Definition

The term gene therapy was previously used to describe replacement of a defective gene with a functional copy by means of gene transfer. The diseases originally targeted for gene therapy were classic, heritable genetic disorders. The term now broadly defines therapy involving the transfer of exogenous genes (complementary DNA [cDNA]) encoding therapeutic proteins into cells to treat disease.¹ The genetically altered cells are made into protein-producing “factories” churning out disease-altering gene products. Specifically, the host cell transcribes the exogenous gene (or transgene) into messenger RNA (mRNA); cytoplasmic ribosomes translate mRNA into the protein product. These products can affect not only the metabolism of cells from which they were made but also the metabolism of adjacent non–genetically altered cells via paracrine mechanisms (Fig. 9–1).

FIGURE 9–1 DNA encoding the gene of interest is constructed into a viral vector that is rendered incapable of replication. The vector is exposed to host cells, attaches to their surface, and is internalized. The released genetic information can either travel to the nucleus, where it may become integrated into the host genome, or remain episomal. It commandeers the normal protein-making machinery of the cell and produces large quantities of transgene.

Exogenous genes are produced and packaged in the laboratory in the following manner. First, cDNA of a gene of interest is constructed by the enzyme reverse transcriptase from mRNA. The cDNA is incorporated into a plasmid, a circular piece of DNA that is self-replicating and capable of delivering exogenous genes into cells, albeit at an inefficient rate. The cDNA plasmid is next integrated into a larger plasmid with a promoter sequence, assembling an expression plasmid. The promoter sequence initiates transcription of the gene of interest by target cells after gene transfer has occurred. The cytomegalovirus promoter is commonly used in gene transfer experiments. This promoter is constitutive, meaning it consistently initiates transcription throughout the life of the gene. The expression plasmid is integrated into either a viral or nonviral molecular vehicle that facilitates transfer of the exogenous gene to cells. These vectors are discussed in the following section.

There are two basic strategies for delivering exogenous genes to target cells. The first is the in vivo method, in which a gene-carrying vector is directly transferred to an intended population of cells within the host. The second approach, known as ex vivo gene therapy, involves removing target cells from the body, genetically altering them in vitro, and reimplanting them in the body (Fig. 9–2). Ex vivo methods are more complex and involve multiple, time-intensive steps. This approach is relatively safer, however, because the genetically altered cells may be observed for abnormal behavior before implantation. The ex vivo strategy allows the opportunity for in vitro selection of cells that express the gene of interest at high levels. Gene transfer via the in vivo method is technically simpler. There are relative advantages and disadvantages to both approaches that depend on the anatomy and physiology of the target organs, the pathophysiology of the disease being treated, the vector of choice, and safety considerations.²

FIGURE 9–2 A, In vivo gene therapy involves direct injection of vector-gene constructs into target tissues within the host. B, In ex vivo method, target cells are harvested from the host and then transduced, expanded, and propagated before reimplantation.

Overview of Vectors

Successful gene therapy generally depends on the efficient transfer of genes to target cells with subsequent expression. Generally, the duration of transgene production to treat disease successfully depends on the disease being targeted. Sustained expression is necessary for chronic conditions such as disc degeneration, whereas brief expression may be sufficient for acute conditions such as bone healing. With few exceptions, naked plasmid DNA is not taken up and expressed by cells effectively. Consequently, vectors are often necessary to package and insert genes into cells in such a way that the genetic information can be expressed. There are two broad categories of vectors, viral and nonviral. Gene delivery involving viral vectors is termed transduction, whereas transfer using nonviral vectors is termed transfection.

Nonviral Vectors

Nonviral vectors include liposomes, DNA-ligand complexes, gene guns, and microbubble-enhanced ultrasound. Liposomes are phospholipid vesicles that deliver genetic material into a cell by fusing with the cell membrane. Liposome vectors are simple, inexpensive, and safe, but drawbacks include transient expression of the transgene, cytotoxicity at higher concentrations, and low efficiency of transfection. DNA-ligand complexes and gene gun are nonpathogenic and relatively inexpensive to construct, but there is concern with lower transfer efficiencies and limited persistence of gene expression. Nishida and colleagues³ showed that ultrasound transfection with microbubbles significantly enhanced the transfection efficiency of plasmid DNA into the nucleus pulposus cells of rats in vivo, observing transgene expression up to 24 weeks. The overall transfection efficiency and level of gene expression of these nonviral vectors are generally inferior, however, to that of viral-mediated gene transfer. Consequently, most current studies involving gene therapy employ viral vectors.⁴

Viral Vectors

Viral vectors take advantage of the natural ability of viruses to infect and deliver genetic information efficiently to specific cell populations. The most commonly used viral vectors are derived from retroviruses, herpes simplex viruses (HSV), adenoviruses, and adeno-associated viruses (AAV). These viruses are often rendered incapable of replication before gene therapy application in an effort to make them less pathogenic. There are inherent merits and drawbacks associated with each viral vector, which are discussed in the following section. The choice of viral vector for gene transfer experiments is based on multiple considerations, including the gene to be delivered, the disease to be treated, and safety considerations.

Retroviruses are small RNA viruses that replicate their genomic RNA into double-stranded DNA (dsDNA) via the action of reverse transcriptase. The dsDNA is integrated into the host genome at a random location where it is able to express transgene for the life of the cell. Exogenous dsDNA is replicated by the transduced cell and passed on to all progeny cells during cell division. Gene delivery with retroviral vectors results in stable, long-term expression because the gene is integrated into the cell’s genome. Because the integration is at a random site, however, the risk of potential mutagenesis of oncogenes exists. Until more recently, this risk was considered only a theoretical possibility, but preliminary reports from a gene therapy trial involving retroviral vectors suggest one of the enrolled subjects developed leukemia as a result of oncogene mutation.⁵ Another disadvantage of the most commonly used retroviral vector, the murine leukemia virus (MLV), is that it infects and transduces only actively dividing cells. For these reasons, MLV is most suited for ex vivo applications. Lentiviruses, another class of retroviruses, are capable of infecting nondividing cells. Their drawbacks lie in their wild-type pathogenicity and complex genomic configuration, which make molecular processing for gene transfer much more intricate.

HSV vectors are dsDNA viruses, and the wild-type virus is a human pathogen that is trophic for sensory neurons. The replication-deficient vectors can infect dividing and nondividing cells of almost all types in vitro and in vivo. In addition, HSV vectors have the capacity to carry large amounts of exogenous DNA. This large carrying capacity allows for the production of HSV vectors that are capable of expressing multiple transgenes, which may be highly desirable for gene therapy applications involving complex disease pathophysiology, as in cancer or arthritis. HSV vectors do not integrate the genes they are carrying into the genome of the target cell. A disadvantage of HSV vectors is that they result in transient expression of the transgene despite efficient transduction. Consequently, these vectors would be insufficient for chronic conditions, such as intervertebral disc degeneration.

Adenoviruses are dsDNA viruses capable of infecting many cell types, including nondividing cells. There are 47 known human serotypes of adenoviruses, with serotypes 2 and 5 most commonly used for gene therapy studies. Wild-type adenoviral infections result in mild respiratory and gastrointestinal illnesses. The ability of adenoviral vectors to transfer genes to target cells is particularly efficient. Consequently, the adenovirus is an appealing option for in vivo gene delivery to quiescent, nondividing cell populations. The adenovirus genome exists as an episome within the nucleus of the infected cell and is not integrated into the genome of the host cell, so the risk of insertional mutagenesis does not exist.

The vectors are relatively easy to engineer in very high titers, in contrast to the HSV vector. A major disadvantage with the adenoviral vector is its short duration of transgene expression in most tissues. The transient expression of gene product is thought to occur because of low-level production of adenoviral antigens by the infected cell, resulting in an immune response directed against these cells. The episomal location of the vector genome is also thought to contribute to the short duration of expression. During cell division, the viral episome is not replicated and instead ultimately is degraded. Research is ongoing to engineer adenoviral vectors to minimize viral protein expression and consequently be less immunogenic.

AAV is a parvovirus with a 4.7-kb single-stranded DNA genome. Wild-type AAV lacks the viral machinery to self-replicate and can reproduce only in association with concomitant viral infection, usually adenovirus. AAV is also capable of infecting many different cell types, dividing and nondividing, but its level of infection efficiency is varied. The wild type is not known to cause disease.

The AAV vector differs from the adenoviral vector in several important ways. First, the AAV vector integrates reliably into a specific site on chromosome 19 in a nonpathogenic manner. Second, AAV does not provoke a significant immune response because the vector fails to express viral gene products after infection of target cells. Wild-type AAV has only two genes, Rep and Cap, which cannot be replicated without the presence of a helper virus. There is no expression of AAV gene products after transduction, theoretically leading to minimal host cell–mediated immune reaction. Although nearly 80% of the population has circulating antibodies against AAV2 (serotype 2) as a result of silent infections, titers of these neutralizing antibodies are usually low. For these reasons, sustained transgene expression can be achieved for 1 year in an immunocompetent host. The main shortcoming of AAV vectors is that they are capable of carrying only small amounts of foreign DNA. In addition, these vectors are difficult to construct and purify in the laboratory without helper virus contamination. There are multiple serotypes of AAV, but AAV2 has been most thoroughly studied for musculoskeletal applications, including degenerative disc disease.

There is a wide range of vector systems with different profiles for delivery efficiency, duration of expression, technical feasibility, and safety. Ongoing investigations are attempting to improve these profiles, and this research is likely to result in enhanced vectors with inducible promoters, tissue-specific promoters, or tissue-specific tropism. As mentioned, the appropriate vector system for a gene therapy application depends on multiple factors, including the method of delivery, pathophysiology of the disease targeted, and the gene selected for transfer.

Clinical Problem

Disorders of the spine often necessitate intervertebral fusion. Although internal fixation devices can successfully achieve temporary stabilization at practically all levels, long-term stability requires osseous consolidation. In contrast to fracture healing, spinal arthrodesis involves deposition of new bone in intersegmental locations that are not biologically structured for bone formation. Consequently, the nonunion rate is 40%^6,7 with single-level fusions and higher when multiple levels are attempted. Although instrumentation has improved the rate of bony union, pseudarthrosis remains a considerable clinical problem. Autogenous bone graft may be scarce in volume in cases such as pediatric fusions and revision surgery. It is associated with substantial donor site morbidity. Owing to these significant obstacles to clinical success, extensive research has been directed at developing molecular therapies to facilitate intersegmental fusion.

Augmentation of Spinal Fusion with Bone Morphogenetic Proteins

Bone morphogenetic proteins (BMPs) are a group of osteoinductive cytokines that play an essential role in the formation and maturation of osseous tissues. Urist and colleagues^8,9 originally recognized these proteins for their ability to form ectopic bone by inducing mesenchymal stem cell differentiation into chondrocytes and osteoblasts. Numerous subsequent animal studies have shown the ability of BMPs to enhance bone deposition at fusion sites.^10–19 This was followed by several clinical trials that validated these preclinical findings. In a study by Patel and colleagues,²⁰ patients undergoing posterolateral lumbar fusion augmented with iliac crest autograft and recombinant human BMP-7 (rhBMP-7) had better outcomes as measured by the Oswestry score and radiographic analysis than patients who received iliac crest autograft alone. In another human trial using rhBMP-2 in interbody fusion cages for single-level lumbar degenerative disc disease, patients who received cages filled with collagen sponge–delivered rhBMP-2 had superior clinical and radiographic results compared with control patients who received cages filled with autogenous bone graft.²¹ Various other clinical trials have similarly shown the efficacy of BMPs to enhance spinal arthrodesis.^22,23

Gene Therapy for Spinal Fusion

Gene therapy represents a potential next step in the evolution of therapies directed at promoting a spinal fusion. Gene therapy techniques to deliver various BMP genes could overcome the barriers of high dosing and complex carrier systems and achieve long-term, controllable BMP expression. The transduced cells would secrete the BMP extracellularly, delivering it to the environment at physiologically appropriate doses for a sustained period, maximizing the osteoinductive potential of these growth factors. In addition, BMP expression could be regulated in a temporal fashion by using vectors with inducible promoters, which would allow the ability to control the activity of the protein tightly to the clinical setting. Another potential advantage is the capacity to deliver gene therapy for spine arthrodesis in a minimally invasive procedure with percutaneous injections to the spine. Lieberman and colleagues²⁴ found an increase in the total volume of new bone with improved histologic quality when the gene for BMP-2 was delivered compared with rhBMP-2 protein alone.

Several studies have shown the feasibility of using gene therapy to enhance spinal fusion. Alden and colleagues²⁵ showed new enchondral bone formation in paraspinal muscles injected with adenoviral BMP-2 constructs (Ad-BMP-2). Important observations made by this study included the absence of bone deposition distant from the injection site and the absence of neural compromise, suggesting that this approach may be safe for the clinical setting. In addition, Helm and colleagues²⁶ documented that the direct injection of Ad-BMP-9 resulted in fusion in a rodent model without the development of nerve root compression or systemic side effects. In a rabbit model, Riew and associates²⁷ showed that mesenchymal cells transduced with BMP-2 can promote spinal arthrodesis.

Many of these studies used first-generation adenoviral vectors for gene delivery to immunocompromised animals, allowing for sustained expression. Gene therapy experiments with immunocompetent animals have led to a relative paucity of bone formation,²⁷ however, owing to the immune response elicited by adenoviral vectors. Further studies using second-generation adenoviral vectors or other vectors such as lentivirus could minimize these responses and maximize gene expression.

Another molecular avenue for bypassing the limitation of adenoviral immunogenicity is to deliver a gene for a factor that is “upstream” from the actions of BMP cytokines. In this way, neither efficient transduction nor sustained duration would be necessary because this factor would start a cascade of BMP activity after only a short period of expression. This intriguing strategy has been developed by Boden and colleagues,^28,29 who showed that a novel intracellular transcription factor, LIM mineralization protein-1 (LMP-1), could be used to upregulate the expression of BMPs and their receptors. LMP-1 initiates a cascade of events intracellularly, which stimulates the secretion of osteoinductive factors, which increases BMP activity. All of the study animals that were implanted with peripheral blood buffy coat cells genetically modified with Ad-LMP-1 showed successful lumbar fusion.²⁸ None of the 10 controlled rabbits had evidence of any bone formation, and the investigators concluded that local gene therapy could reliably induce spinal fusion in an immunocompetent animal.

Future Directions

With further development, gene therapy techniques will likely be able to induce bony union between vertebral bodies, transverse processes, facets, laminae, and spinous processes in a minimally invasive fashion. Although the studies mentioned earlier establish the potential of gene therapy for enhancing spinal fusion, many issues related to safety, efficacy, and cost remain to be resolved before translation into clinical success.

Clinical Problem

Degenerative disc disease is a chronic process that can clinically manifest in multiple disorders, such as idiopathic low back pain, disc herniation, radiculopathy, myelopathy, and spinal stenosis. It is a significant source of patient pain and morbidity, using a large portion of health care resources.³⁰ Available treatment options include conservative measures such as bed rest, anti-inflammatory agents, analgesic medications, and physical therapy. When conservative measures fail, invasive surgical procedures such as discectomy, instrumentation, or fusion, with their inherent risks and expenses, are often required. Conservative and surgical treatment modalities focus on the clinical symptoms of intervertebral disc degeneration without addressing the underlying pathologic processes occurring throughout the course of degeneration.

Although the precise pathophysiology of degenerative disc disease remains to be delineated, it is known to involve a complex interaction of biologic, genetic, and biomechanical factors. The progressive decline in aggrecan, the primary proteoglycan of the nucleus pulposus, is known to be a significant and characteristic factor.^31–33 At the biochemical level, aggrecan homeostasis is altered by various combinations of decreased synthesis and increased breakdown. With reductions in proteoglycan content, the nucleus pulposus dehydrates, decreasing disc height and its load-bearing capacity.^34–36 This situation may directly affect biomechanical function by altering the loads experienced by the facet joints, leading to degenerative changes. Although disc degeneration most probably evolves in response to a complex interplay of multiple biochemical and biomechanical factors,³⁷ the ability to restore proteoglycan content may have therapeutic benefit by increasing disc hydration and potentially improving biomechanics.

The development of newer biologic therapies may allow for the treatment of intervertebral disc degeneration on a molecular level, without the need for disc excision or fusion surgery. In the last decade, numerous biologic factors involved in the regulation of disc extracellular matrix production, cell proliferation, and cell death have been identified. Many of these growth factors and cytokines have been shown to be present or involved in the degenerating intervertebral disc, and such growth factors may have therapeutic potential for regulating matrix production. The goal of gene therapy for disc degeneration is to transfer the gene of interest to the cells of the nucleus pulposus, allowing sustained transgene expression and upregulation of proteoglycan synthesis or inhibition of catabolic activity, increasing water content and maintaining or improving disc biology and biomechanics.

Growth Factors

The ability to increase proteoglycan synthesis in the intervertebral disc was shown by Thompson and colleagues,³⁸ who showed that the exogenous application of human transforming growth factor (TGF)-β1 to canine disc tissue in culture stimulated in vitro proteoglycan synthesis. The authors suggested that growth factors may be useful for the treatment of disc degeneration. Subsequent studies with other growth factors, such as insulinlike growth factor (IGF)-I, BMP-2, and osteogenic protein (OP)-1 (OP-1) also exhibited the ability to upregulate proteoglycan content in intervertebral disc cells.^39,40 Owing to the relatively brief half-life of these factors, however, practical application of growth factor therapy to chronic conditions such as degenerative disc disease would necessitate repeated administrations. Consequently, efforts were directed at developing approaches to induce endogenous synthesis of growth factors via gene therapy such that genetically modified disc cells manufacture the desired growth factors on a continuous basis, enabling long-term regulation of matrix synthesis with the potential to prevent or delay degenerative disc disease.

Previous Studies of Intradiscal Gene Therapy

The notion of using gene transfer for intervertebral disc applications was initially introduced by Wehling and colleagues.⁴¹ In an in vitro study, these investigators reported on a retroviral mediated transfer of two different genes to cultured chondrocytic cells from bovine intervertebral endplates: (1) the bacterial β-galactosidase marker gene (LacZ) and (2) the cDNA of the human interleukin-1 receptor antagonist (IL-1Ra). Transfer of LacZ resulted in transduction of approximately 1% of the cell population. Transfer of the IL-1Ra cDNA resulted in significant levels of IL-1Ra protein by 48 hours. The authors concluded that this ex vivo approach, involving harvesting of endplate tissue from a degenerating disc, transducing these cultured cells with therapeutic genes, and reimplanting the genetically modified cells into the disc, could provide a novel strategy for treating degenerative diseases of the spine.

Nishida and colleagues⁴² reported the first successful in vivo gene transfer to the intervertebral disc using an adenoviral vector to deliver the LacZ marker gene to the rabbit lumbar disc. The authors were able to show sustained transgene production with no significant reduction in expression 3 months after transduction (Fig. 9–3). The rabbits used in these studies showed no signs of systemic illness in response to the adenoviral vector and its transgene synthesis. In addition, no histologic changes suggesting a cellular immune response were observed.

FIGURE 9–3 Qualitative analysis of intradiscal lacZ transgene expression up to and including 1 year after injection of Ad-lacZ into lumbar intervertebral discs of adult New Zealand White rabbits. Serial histologic sections were stained with X-Gal and counterstained with eosin. A-F, Representative sections of lumbar discs at 3 weeks (A and B), 6 weeks (C and D), and 24 weeks (E and F) after injection are shown. All of the discs injected with Ad-LacZ exhibited positive X-Gal staining. G, At 52 weeks after injection, positive X-Gal staining was observed in the discs from two of three rabbits. The intensity of positive staining was less than in discs from the other time periods, however. (Original magnifications 40× [A, C, E], 200× [B, D, F], and 600× [G].)

Encouraged by these results with a marker gene, the successful in vivo transduction of the intervertebral disc with a therapeutic gene, human TGF-β1, was soon accomplished.⁴³ This study showed a 30-fold increase in active TGF-β1 synthesis and a 5-fold increase in total TGF-β1 production in discs injected with the adenoviral–growth factor construct (Fig. 9–4). Biologic modulation was also documented by a 100% increase in proteoglycan synthesis (Fig. 9–5). As in the previous studies, no signs of local or systemic immune response were noted.

FIGURE 9–4 A, Active transforming growth factor (TGF)-β1 production in rabbit nucleus pulposus tissue 1 and 6 weeks after in vivo injection of Ad-TGF-β1 compared with intact control discs and discs injected with saline and Ad-luciferase. B, Total (active and latent) TGF-β1 production in rabbit disc tissue 1 and 6 weeks after in vivo injection. There were no significant differences in either active or total TGF-β1 production at 1 week and 6 weeks, indicating that the therapeutic gene expression was sustained. Asterisk denotes significant increase over corresponding intact, saline, and viral (Ad-luciferase) control groups (P < .05).

FIGURE 9–5 Proteoglycan synthesis in rabbit nucleus pulposus tissue 1 and 6 weeks after in vivo injection of Ad-TGF-β1 compared with intact control discs and discs injected with saline and Ad-luciferase. There were no significant differences in proteoglycan synthesis at 1 week and 6 weeks, indicating that the biologic effect of transgene synthesis was sustained. Asterisk denotes significant increase over corresponding intact, saline, and viral (Ad-luciferase) control groups (P < .05).

Additional in vitro studies with cultured human nucleus pulposus cells yielded similar promising results. Successful transduction of the LacZ marker gene delivered with adenoviral vectors was achieved in human cells from degenerated discs.⁴⁴ The response of human cells from degenerated discs to adenoviral-mediated delivery of TGF-β1 was subsequently assessed.⁴⁵ Increased production of TGF-β1, proteoglycan, and collagen was shown in cells receiving gene therapy compared with controls. Cells receiving the adenovirus–TGF-β1 construct showed increased proteoglycan and collagen synthesis compared with cells receiving exogenous TGF-β1 protein, presumably in response to the sustained expression of this growth factor with gene transfer.

The viral load required to increase proteoglycan synthesis was significantly less than the load necessary for transduction of the entire cell population, perhaps highlighting the ability of a transduced cell to influence the biologic activity of non–genetically altered neighboring cells. The concept that successfully transduced cells exert a paracrinelike effect on their nontransduced neighboring cells implies that significant alteration in protein synthesis can be achieved with only a few transduced cells.² A better understanding of this paracrine effect may enable the use of decreased viral loads to achieve a therapeutic effect, minimizing potential viral toxicity.

Additional in vitro studies with other promising growth factors such as BMP-2 and IGF-I documented the potential of adenoviral delivery of these factors to increase proteoglycan synthesis in a viral dose–dependent manner.^46,47 Adenoviral delivery of tissue inhibitor of metalloproteinase (TIMP)-1 showed the same ability.⁴⁷ TIMP-1 is an endogenous inhibitor of matrix metalloproteinases, enzymes capable of degrading the extracellular matrix of the intervertebral disc. This finding established a second gene therapy strategy to modify the disrupted balance of synthesis and catabolism occurring in the degenerated intervertebral disc: inhibition of matrix degradation with ensuing net increase in proteoglycan content.

Considering the potential adverse effects of viral vectors, studies have been undertaken to develop strategies to minimize viral loads while maintaining the same biologic effects. Experiments with combination gene therapy involving TGF-β1, IGF-I, and BMP-2 suggested that these growth factors are synergistic in amplifying matrix synthesis.⁴⁶ Adenoviral delivery of a single growth factor increased proteoglycan synthesis by a range of 180% to 295%, whereas combination gene therapy with two agents resulted in increases of 322% to 398%. When all three growth factors were combined, proteoglycan synthesis was increased by 471% (Fig. 9–6). It remains to be determined if combination gene therapy with an anabolic growth factor and a catabolic inhibitor such as TIMP-1 would have a similar synergistic effect.

FIGURE 9–6 Proteoglycan synthesis in human intervertebral disc cells treated with different combinations of therapeutic adenoviral vectors (Ad-TGF-β1, Ad-IGF-I, Ad-BMP-2). All groups showed significant increase in synthesis compared with saline and viral (Ad-luciferase) control groups. Asterisk indicates P < .05.

Lattermann and colleagues⁴⁸ investigated the transduction efficiency of the AAV vector on nucleus pulposus cells compared with an adenoviral vector in vitro and in vivo. This study showed that the transduction efficacy of the AAV vector on human nucleus pulposus cells in vitro was high, but 48% lower than adenovirus (Fig. 9–7). Next, in vivo gene expression in rabbits after transduction with an AAV vector carrying the luciferase marker gene was achieved at all time points up to 6 weeks (Fig. 9–8). Similar to the in vitro results, the maximum transgene expression using the AAV-luciferase was approximately 50% of the maximum obtained after transduction with the adenoviral vector. Although transgene expression in vivo was decreased compared with levels achieved after adenoviral vector transduction, the overall amount of luciferase was high, likely exceeding any potential therapeutic dose. The authors concluded these experiments showed the feasibility of the AAV vector, as these levels of gene expression may be sufficient for the sustained delivery of a growth factor gene to the nucleus pulposus.

FIGURE 9–7 A, Luciferase activity in human nucleus pulposus cells after transfection with AAV-luc and Ad-luc after 8 days. Transduction with 100,000 particles/cell AAV-luc yields 23.74 ± 4.62 ng (SEM) luciferase protein. This is exactly 52% of 45.23 ± 9.34 ng (SEM) luciferase protein measured for 100,000 particles of Ad-luc. With decreasing virus concentrations, gene expression decreases in a linear fashion in both groups. Asterisk refers to t test with Bonferroni correction. B, Human nucleus pulposus (hNP) cells show very high transgene expression after transduction with AAV viral vectors compared with other orthopaedically relevant cell types. Although human synovial fibroblasts (HIG82) generally show low transgene expression with both vector systems, human bone marrow–derived (hBMDC) cells show comparable response to human nucleus pulposus cells after transduction with adenoviral vectors. This may make hNP cells an ideal target for AAV viral vectors.

(From Lattermann C, Oxner W, Ixiao X, et al: The adeno-associated viral vector as a strategy for intradiscal gene transfer in immune competent and pre-exposed rabbits. Spine 30:500, 2005.)

FIGURE 9–8 Luciferase expression after in vivo gene delivery of luciferase gene using AAV-luc vector. At 2 weeks, there is very little gene expression in intervertebral discs. At 4 weeks, there is a significant increase in gene expression. This sustained level is maintained through 6 weeks. Asterisk refers to t test with Bonferroni correction.

(From Lattermann C, Oxner W, Ixiao X, et al: The adeno-associated viral vector as a strategy for intradiscal gene transfer in immune competent and pre-exposed rabbits. Spine 30:501, 2005.)

Other studies have focused on the role of LIM mineralization protein (LMP)-1 and Sox9 in the intervertebral disc. LMP-1 is an intracellular regulatory protein that upregulates expression and enhances anabolic activity of the BMP family of proteins. Yoon and colleagues⁴⁹ showed an increase in total proteoglycan and aggrecan synthesis in vitro and in vivo after transduction of rat intervertebral disc cells with an adenoviral vector construct containing LMP-1. They found significant increases in expression of BMP-2 and BMP-7 mRNA after in vitro transduction with LMP-1. Sox9 is a transcription factor responsible for chondrogenesis and type II collagen expression. Paul and colleagues⁵⁰ treated human intervertebral disc cells with an adenoviral vector construct containing Sox9 and found an increase in type II collagen production compared with controls. These studies make LMP-1 and Sox9 attractive candidates for further investigation as effective tools for intervertebral disc degeneration gene therapy.

Gene Expression Time Frame

Adenoviral vectors have been frequently used in gene therapy studies for the intervertebral disc owing to their ability to transduce efficiently highly differentiated, nondividing cells such as the cells of the nucleus pulposus. Successful gene therapy for the degenerated disc depends not only on efficient gene transfer but also on the expression of transgene for sufficiently long periods. The duration of gene expression after adenoviral transfer to an immunocompetent animal is limited in most organs and tissues by immune reactions to viral proteins and to foreign proteins encoded by the transgene. Specifically, expression for longer than 12 weeks has been difficult to achieve in most musculoskeletal tissues after adenoviral delivery, owing to brisk immune responses. Gene expression can occur for longer periods, however, in an “immune privileged” site. In studies by Nishida and colleagues,^42,43 intradiscal marker gene expression was achieved in the rabbit lumbar disc for 1 year after transduction by adenovirus. Because the intervertebral disc is well encapsulated and avascular, it seems to act as an “immune privileged” organ, permitting longer durations of transgene expression compared with other musculoskeletal tissues.

Compared with the adenoviral vector, the AAV vector has a relatively slower initial gene expression after transduction, likely attributable to an increased latency period. This delay in gene expression was also observed in the previously described in vivo experiments involving the intervertebral disc.⁴⁸ At 2 weeks, transgene expression was present but low compared with the adenovirus. At 4 weeks, transgene expression increased 60-fold, however, and that level was maintained at 6 weeks. This characteristic delay in gene expression could potentially preclude the use of AAV vectors in acute processes such as fracture healing, but there is no theoretical disadvantage when treating more chronic diseases such as degenerative disc disease.

Future Directions

The aforementioned studies documented the feasibility of in vivo gene transfer to the intact intervertebral disc. There are reasonable concerns, however, regarding the feasibility of efficient gene transfer to a degenerated disc in which the cell population and extracellular environment are distorted. More recent studies have identified possible genetic causes for disc degeneration.^47–49 Gene transfer to replace the defective gene with a normal copy may have great therapeutic benefit in susceptible populations. Although the intervertebral disc seems to be an “immune privileged” organ, the issue of viral vector immunogenicity is still crucial in light of a reported death of a patient undergoing a clinical trial with the adenovirus. Consequently, other vectors, such as the AAV vector, have been rigorously investigated because of their decreased side-effect profile.

To address the issue of transgene product toxicity, regulatory control systems may be necessary to control transgene expression, including the ability to turn on or off expression in the event of an errant injection or leakage of the injection contents into surrounding tissues.⁵¹ The ability to turn the transgene on or off based on the application of an externally applied stimulus (i.e., oral pharmaceutical) is an advantageous safety feature and a method to modify treatment by titrating transgene expression. Numerous inducible gene expression systems have been investigated, including systems using heat shock proteins, metallothionein, steroid regulatory promoters, tetracycline, and, most recently, the EcR insect receptor. Regulation systems with tissue-specific promoters would further optimize efficacy and minimize treatment side effects.

The length of transgene expression that is necessary for the treatment of a chronic condition such as intervertebral disc degeneration also requires further investigation. The persistence of gene expression after transduction has not been adequately defined in animal models, and the time course necessary for continued treatment is unknown. In addition, the possibility remains that the host immune response will eventually detect and destroy cells that have been transduced.

Another strategy for gene modulation that has emerged is RNA interference (RNAi). RNAi is mediated by small interfering RNA (siRNA) molecules that bind in a sequence-specific manner to target mRNAs, marking them for degradation and preventing translation of the target gene. The efficacy of RNAi mediated by siRNAs has been reported in nucleus pulposus cells in vitro using an exogenous reporter gene and an endogenous gene.⁵² To overcome the short half-life of the siRNA, Nishida and colleagues⁵³ used DNA vector–based RNAi and showed a stable downregulation of specific gene expression. RNAi has potential use as a novel strategy of gene therapy for treatment of degenerative disc disease by silencing expression of catabolic genes.