biological effect making it potentially suitable in the treatment of chronic diseases such as spinal disorders, particularly those disorders associated with disc degeneration.
2. Intradiscal gene therapy
2.1. Previously reported intradiscal gene therapy
2.1.1. Virus vector-mediated intradiscal gene therapy
In 1997, Wehling et al. reported gene transfer to the chondrocytic cells from bovine intervertebral endplates using retrovirus vector
In 1998, Nishida and Kang et al. reported intradiscal direct gene transfer to the disc using an adenoviral vector
In their next study, Nishida and Kang et al. reported an
Recently, Liang et al. reported the successful curative effects of gene therapy for the treatment of the degenerated disc in a novel mice model by adenoviral mediation of the human growth and differentiation factor-5 (GDF-5) gene (Liang et al., 2010). Their study confirmed the long-term expression of the target protein in the disc
Meanwhile, Lattermann et al. reported adeno-associated virus (AAV) vector-mediated gene transfer to intervertebral disc
Liu et al. reported the usefulness of baculovirus for the disc (Liu et al., 2006). Baculovirus is an insect virus able to deliver exogenous genes to mammalian cells including nondividing cells with no cell toxicity in vitro or in vivo. They concluded that baculovirus could transfer exogenous genes into rabbit nucleus pulposus cells safely and with high efficiency both
2.1.1. Nonvirus vector-mediated intradiscal gene therapy
Although the development of more sophisticated viral vectors such as non-toxic and safer vectors is still being investigated, progress in intradiscal gene therapy has been delayed by worries over the safety and potential costs of using recombinant viral vectors. Therefore, a number of non-virus mediated gene transfer techniques have been developed. However, the lower transfection efficiency compared with viral vector-mediated methods has been a major limitations of the non-virus mediated method, making the
In 2006, Nishida et al. reported the efficacy of so-called “microbubble-enhanced ultrasound gene therapy” to the disc. More recent evidence suggested an appropriate intensity of ultrasound exposure could make a small transient hole on the cell membrane in a phenomenon called sonoporation, without causing cell toxicity. Additionally, it was reported that a kind of ultrasonography contrast agent called “micro-bubble” makes a cavitation with the exposure of ultrasound and this results in the bursting of the microbubbles leading to a distribution of material over a specific area of interest. Both these phenomenon, sonoporation and cavitation, are thought to have synergistic effects for increased transfection efficiency (Lawrie et al., 1999, 2000).
They demonstrated this micro-bubble enhanced ultrasound gene therapy in the intervertebral disc
2.2. New strategy for intradiscal gene therapy
Despite much research effort, the successful biological treatment of degenerated discs is still limited to the injury induced disc degeneration (stub injury) model using relatively small animals. There are considerable limitations arising from the unique anatomy and physiology of the disc.
2.2.1. Limitations of biological approaches for degenerated disc
The lumbar intervertebral disc is known to be the largest avascular organ in the human body and the main path of nutrition or oxygen supply is passive diffusion via the endplates, resulting in poor nutrition and low oxygen tension, especially near the center of the disc (Grunhagen et al., 2006). Therefore, the metabolism in this area has to be relatively anaerobic, leading to the production of lactate, which in turn produces low pH inside of the disc (Nachemson, 1969). Low nutrition and oxygen tension, low pH, and high positive pressure make the interior of the intervertebral disc a harsh biological environment. Therefore, NP cells existing in this environment must be highly differentiated to survive, which results in relative stability in terms of cell proliferation. Moreover, probably due to the low nutrition supply, the NP has a very low cell number compared with the rich extra-cellular matrix and a significantly low cellular metabolism.
Up to now, the main focus of intradiscal gene therapy and growth factor injection or cell therapy approaches has been to stimulate matrix synthesis. However, when taking the disc environment into consideration, methods requiring more resources or energy to up-regulate or stimulate matrix synthesis as well as cell proliferation seem less likely to result in successful disc regeneration. For successful biological treatment, taking into consideration of the characteristics of the target organ/tissue, and careful selection of the treatment strategy is necessary. As such, a different approach requiring less energy or fewer resources may have a better chance of promoting disc regeneration.
One of these approaches is the down-regulation of gene expressions that are potentially harmful for the physiological condition of the disc, and thus may induce degenerative change in the disc. This kind of approach focuses more on prophylactic treatment of disc degeneration. However, theoretically, it could become a regenerative treatment over an extended period (Figure 3). RNA interference (RNAi) is known to be a powerful means of sequence-speciﬁc gene silencing and would thus be a good candidate for this new strategy.
2.1.2. Potential application of RNA interference for treatment degenerated disc associated disorders
RNAi was ﬁrst reported in 1998 by Fire et al. who demonstrated that double-stranded RNA induced sequence-speciﬁc silencing of gene expression in nematode cells (Fire et al., 1998). Elbashir et al. demonstrated that RNAi can be achieved in mammalian cells using oligoribonucleotide duplex 21 or 22 bases in length (small interfering RNA; siRNA) (Elbashir et al., 2001). Since then, a numbers of RNAi applications for a variety of organs/diseases have been intensively investigated.
In 2006, Kakutani and Nishida et al. reported for the first time that RNAi targeting exogenous reporter gene is effective in silencing transgene expression in nucleus pulposus cells in humans and rats
In their follow-up study, Suzuki and Nishida et al. reported the effectiveness of the DNA vector-based RNAi technique
More recently, Sudo and Minami reported successful RNAi in the degenerative process of the disc by down regulating Apotosis related factor caspase 3 (Sudo & Minami, 2011). In their report, they investigated the effects of siRNA targeting caspase 3 on rabbit nucleus pulposus (NP) cells
The gene therapy approach for the degenerated disc initially used a viral vector to transfer genes to stimulate important matrix synthesis for the disc such as proteoglycan. However, due to problems associated with a virus vector, a non-virus mediated gene transfer technique has been explored while the development of more sophisticated virus vectors remain a subject of investigation.
The difficulties of disc regeneration using a biological approach originate from the unique anatomical and physiological character of the disc. However, if we disregard these specific properties and the associated limitations of the disc, there may be little chance for successful regeneration. The successful treatment strategy for osteoporosis patients suggests us that the down-regulation of catabolic factors may be a more suitable strategy especially for organs/tissues with low cell metabolism and extensive extra-cellular matrix including bone or interverteral disc. As such, RNAi has been applied to the disc to down-regulate catabolic factors in the disc and correct imbalance between anabolic and catabolic factors with minimal nutrition requirements. This kind of approach would focus more on prophylactic treatment of disc degeneration. However, the potential exists for it to become a regenerative treatment over an extended period.
There are many issues to be overcome before the clinical use of RNAi can be permitted in patients suffering from degenerated discs and associated diseases. However, the demonstration that siRNA transfected by a nonvirus-mediated method can effectively inhibit specific gene expression in nucleus pulposus cells
The authors thank Mrs. Tubby for their help in preparing the manuscript. Partial of this work was supported by Grant-in-Aid for Scientiﬁc Research (B) (19300184 and 21300189) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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