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Biochemistry of intervertebral disc degeneration and the potential for gene therapy applications
The Spine Journal 1 (2001) 205–214
Biochemistry of intervertebral disc degeneration and the potential for gene therapy applications Ezequiel H. Cassinelli, MD, Ronald A. Hall, MD, James D. Kang, MD* Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, 1010 Kaufmann Building, 3471 Fifth Avenue, Pittsburgh, PA 15213-3221, USA Received 28 June 2000; revised 4 December 2000; second revision 22 January 2001; accepted 1 March 2001
Abstract
Background context: Low back pain continues to be a major cause of morbidity in the United States and the world. Although the exact cause has yet to be defined, the intervertebral disk and its age-related changes have been most frequently implicated. Purpose: This article represents a brief summary of intervertebral disk structure and function, both in the “normal” and degenerative states. Study design/setting: Review article. A Medline search from 1966 to present was performed to identify pertinent articles related to the topic of the intervertebral disc and degeneration. Methods: This review article describes the pertinent anatomy, as well as the biochemical and biomechanical changes that occur in the intervertebral disc over time. It presents many of the current theories implicated as causing these changes. Results: Recent studies have shown that gene therapy (the transfer of therapeutic gene[s] into a cell), may have promise as a method of slowing down, or preventing some of the changes seen in the intervertebral disc. Conclusion: Intervertebral disc degeneration is a complex phenomenon, likely the result of a combination of biochemical and biomechanical factors that are known to occur in the disk. Ongoing research efforts in the area of gene therapy show promise as a way to prevent, or even reverse, some of these changes. © 2001 Elsevier Science Inc. All rights reserved.
Keywords:
Intervertebral disk; Degeneration; Gene therapy
Introduction Low back pain affects the majority of the US public at some time [1–3], with an estimated cost of up to $50 billion [4,5]. The cause of low back pain is not known, but it is the intervertebral disc and the inevitable, age-related deterioration it undergoes that has been most commonly associated with it. The frequency of low back pain, stiffness, and intervertebral disc changes increase with age [6]. Intervertebral disk alterations for the most part also precede other degenerative changes in the spine [7–9]. What is not known, however, is what causes disc degeneration and how it is associated with the clinical phenomena seen by physicians. This article is a brief overview of what is known about the structure and function of the lumbar intervertebral disc No conflict of interest identified. Nothing of value received from a commercial party. * Corresponding author. Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, 1010 Kaufmann Building, 3471 Fifth Avenue, Pittsburgh, PA 15213-3221, USA. Tel.: 1-412-605-3241. E-mail address: [email protected] (J.D. Kang)
and the changes it undergoes with time. It will provide the clinician with a brief description of the anatomy and the biochemistry of the intervertebral disc, followed by the changes noted in an aging or degenerated disc. Current factors implicated as possible causes of disc degeneration will then be addressed. Finally, the use of new molecular genetics techniques, such as gene therapy, and their potential role in this disease process will then be presented. Structure and function of the intervertebral disc The intervertebral disc is a fibrocartilagenous structure made of four concentrically arranged tissues enclosed by the cartilagenous end plates of adjacent vertebral bodies. The outermost layer is the outer annulus fibrosus, which is made of dense, highly oriented collagen, which blends with the posterior longitudinal ligament as well as inserts into the vertebral bodies. The inner annulus fibrosus is a less dense collagenous structure, which lacks the organization of the outer annulus. In young discs, the annulus has a smooth,
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distinct appearance. The transition zone is an area of thin fibrous tissue, which separates the inner annulus from the centrally located, gelatinous nucleus pulposus [10]. Intervertebral discs are relatively hypovascular and have limited innervation. Diffusion of nutrients and metabolites is the main way that adult discs receive nourishment. A variety of nerve endings have been shown to penetrate only a short distance into the annulus, leaving the inner regions of the disc without innervation [11–13]. Discs contain relatively few cells interspersed in an abundant extracellular matrix, which is made predominantly of water, proteoglycans, collagens, and noncollagenous proteins. Disc cells synthesize these macromolecules as well as maintain and repair the framework they create. In young discs, the nucleus pulposus contains two populations of cells: chondrocyte-type cells and notochordal cells. Notochordal cells, when present, allow for increased proteoglycan production [14]. These disappear completely by adulthood and leave the chondrocytic cells in its place [10]. The inner annulus fibrosus, the transition zone, and the cartilage end plates also contain these chondrocytetype cells, whereas the outer annulus has a predominant amount of fibroblast-type cells [10]. Proteoglycans are made of a protein core attached to at least one glycosaminoglycan (GAG) chain. The predominant GAGs in the intervertebral disc are chondroitin-6-sulfate and keratin sulfate. The main proteoglycans found in the intervertebral disc are similar to the large aggrecan molecule found in articular cartilage and are able to form large aggregates by means of link proteins and hyaluronan [15,16]. Proteoglycans are unevenly distributed in the disc, making up a small percentage of the dry weight of the outer annulus while constituting 50% of the dry weight of the nucleus in young discs [10,17]. Large proteoglycan aggregates and their interaction with water have a major effect on the properties of the intervertebral disc. The hydrophilic nature of the GAGs attract and hold water in the intervertebral disc, absorbing much of the compressive loads seen in the spine while evenly distributing the remaining force around the circumference of the annulus [18]. The concentration of proteoglycans also changes tissue permeability and diffusion rates in the disc, which affects the passage of nutrients, chemical mediators, and cellular waste products [18]. Collagen is the other major structural component of the extracellular matrix. It provides the tensile strength of the intervertebral disc, allows for stability between vertebrae, and resists excessive disc bulging in response to loads. In younger discs, collagen makes up 67% of the dry weight of the annulus fibrosus and 25% of the nucleus pulposus. The distribution of collagen is also uneven, with decreasing amounts from the outer annulus in toward the nucleus pulposus. The types of collagen present in the disc are many [17,19,20], and their distributions vary as well [10,17]. Types I and II make up 80% of the collagen content of the disc. Type I collagen is most abundant in the outer annulus and decreases toward the center of the disc, with no type I collagen present in the nucleus. Type II collagen follows an
opposite trend, accounting for 80% of the collagen in the nucleus pulposus. Collagen types III, V, VI, IX, and XI are also present in smaller quantities, and play a role in collagen fibril organization. Aging and degeneration of the intervertebral disc Some degree of disc degeneration occurs in all people. A clear association has been shown between increasing age and disc degeneration. Miller et al. [21], using data from studies addressing the incidence of disc degeneration in cadaveric specimens, found that by age 49, 97% of lumbar intervertebral discs showed at least some evidence of disc degeneration. Degeneration was seen as early as the second decade of life. It is also known that asymptomatic disc degeneration is not uncommon [22,23] and that low back pain and radiographic evidence of degeneration do not correlate well [24]. This raises the issue of whether lumbar disc degeneration is a “disease” or rather a natural part of aging. Discs from both asymptomatic and symptomatic individuals exhibit similar chemical, structural, and radiographic findings. People who do have low back pain, however, have more diffuse and severe degeneration than those who are asymptomatic [25,26]. Biochemical analyses of herniated discs in a middle-aged population resemble those of more aged discs [27,28]. This indicates that the discs of symptomatic individuals undergo either an acceleration of the normal aging process, an exacerbation of the aging process as a result of some combination of environmental and genetic factors, or both of the above. It is probably best to view disc degeneration as an inevitable, natural part of aging, which in some people occurs at an accelerated rate for reasons that are currently unknown. The poorly understood causes and the clinical variability of disc degeneration make it difficult to assess accurately who is at risk for the development of more severe, earlieronset disc degeneration. Male discs degenerate a decade earlier than female discs and also have more degeneration than their age-matched female counterparts [21]. Environmental factors associated with increased disc degeneration include smoking, exposure to whole body vibration, and heavy lifetime occupational and leisure physical loading [29–31]. Many have also implicated a genetic predisposition to disc degeneration [30,32]. Recent studies have suggested a link between a specific aggrecan gene polymorphism and early, more severe disc degeneration [33]. It is likely that the risk of developing degenerative disc “disease” is multifactorial, with both environmental and genetic factors playing a role. Disc alterations seen with aging The intervertebral disc undergoes substantial chemical and structural changes with aging, which are in general progressive and irreversible. It is known that many of the alterations seen in disc composition occur well before morpho-
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logic evidence of degeneration is apparent. These can affect spinal mobility and change the loads seen by nearby structures (i.e., facet joints, vertebrae, spinal ligaments). As the intervertebral disc ages, blood vessels in the vertebral end plates become less and less numerous, disappearing by the third decade of life. The number of viable cells in the inner regions of the disc decreases [34], and the normal collagen fibril organization begin to disappear. Granular material, which is likely made of degraded matrix molecules, is seen throughout the matrix of the nucleus pulposus, particularly in the immediate vicinity of the chondrocyte-type cells [10,34,35]. The biochemical constituents of the intervertebral disc that have received the most attention in degeneration are collagen and proteoglycan. Most have shown there to be few changes in collagen content with age [28,36,37]. The ratio of type I to type II collagen, however, changes in the anterior and posterior segments of the outer annulus [38], with an increase in collagen type I and III being seen as well [28]. Collagen cross-links formed by enzymatically mediated glycosylation, which are believed to play an important role in the normal functioning of collagen, decrease with age [39]. An increase in the amount of nonenzymatically mediated collagen cross-links is also seen, and this may contribute to degeneration by making the tissue more susceptible to mechanical failure [40,41]. In contrast to collagen, proteoglycan content in the nucleus pulposus decreases noticeably with age. The size of aggrecan and the amount of proteoglycans that form aggregates decreases with time [27,42,43]. Loss of these large molecules reduces the amount of hydration in the disc, changing its shape and volume, likely affecting the ability to absorb and distribute loads effectively. Recently, fibromodulin, a proteoglycan known to affect the structure of collagen fibrils, has also been found to undergo structural alterations in the aging disc [44]. The end result of these alterations in collagen and proteoglycan is a decrease in the proteoglycan/collagen ratio of degenerated discs, particularly in the nucleus pulposus, where much of the proteoglycan is lost [45]. What results is an increasingly fibrotic nucleus pulposus that exhibits altered load-bearing capabilities.
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The changes in the biochemical composition of the intervertebral disc likely play a role in the altered mechanical behavior of the disc tissue components. The tensile properties undergo small changes with aging and degeneration. [46] The effects of shear stresses on the mechanical behavior of the aging nucleus pulposus suggests that with increasing age, the nucleus becomes more stiff and less able to dissipate energy, acting more like a solid substance than a gelatinous one [47]. Intradiscal pressure measurements in degenerated discs reveal a decrease in magnitude compared with normal discs. They have also shown that the pressure within the degenerated disc is unevenly distributed (nonisotropic) in the vertical and lateral directions [48]. These alterations in load-bearing capability and distribution could cause unusually high stresses at specific points in the disc, causing the development of localized tissue damage. Anatomic changes in the degenerating intervertebral disc can be grouped into stages [49,50]. The nucleus initially becomes increasingly dehydrated and fibrotic, making the distinction between the nucleus and the annulus more difficult. Circumferential tears of the annulus are seen, which may then progress to radial tears. Further degeneration of the annulus leads to loss of disc height and disc resorption. Thompson et al. [51] proposed a classification scheme for degenerative disc disease using sagittal sections of the vertebral column and grading degeneration based on the morphologic appearance of the nucleus, annulus, end plate, and vertebral body (Table 1). This grading scheme allows for comprehensive evaluation of all disc structures while maintaining good inter- and intraobserver reliability.
Factors implicated in disc degeneration There is increasing evidence that suggests that the biochemical events occurring in the disc may have an important role in disc degeneration. Many factors have been implicated and can be grouped into a few general categories: cell nutrition and viability; degradation and modification of extracellular matrix components; and disc matrix alterations resulting from mechanical loading. It is essential to note that
Table 1 Thompson’s classification scheme for disc degeneration Grade Nucleus
Annulus
End plate
I II III
Bulging gel White fibrous tissue Consolidated fibrous tissue
Hyaline, uniformly thick Thickness irregular between lamellas Focal defects in cartilage
IV
Horizontal clefts parallel to end plate
Discrete fibrous lamellas Mucinous material peripherally Extensive mucinous infiltration; loss of annular-nuclear demarcation Focal disruptions
V
Clefts extend through nucleus and annulus Reprinted with permission from [51].
Vertebral body
Margins rounded Margins pointed Early chondrophytes or osteophytes at margins Fibrocartilage extending from subchondral Osteophytes less than 2 mm bone; irregularity and focal sclerosis in subchondral bone Diffuse sclerosis Osteophytes greater than 2 mm
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the relative importance each one has in degeneration is not currently known. Cell nutrition and viability Many studies have implicated decreased cell nutrition as the main cause of disc degeneration [17,52]. Diffusion, being the primary mechanism of nutrient transport to the center of the adult disc, has been shown to slow down once the end plates become calcified with age [53,54]. Smoking and vibration have both also been shown to decrease solute transport into the central disc [55,56]. The decrease in diffusion also affects the passage of cellular waste products out of the disc and results in their accumulation in the matrix. These findings, coupled with the increasingly fibrotic nature of the nucleus and the decrease in nuclear water concentration, may additionally inhibit cellular nutrition. The decrease in cellular nutrition has an effect on various aspects of cellular metabolism. The concentrations of oxygen and lactate and the extracellular pH in the intervertebral disc have been studied. Steep oxygen concentration gradients are found in the disc [57], leading to high lactate concentrations (and low pH values) in the nucleus. Low oxygen concentration affects cellular metabolism directly by decreasing proteoglycan synthesis, particularly in the nucleus pulposus [58]. High lactate and low pH levels, which have been found in degenerated disc surgical specimens [59], also affect cellular metabolism [60]. These studies implicate decreased cellular nutrition as a cause of disc degeneration through its eventual effect on matrix production. This decrease in cellular nutrition also likely hinders cell viability. Other possibilities include apoptosis, or programmed cell death, of intervertebral disc cells. Gruber and Hanley [61] found a high incidence of apoptosis in the intervertebral disc and thought that it might be related to specimen age. It is not understood why these cells undergo apoptosis, but a variety of stimuli have been implicated [62]. Recent in vitro studies have shown that intervertebral disc cells undergo a significantly higher rate of apoptosis when subjected to prolonged mechanical compression [63]. Although it is not known how cell death might ultimately affect disc degeneration, a decrease in the number of matrix-producing cells with age is likely to affect disc composition. Along with the decrease in the amount of viable disc cells, the type of matrix they produce changes with age. Few studies have looked at metabolic alterations in the intervertebral disc, but age-related changes in cell function do affect other tissues [42]. Bayliss et al. [64] found the rate of proteoglycan production in neonatal and infant discs to be greater than in adult discs. In a separate study, they found that in the adult disc, newly synthesized proteoglycans were less stable and less able to aggregate than pre-existing proteoglycans [65]. What is seen, in addition to a decrease in matrix synthesis, is a notable change in the type of matrix synthesized, which produces an environment that is less able to maintain the normal properties of the disc.
Matrix degradation and modification Another important factor thought to play a role in disc degeneration is the alteration of the already existing extracellular matrix of the nucleus pulposus. Changes in the type of collagen cross-links occur, which alter collagen properties, including solubility and mechanical strength [40,41]. A variety of mediators have been identified that degrade or modify matrix constituents. These include the neutral proteinases (including serine proteinases and matrix metalloproteinases), cytokines (particularly interleukin-1 [IL-1] and interleukin-6), nitric oxide, and prostaglandins. All are involved in the matrix breakdown of articular cartilage, and are also suspected of having a similar effect in the intervertebral disc [66,67]. Kang et al. [68] found that degenerated herniated discs from both the cervical and lumbar spine spontaneously made increased amounts of all of these mediators. They also found that IL-1 was able to increase the production of the other mediators in intervertebral disc cells taken from both normal and herniated disc specimens. Others have found high proteinase activity in the nucleus pulposus and the end plates of degenerated discs, with no detection in normal disc tissue. Although the ultimate role these mediators in disc degeneration is not yet clear, the evidence suggests that they may play an important one. They may also be involved in the clinical sequelae of disc degeneration [68]. It is possible that with an acute disc herniation, the body’s inflammatory response may further stimulate disc cell production of these mediators. Each patient’s individual inflammatory response may be partially responsible for the clinical variability seen with herniation. It can also be hypothesized that the inflammatory response may produce “idiopathic” low back pain [69]. It is possible that the increased presence of these mediators in degenerated discs might irritate and stimulate the nerve endings near the outer annulus and longitudinal ligaments, as well as the dorsal root ganglia and spinal nerves. Mechanical loading effects on the disc matrix The effect of mechanical loads on the intervertebral disc matrix may also cause disc degeneration. Disc matrix synthesis rates decrease abruptly if the tissue either swells excessively or loses fluid [18,70]. Static loads on the disc result in fluid extrusion, which is in turn dependent on the size of the load and the composition of the tissue [37]. Discs with low proteoglycan content have a higher permeability and thus a larger amount of fluid loss under mechanical loads. Recent in vitro studies have also shown that prolonged mechanical loading causes a higher rate of apoptosis in intervertebral disc cells [63]. The effects of increased mechanical loads on matrix synthesis and cell survival could help explain the increased rate of disc degeneration seen in people who are exposed to increased physical loading. The exact role that the above theories play in intervertebral disc degeneration is not entirely clear, but it is likely that they are interrelated. Whatever the inciting event(s)
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may be, there is probably a complex interplay between them. A hypothetical scenario could be envisioned whereby decreased cellular nutrition and increased apoptosis cause a decrease in cell viability. The decrease in cell number could, along with prolonged mechanical loading, result in a decrease in matrix production. Also, whether as a result of a more “stressed” environment or some other extrinsic factors, the cells alter the type of matrix synthesized and secrete mediators that degrade the pre-existing matrix. This would result in a matrix that is less able to maintain the hydrostatic properties of the disc and is more difficult for solute diffusion. A vicious cycle could result with more cell death from further decreased nutrition, along with a higher rate of apoptosis and decreased matrix synthesis from increased loads seen by the cells. It is possible that in the process of “normal” disc aging, fewer factors are present at any one time, allowing the disc to adapt to changes in its environment. The accelerated degeneration seen in some people could occur when all factors are present concurrently, producing simultaneous “hits” to the system and overwhelming any adaptive mechanism the disc may have. Gene therapy and disc degeneration Given the high incidence of low back pain and disc degeneration, many investigators have sought for ways to try
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to prevent or halt the biochemical changes that the disc undergoes. Advances in the field of molecular genetics, such as gene therapy, have made it an attractive candidate to be used as a potential therapeutic intervention for spinal disorders. The unique characteristics of the intervertebral disc make it an ideal site for gene transfer. The isolated and encapsulated nature of the intervertebral disc helps to maintain high intradiscal concentrations of locally delivered vectors while limiting potential systemic side effects. It also can protect the vectors from the body’s own immunosurveillance mechanisms, prolonging therapeutic gene expression. Gene transfer is typically performed by introduction of the gene of interest (see following discussion of vectors), either directly into the target cells or by removal of the target cells, followed by genetic alteration with subsequent reimplantation. The former strategy is known as direct, or in vivo gene therapy, and the latter, as indirect, or ex vivo gene therapy (Fig. 1) [71]. Additional attention should be directed toward the route of administration. The direct delivery of genes to the site of disease is referred to as local gene therapy, whereas gene transfer that occurs distant from the disease site and uses the circulation system to deliver genetically modified cells and/or gene product is termed systemic gene therapy. These processes will hopefully result in the formation of protein products, which not only affect the
Fig 1. Proposed scheme of the effects of various factors on intervertebral disc degeneration.
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Fig. 2. In vivo gene therapy introduces vectors containing the appropriate gene directly into the target organ/tissue. Ex vivo gene therapy requires the removal of target cells from body, genetic alteration of these cells in vitro, followed by reimplantation of the genetically modified cells into the target organ/tissue. Reprinted with permission from [71].
cells’ own metabolism, but the metabolism of surrounding, nongenetically altered cells. With few exceptions [72–74], naked DNA (fragments of DNA not linked to any transporters) is not well incorporated by cells. Therefore, to enable the cellular uptake of genetic material in a manner that allows the expression of its genetic information, a transporter system (vector) is required. There are two general classes of vectors: viral and nonviral. Under normal circumstances, gene expression requires the genetic material to be delivered into the nucleus of the cell, where the transcriptional machinery resides. DNA may integrate into the chromosomes of the target cell, or it may remain extrachromosomal (episomal). Chromosomal integration of the delivered gene stabilizes it and guarantees its transmission to daughter cells upon cell division. Episomal DNA, although expressed at higher levels than integrated genes, often has gene expression wane with time, especially as the cell divides. Tables 2 and 3 summarize many of the advantages and disadvantages of a variety of vectors currently being used [75]. Viruses are efficient vectors, because their normal life cycles have the enhanced ability to enter the cell and initiate the expression of virally encoded genes. When using viral vectors, it is customary for the noxious or detrimental elements of the viral genome to be removed (or inactivated) and replaced by the genes of interest. Additional genetic modifications are often performed to prevent the replication and pathogenesis of the virus after infection of the target cells. Nonviral vectors are comparatively easier to produce than their viral counterparts, have increased chemical stability, have a virtually infinite gene capacity, and have no infectious or mutagenic capability [75]. All current nonviral
vector systems deliver DNA episomally, presenting the disadvantage of a limited duration of gene expression. Several early investigations evaluated the use of growth factors as a possible therapeutic intervention for intervertebral disc degeneration. Thompson et al. [76] demonstrated that the addition of human transforming growth factor beta I (TGF-1) to canine intervertebral disc tissue stimulated an increase in in vitro proteoglycan synthesis, suggesting that this growth factor may be useful in the treatment of disc degeneration. Various other mediators have been shown to increase proteoglycan synthesis in vitro, including interleukin-I [77], insulin-like growth factor-I [78], and osteogenic protein-I [79]. One of the major shortcomings of growth factor usage however, is its characteristic short-lived duration. Gene therapy is a novel approach that could overcome this problem while maintaining many of the growth factor’s potentially beneficial properties. Few investigators have had success with nonviral transduction of intervertebral disc cells. Chang et al. [80] recently reported successful marker gene transfer using the gene gun in cultured rabbit nucleus pulposus and annulus fibrosus cells. The transfection efficiency was noted to be approximately 10%. Wehling et al. [81] reported retrovirus-mediated transfer of both bacterial -galactosidase (LacZ) and human interleukin-1 receptor antagonist (IL-1Ra) to cultured chondrocytic bovine intervertebral end plate cells. The authors suggested the possibility of an ex vivo approach to treatment of disc degeneration by transduction of the cells in vitro using therapeutic genes with subsequent reinjection of the cells back into the disc. Nishida et al. [82] reported adenovirus- mediated transfer of the LacZ marker gene to the rabbit intervertebral disc cells both in vitro and in vivo. In vivo gene expression continued
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Table 2 Viral vectors for gene delivery Vector
Integration
Advantages
Disadvantages
Retrovirus
Yes
Well developed Ease in production
Adeno-associated virus
Yes
Adenovirus
No
Herpes simplex virus
No
Nonpathogenic Site-selective integration Stable expression Infects nondividing cells Broad range of target cells High titer production High infectivity Ease in production Infects nondividing cells Broad range of target cells High titer production Infects nondividing cells Large capacity (35 kb) Establishment of latency
Random insertion Infects only dividing cells Variable long-term expression Small capacity (4 kb) Difficult to produce Not as well developed
DNA remains episomal Toxicity Immunogenicity Persistent viral protein expression Poorly developed Toxicity Transient expression
Reprinted with permission from [75].
at undiminished levels for at least 12 weeks, with no expression seen in the control discs. Because of the immunogenic nature of the adenoviral vector, Nishida et al. [83] also performed in vivo safety studies in the rabbit by looking at the immune response and marker gene expression under various conditions. Three groups were employed: immunizing injection into the subcutaneous tissues, subcutaneous injection with simultaneous intradiscal injection, and immunizing subcutaneous injection followed 2 weeks later by intradiscal injection. Neutralizing antibodies were confirmed in all three groups up to 6 weeks after injection. However, both of the intradiscal injection groups demonstrated strong gene expression despite the presence of peripherally circulating neutralizing antibodies, indicating that the disc protected the virus from the host’s immune system.
The successful long-term gene expression of intervertebral disc cells using a marker gene and the unique immunologic characteristics of the disc suggested that the adenoviral vector system might be a suitable delivery mechanism for the transfer of therapeutic genes for the treatment of spinal disorders. Nishida et al. [84] then evaluated the feasibility of in vivo adenovirus-mediated therapeutic gene transfer to the intervertebral disc of the rabbit. The therapeutic gene chosen was the one for human TGF-1. After 1 week, total TGF-1 production increased nearly six-fold compared with controls and exhibited a significant increase in proteoglycan synthesis, demonstrating the in vivo efficacy of adenovirus-mediated transfer of a therapeutic gene to the intervertebral disc and its potential to modulate the complex biologic activity within the disc.
Table 3 Nonviral vectors for gene delivery Vector
Advantages
Disadvantages
Naked DNA/plasmid
Nonimmunogenic Inexpensive Simple to use Nonimmunogenic Targeted delivery Noninfectious Inexpensive Large carrying capacity High efficiencies claimed
DNA remains episomal Limited number of target cells Poor expression DNA remains episomal Poor expression DNA remains episomal Limited number of target cells
DNA-ligand complex (molecular conjugates) Liposomes
Gene gun (colloidal gold)
Reprinted with permission from [75].
DNA remains episomal In vivo access difficult Long-term metal particle effects unknown Tissue damage effects unknown
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Conclusion This article describes the basic structure and function of the intervertebral disc and the changes they undergo with time. It is clear that the biochemical changes that occur in the disc play a major role in the pathogenesis of disc degeneration. What remains uncertain is what causes these changes and how, if at all, they can be prevented or even reversed. Gene therapy shows promise as a tool to better address these issues. Undoubtedly, several barriers must be overcome before gene therapy can be used clinically in humans for the treatment of spinal disorders. Two primary concerns, which genes should be administered and what the best method of delivery is, need to be elucidated before this technology can be applied. Recent studies continue to bring forth attractive candidates for gene transfer [85]. Current studies in our center are evaluating different viral vectors in order to optimize gene delivery while minimizing any potential risks to the recipient. With continued, dedicated research efforts, gene therapy has the potential to become an effective treatment modality for intervertebral disc degeneration in the twenty-first century.
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Biochemistry of intervertebral disc degeneration and the potential for gene therapy applications Ezequiel H. Cassinelli, MD, Ronald A. Hall, MD, James D. Kang, MD* Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, 1010 Kaufmann Building, 3471 Fifth Avenue, Pittsburgh, PA 15213-3221, USA Received 28 June 2000; revised 4 December 2000; second revision 22 January 2001; accepted 1 March 2001
Abstract
Background context: Low back pain continues to be a major cause of morbidity in the United States and the world. Although the exact cause has yet to be defined, the intervertebral disk and its age-related changes have been most frequently implicated. Purpose: This article represents a brief summary of intervertebral disk structure and function, both in the “normal” and degenerative states. Study design/setting: Review article. A Medline search from 1966 to present was performed to identify pertinent articles related to the topic of the intervertebral disc and degeneration. Methods: This review article describes the pertinent anatomy, as well as the biochemical and biomechanical changes that occur in the intervertebral disc over time. It presents many of the current theories implicated as causing these changes. Results: Recent studies have shown that gene therapy (the transfer of therapeutic gene[s] into a cell), may have promise as a method of slowing down, or preventing some of the changes seen in the intervertebral disc. Conclusion: Intervertebral disc degeneration is a complex phenomenon, likely the result of a combination of biochemical and biomechanical factors that are known to occur in the disk. Ongoing research efforts in the area of gene therapy show promise as a way to prevent, or even reverse, some of these changes. © 2001 Elsevier Science Inc. All rights reserved.
Keywords:
Intervertebral disk; Degeneration; Gene therapy
Introduction Low back pain affects the majority of the US public at some time [1–3], with an estimated cost of up to $50 billion [4,5]. The cause of low back pain is not known, but it is the intervertebral disc and the inevitable, age-related deterioration it undergoes that has been most commonly associated with it. The frequency of low back pain, stiffness, and intervertebral disc changes increase with age [6]. Intervertebral disk alterations for the most part also precede other degenerative changes in the spine [7–9]. What is not known, however, is what causes disc degeneration and how it is associated with the clinical phenomena seen by physicians. This article is a brief overview of what is known about the structure and function of the lumbar intervertebral disc No conflict of interest identified. Nothing of value received from a commercial party. * Corresponding author. Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, 1010 Kaufmann Building, 3471 Fifth Avenue, Pittsburgh, PA 15213-3221, USA. Tel.: 1-412-605-3241. E-mail address: [email protected] (J.D. Kang)
and the changes it undergoes with time. It will provide the clinician with a brief description of the anatomy and the biochemistry of the intervertebral disc, followed by the changes noted in an aging or degenerated disc. Current factors implicated as possible causes of disc degeneration will then be addressed. Finally, the use of new molecular genetics techniques, such as gene therapy, and their potential role in this disease process will then be presented. Structure and function of the intervertebral disc The intervertebral disc is a fibrocartilagenous structure made of four concentrically arranged tissues enclosed by the cartilagenous end plates of adjacent vertebral bodies. The outermost layer is the outer annulus fibrosus, which is made of dense, highly oriented collagen, which blends with the posterior longitudinal ligament as well as inserts into the vertebral bodies. The inner annulus fibrosus is a less dense collagenous structure, which lacks the organization of the outer annulus. In young discs, the annulus has a smooth,
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distinct appearance. The transition zone is an area of thin fibrous tissue, which separates the inner annulus from the centrally located, gelatinous nucleus pulposus [10]. Intervertebral discs are relatively hypovascular and have limited innervation. Diffusion of nutrients and metabolites is the main way that adult discs receive nourishment. A variety of nerve endings have been shown to penetrate only a short distance into the annulus, leaving the inner regions of the disc without innervation [11–13]. Discs contain relatively few cells interspersed in an abundant extracellular matrix, which is made predominantly of water, proteoglycans, collagens, and noncollagenous proteins. Disc cells synthesize these macromolecules as well as maintain and repair the framework they create. In young discs, the nucleus pulposus contains two populations of cells: chondrocyte-type cells and notochordal cells. Notochordal cells, when present, allow for increased proteoglycan production [14]. These disappear completely by adulthood and leave the chondrocytic cells in its place [10]. The inner annulus fibrosus, the transition zone, and the cartilage end plates also contain these chondrocytetype cells, whereas the outer annulus has a predominant amount of fibroblast-type cells [10]. Proteoglycans are made of a protein core attached to at least one glycosaminoglycan (GAG) chain. The predominant GAGs in the intervertebral disc are chondroitin-6-sulfate and keratin sulfate. The main proteoglycans found in the intervertebral disc are similar to the large aggrecan molecule found in articular cartilage and are able to form large aggregates by means of link proteins and hyaluronan [15,16]. Proteoglycans are unevenly distributed in the disc, making up a small percentage of the dry weight of the outer annulus while constituting 50% of the dry weight of the nucleus in young discs [10,17]. Large proteoglycan aggregates and their interaction with water have a major effect on the properties of the intervertebral disc. The hydrophilic nature of the GAGs attract and hold water in the intervertebral disc, absorbing much of the compressive loads seen in the spine while evenly distributing the remaining force around the circumference of the annulus [18]. The concentration of proteoglycans also changes tissue permeability and diffusion rates in the disc, which affects the passage of nutrients, chemical mediators, and cellular waste products [18]. Collagen is the other major structural component of the extracellular matrix. It provides the tensile strength of the intervertebral disc, allows for stability between vertebrae, and resists excessive disc bulging in response to loads. In younger discs, collagen makes up 67% of the dry weight of the annulus fibrosus and 25% of the nucleus pulposus. The distribution of collagen is also uneven, with decreasing amounts from the outer annulus in toward the nucleus pulposus. The types of collagen present in the disc are many [17,19,20], and their distributions vary as well [10,17]. Types I and II make up 80% of the collagen content of the disc. Type I collagen is most abundant in the outer annulus and decreases toward the center of the disc, with no type I collagen present in the nucleus. Type II collagen follows an
opposite trend, accounting for 80% of the collagen in the nucleus pulposus. Collagen types III, V, VI, IX, and XI are also present in smaller quantities, and play a role in collagen fibril organization. Aging and degeneration of the intervertebral disc Some degree of disc degeneration occurs in all people. A clear association has been shown between increasing age and disc degeneration. Miller et al. [21], using data from studies addressing the incidence of disc degeneration in cadaveric specimens, found that by age 49, 97% of lumbar intervertebral discs showed at least some evidence of disc degeneration. Degeneration was seen as early as the second decade of life. It is also known that asymptomatic disc degeneration is not uncommon [22,23] and that low back pain and radiographic evidence of degeneration do not correlate well [24]. This raises the issue of whether lumbar disc degeneration is a “disease” or rather a natural part of aging. Discs from both asymptomatic and symptomatic individuals exhibit similar chemical, structural, and radiographic findings. People who do have low back pain, however, have more diffuse and severe degeneration than those who are asymptomatic [25,26]. Biochemical analyses of herniated discs in a middle-aged population resemble those of more aged discs [27,28]. This indicates that the discs of symptomatic individuals undergo either an acceleration of the normal aging process, an exacerbation of the aging process as a result of some combination of environmental and genetic factors, or both of the above. It is probably best to view disc degeneration as an inevitable, natural part of aging, which in some people occurs at an accelerated rate for reasons that are currently unknown. The poorly understood causes and the clinical variability of disc degeneration make it difficult to assess accurately who is at risk for the development of more severe, earlieronset disc degeneration. Male discs degenerate a decade earlier than female discs and also have more degeneration than their age-matched female counterparts [21]. Environmental factors associated with increased disc degeneration include smoking, exposure to whole body vibration, and heavy lifetime occupational and leisure physical loading [29–31]. Many have also implicated a genetic predisposition to disc degeneration [30,32]. Recent studies have suggested a link between a specific aggrecan gene polymorphism and early, more severe disc degeneration [33]. It is likely that the risk of developing degenerative disc “disease” is multifactorial, with both environmental and genetic factors playing a role. Disc alterations seen with aging The intervertebral disc undergoes substantial chemical and structural changes with aging, which are in general progressive and irreversible. It is known that many of the alterations seen in disc composition occur well before morpho-
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logic evidence of degeneration is apparent. These can affect spinal mobility and change the loads seen by nearby structures (i.e., facet joints, vertebrae, spinal ligaments). As the intervertebral disc ages, blood vessels in the vertebral end plates become less and less numerous, disappearing by the third decade of life. The number of viable cells in the inner regions of the disc decreases [34], and the normal collagen fibril organization begin to disappear. Granular material, which is likely made of degraded matrix molecules, is seen throughout the matrix of the nucleus pulposus, particularly in the immediate vicinity of the chondrocyte-type cells [10,34,35]. The biochemical constituents of the intervertebral disc that have received the most attention in degeneration are collagen and proteoglycan. Most have shown there to be few changes in collagen content with age [28,36,37]. The ratio of type I to type II collagen, however, changes in the anterior and posterior segments of the outer annulus [38], with an increase in collagen type I and III being seen as well [28]. Collagen cross-links formed by enzymatically mediated glycosylation, which are believed to play an important role in the normal functioning of collagen, decrease with age [39]. An increase in the amount of nonenzymatically mediated collagen cross-links is also seen, and this may contribute to degeneration by making the tissue more susceptible to mechanical failure [40,41]. In contrast to collagen, proteoglycan content in the nucleus pulposus decreases noticeably with age. The size of aggrecan and the amount of proteoglycans that form aggregates decreases with time [27,42,43]. Loss of these large molecules reduces the amount of hydration in the disc, changing its shape and volume, likely affecting the ability to absorb and distribute loads effectively. Recently, fibromodulin, a proteoglycan known to affect the structure of collagen fibrils, has also been found to undergo structural alterations in the aging disc [44]. The end result of these alterations in collagen and proteoglycan is a decrease in the proteoglycan/collagen ratio of degenerated discs, particularly in the nucleus pulposus, where much of the proteoglycan is lost [45]. What results is an increasingly fibrotic nucleus pulposus that exhibits altered load-bearing capabilities.
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The changes in the biochemical composition of the intervertebral disc likely play a role in the altered mechanical behavior of the disc tissue components. The tensile properties undergo small changes with aging and degeneration. [46] The effects of shear stresses on the mechanical behavior of the aging nucleus pulposus suggests that with increasing age, the nucleus becomes more stiff and less able to dissipate energy, acting more like a solid substance than a gelatinous one [47]. Intradiscal pressure measurements in degenerated discs reveal a decrease in magnitude compared with normal discs. They have also shown that the pressure within the degenerated disc is unevenly distributed (nonisotropic) in the vertical and lateral directions [48]. These alterations in load-bearing capability and distribution could cause unusually high stresses at specific points in the disc, causing the development of localized tissue damage. Anatomic changes in the degenerating intervertebral disc can be grouped into stages [49,50]. The nucleus initially becomes increasingly dehydrated and fibrotic, making the distinction between the nucleus and the annulus more difficult. Circumferential tears of the annulus are seen, which may then progress to radial tears. Further degeneration of the annulus leads to loss of disc height and disc resorption. Thompson et al. [51] proposed a classification scheme for degenerative disc disease using sagittal sections of the vertebral column and grading degeneration based on the morphologic appearance of the nucleus, annulus, end plate, and vertebral body (Table 1). This grading scheme allows for comprehensive evaluation of all disc structures while maintaining good inter- and intraobserver reliability.
Factors implicated in disc degeneration There is increasing evidence that suggests that the biochemical events occurring in the disc may have an important role in disc degeneration. Many factors have been implicated and can be grouped into a few general categories: cell nutrition and viability; degradation and modification of extracellular matrix components; and disc matrix alterations resulting from mechanical loading. It is essential to note that
Table 1 Thompson’s classification scheme for disc degeneration Grade Nucleus
Annulus
End plate
I II III
Bulging gel White fibrous tissue Consolidated fibrous tissue
Hyaline, uniformly thick Thickness irregular between lamellas Focal defects in cartilage
IV
Horizontal clefts parallel to end plate
Discrete fibrous lamellas Mucinous material peripherally Extensive mucinous infiltration; loss of annular-nuclear demarcation Focal disruptions
V
Clefts extend through nucleus and annulus Reprinted with permission from [51].
Vertebral body
Margins rounded Margins pointed Early chondrophytes or osteophytes at margins Fibrocartilage extending from subchondral Osteophytes less than 2 mm bone; irregularity and focal sclerosis in subchondral bone Diffuse sclerosis Osteophytes greater than 2 mm
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the relative importance each one has in degeneration is not currently known. Cell nutrition and viability Many studies have implicated decreased cell nutrition as the main cause of disc degeneration [17,52]. Diffusion, being the primary mechanism of nutrient transport to the center of the adult disc, has been shown to slow down once the end plates become calcified with age [53,54]. Smoking and vibration have both also been shown to decrease solute transport into the central disc [55,56]. The decrease in diffusion also affects the passage of cellular waste products out of the disc and results in their accumulation in the matrix. These findings, coupled with the increasingly fibrotic nature of the nucleus and the decrease in nuclear water concentration, may additionally inhibit cellular nutrition. The decrease in cellular nutrition has an effect on various aspects of cellular metabolism. The concentrations of oxygen and lactate and the extracellular pH in the intervertebral disc have been studied. Steep oxygen concentration gradients are found in the disc [57], leading to high lactate concentrations (and low pH values) in the nucleus. Low oxygen concentration affects cellular metabolism directly by decreasing proteoglycan synthesis, particularly in the nucleus pulposus [58]. High lactate and low pH levels, which have been found in degenerated disc surgical specimens [59], also affect cellular metabolism [60]. These studies implicate decreased cellular nutrition as a cause of disc degeneration through its eventual effect on matrix production. This decrease in cellular nutrition also likely hinders cell viability. Other possibilities include apoptosis, or programmed cell death, of intervertebral disc cells. Gruber and Hanley [61] found a high incidence of apoptosis in the intervertebral disc and thought that it might be related to specimen age. It is not understood why these cells undergo apoptosis, but a variety of stimuli have been implicated [62]. Recent in vitro studies have shown that intervertebral disc cells undergo a significantly higher rate of apoptosis when subjected to prolonged mechanical compression [63]. Although it is not known how cell death might ultimately affect disc degeneration, a decrease in the number of matrix-producing cells with age is likely to affect disc composition. Along with the decrease in the amount of viable disc cells, the type of matrix they produce changes with age. Few studies have looked at metabolic alterations in the intervertebral disc, but age-related changes in cell function do affect other tissues [42]. Bayliss et al. [64] found the rate of proteoglycan production in neonatal and infant discs to be greater than in adult discs. In a separate study, they found that in the adult disc, newly synthesized proteoglycans were less stable and less able to aggregate than pre-existing proteoglycans [65]. What is seen, in addition to a decrease in matrix synthesis, is a notable change in the type of matrix synthesized, which produces an environment that is less able to maintain the normal properties of the disc.
Matrix degradation and modification Another important factor thought to play a role in disc degeneration is the alteration of the already existing extracellular matrix of the nucleus pulposus. Changes in the type of collagen cross-links occur, which alter collagen properties, including solubility and mechanical strength [40,41]. A variety of mediators have been identified that degrade or modify matrix constituents. These include the neutral proteinases (including serine proteinases and matrix metalloproteinases), cytokines (particularly interleukin-1 [IL-1] and interleukin-6), nitric oxide, and prostaglandins. All are involved in the matrix breakdown of articular cartilage, and are also suspected of having a similar effect in the intervertebral disc [66,67]. Kang et al. [68] found that degenerated herniated discs from both the cervical and lumbar spine spontaneously made increased amounts of all of these mediators. They also found that IL-1 was able to increase the production of the other mediators in intervertebral disc cells taken from both normal and herniated disc specimens. Others have found high proteinase activity in the nucleus pulposus and the end plates of degenerated discs, with no detection in normal disc tissue. Although the ultimate role these mediators in disc degeneration is not yet clear, the evidence suggests that they may play an important one. They may also be involved in the clinical sequelae of disc degeneration [68]. It is possible that with an acute disc herniation, the body’s inflammatory response may further stimulate disc cell production of these mediators. Each patient’s individual inflammatory response may be partially responsible for the clinical variability seen with herniation. It can also be hypothesized that the inflammatory response may produce “idiopathic” low back pain [69]. It is possible that the increased presence of these mediators in degenerated discs might irritate and stimulate the nerve endings near the outer annulus and longitudinal ligaments, as well as the dorsal root ganglia and spinal nerves. Mechanical loading effects on the disc matrix The effect of mechanical loads on the intervertebral disc matrix may also cause disc degeneration. Disc matrix synthesis rates decrease abruptly if the tissue either swells excessively or loses fluid [18,70]. Static loads on the disc result in fluid extrusion, which is in turn dependent on the size of the load and the composition of the tissue [37]. Discs with low proteoglycan content have a higher permeability and thus a larger amount of fluid loss under mechanical loads. Recent in vitro studies have also shown that prolonged mechanical loading causes a higher rate of apoptosis in intervertebral disc cells [63]. The effects of increased mechanical loads on matrix synthesis and cell survival could help explain the increased rate of disc degeneration seen in people who are exposed to increased physical loading. The exact role that the above theories play in intervertebral disc degeneration is not entirely clear, but it is likely that they are interrelated. Whatever the inciting event(s)
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may be, there is probably a complex interplay between them. A hypothetical scenario could be envisioned whereby decreased cellular nutrition and increased apoptosis cause a decrease in cell viability. The decrease in cell number could, along with prolonged mechanical loading, result in a decrease in matrix production. Also, whether as a result of a more “stressed” environment or some other extrinsic factors, the cells alter the type of matrix synthesized and secrete mediators that degrade the pre-existing matrix. This would result in a matrix that is less able to maintain the hydrostatic properties of the disc and is more difficult for solute diffusion. A vicious cycle could result with more cell death from further decreased nutrition, along with a higher rate of apoptosis and decreased matrix synthesis from increased loads seen by the cells. It is possible that in the process of “normal” disc aging, fewer factors are present at any one time, allowing the disc to adapt to changes in its environment. The accelerated degeneration seen in some people could occur when all factors are present concurrently, producing simultaneous “hits” to the system and overwhelming any adaptive mechanism the disc may have. Gene therapy and disc degeneration Given the high incidence of low back pain and disc degeneration, many investigators have sought for ways to try
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to prevent or halt the biochemical changes that the disc undergoes. Advances in the field of molecular genetics, such as gene therapy, have made it an attractive candidate to be used as a potential therapeutic intervention for spinal disorders. The unique characteristics of the intervertebral disc make it an ideal site for gene transfer. The isolated and encapsulated nature of the intervertebral disc helps to maintain high intradiscal concentrations of locally delivered vectors while limiting potential systemic side effects. It also can protect the vectors from the body’s own immunosurveillance mechanisms, prolonging therapeutic gene expression. Gene transfer is typically performed by introduction of the gene of interest (see following discussion of vectors), either directly into the target cells or by removal of the target cells, followed by genetic alteration with subsequent reimplantation. The former strategy is known as direct, or in vivo gene therapy, and the latter, as indirect, or ex vivo gene therapy (Fig. 1) [71]. Additional attention should be directed toward the route of administration. The direct delivery of genes to the site of disease is referred to as local gene therapy, whereas gene transfer that occurs distant from the disease site and uses the circulation system to deliver genetically modified cells and/or gene product is termed systemic gene therapy. These processes will hopefully result in the formation of protein products, which not only affect the
Fig 1. Proposed scheme of the effects of various factors on intervertebral disc degeneration.
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Fig. 2. In vivo gene therapy introduces vectors containing the appropriate gene directly into the target organ/tissue. Ex vivo gene therapy requires the removal of target cells from body, genetic alteration of these cells in vitro, followed by reimplantation of the genetically modified cells into the target organ/tissue. Reprinted with permission from [71].
cells’ own metabolism, but the metabolism of surrounding, nongenetically altered cells. With few exceptions [72–74], naked DNA (fragments of DNA not linked to any transporters) is not well incorporated by cells. Therefore, to enable the cellular uptake of genetic material in a manner that allows the expression of its genetic information, a transporter system (vector) is required. There are two general classes of vectors: viral and nonviral. Under normal circumstances, gene expression requires the genetic material to be delivered into the nucleus of the cell, where the transcriptional machinery resides. DNA may integrate into the chromosomes of the target cell, or it may remain extrachromosomal (episomal). Chromosomal integration of the delivered gene stabilizes it and guarantees its transmission to daughter cells upon cell division. Episomal DNA, although expressed at higher levels than integrated genes, often has gene expression wane with time, especially as the cell divides. Tables 2 and 3 summarize many of the advantages and disadvantages of a variety of vectors currently being used [75]. Viruses are efficient vectors, because their normal life cycles have the enhanced ability to enter the cell and initiate the expression of virally encoded genes. When using viral vectors, it is customary for the noxious or detrimental elements of the viral genome to be removed (or inactivated) and replaced by the genes of interest. Additional genetic modifications are often performed to prevent the replication and pathogenesis of the virus after infection of the target cells. Nonviral vectors are comparatively easier to produce than their viral counterparts, have increased chemical stability, have a virtually infinite gene capacity, and have no infectious or mutagenic capability [75]. All current nonviral
vector systems deliver DNA episomally, presenting the disadvantage of a limited duration of gene expression. Several early investigations evaluated the use of growth factors as a possible therapeutic intervention for intervertebral disc degeneration. Thompson et al. [76] demonstrated that the addition of human transforming growth factor beta I (TGF-1) to canine intervertebral disc tissue stimulated an increase in in vitro proteoglycan synthesis, suggesting that this growth factor may be useful in the treatment of disc degeneration. Various other mediators have been shown to increase proteoglycan synthesis in vitro, including interleukin-I [77], insulin-like growth factor-I [78], and osteogenic protein-I [79]. One of the major shortcomings of growth factor usage however, is its characteristic short-lived duration. Gene therapy is a novel approach that could overcome this problem while maintaining many of the growth factor’s potentially beneficial properties. Few investigators have had success with nonviral transduction of intervertebral disc cells. Chang et al. [80] recently reported successful marker gene transfer using the gene gun in cultured rabbit nucleus pulposus and annulus fibrosus cells. The transfection efficiency was noted to be approximately 10%. Wehling et al. [81] reported retrovirus-mediated transfer of both bacterial -galactosidase (LacZ) and human interleukin-1 receptor antagonist (IL-1Ra) to cultured chondrocytic bovine intervertebral end plate cells. The authors suggested the possibility of an ex vivo approach to treatment of disc degeneration by transduction of the cells in vitro using therapeutic genes with subsequent reinjection of the cells back into the disc. Nishida et al. [82] reported adenovirus- mediated transfer of the LacZ marker gene to the rabbit intervertebral disc cells both in vitro and in vivo. In vivo gene expression continued
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Table 2 Viral vectors for gene delivery Vector
Integration
Advantages
Disadvantages
Retrovirus
Yes
Well developed Ease in production
Adeno-associated virus
Yes
Adenovirus
No
Herpes simplex virus
No
Nonpathogenic Site-selective integration Stable expression Infects nondividing cells Broad range of target cells High titer production High infectivity Ease in production Infects nondividing cells Broad range of target cells High titer production Infects nondividing cells Large capacity (35 kb) Establishment of latency
Random insertion Infects only dividing cells Variable long-term expression Small capacity (4 kb) Difficult to produce Not as well developed
DNA remains episomal Toxicity Immunogenicity Persistent viral protein expression Poorly developed Toxicity Transient expression
Reprinted with permission from [75].
at undiminished levels for at least 12 weeks, with no expression seen in the control discs. Because of the immunogenic nature of the adenoviral vector, Nishida et al. [83] also performed in vivo safety studies in the rabbit by looking at the immune response and marker gene expression under various conditions. Three groups were employed: immunizing injection into the subcutaneous tissues, subcutaneous injection with simultaneous intradiscal injection, and immunizing subcutaneous injection followed 2 weeks later by intradiscal injection. Neutralizing antibodies were confirmed in all three groups up to 6 weeks after injection. However, both of the intradiscal injection groups demonstrated strong gene expression despite the presence of peripherally circulating neutralizing antibodies, indicating that the disc protected the virus from the host’s immune system.
The successful long-term gene expression of intervertebral disc cells using a marker gene and the unique immunologic characteristics of the disc suggested that the adenoviral vector system might be a suitable delivery mechanism for the transfer of therapeutic genes for the treatment of spinal disorders. Nishida et al. [84] then evaluated the feasibility of in vivo adenovirus-mediated therapeutic gene transfer to the intervertebral disc of the rabbit. The therapeutic gene chosen was the one for human TGF-1. After 1 week, total TGF-1 production increased nearly six-fold compared with controls and exhibited a significant increase in proteoglycan synthesis, demonstrating the in vivo efficacy of adenovirus-mediated transfer of a therapeutic gene to the intervertebral disc and its potential to modulate the complex biologic activity within the disc.
Table 3 Nonviral vectors for gene delivery Vector
Advantages
Disadvantages
Naked DNA/plasmid
Nonimmunogenic Inexpensive Simple to use Nonimmunogenic Targeted delivery Noninfectious Inexpensive Large carrying capacity High efficiencies claimed
DNA remains episomal Limited number of target cells Poor expression DNA remains episomal Poor expression DNA remains episomal Limited number of target cells
DNA-ligand complex (molecular conjugates) Liposomes
Gene gun (colloidal gold)
Reprinted with permission from [75].
DNA remains episomal In vivo access difficult Long-term metal particle effects unknown Tissue damage effects unknown
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Conclusion This article describes the basic structure and function of the intervertebral disc and the changes they undergo with time. It is clear that the biochemical changes that occur in the disc play a major role in the pathogenesis of disc degeneration. What remains uncertain is what causes these changes and how, if at all, they can be prevented or even reversed. Gene therapy shows promise as a tool to better address these issues. Undoubtedly, several barriers must be overcome before gene therapy can be used clinically in humans for the treatment of spinal disorders. Two primary concerns, which genes should be administered and what the best method of delivery is, need to be elucidated before this technology can be applied. Recent studies continue to bring forth attractive candidates for gene transfer [85]. Current studies in our center are evaluating different viral vectors in order to optimize gene delivery while minimizing any potential risks to the recipient. With continued, dedicated research efforts, gene therapy has the potential to become an effective treatment modality for intervertebral disc degeneration in the twenty-first century.
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