Gene Transfer Approaches to the Healing of Bone and Cartilage
Authors: Jay R. Lieberman, Steven C. Ghivizzani, Christopher H. Evans
Journal: Molecular Therapy (2002)

Gene therapy is traditionally considered a treatment modality for genetic diseases and a limited number of complex, life-threatening disorders, such as cancer. However, gene therapy also holds promise as a novel means of treating more mundane conditions, including broken bones and damaged cartilages [1Evans C.H. Robbins P.D. Possible orthopaedic applications of gene therapy.J. Bone Joint Surg. Am. 1995; 77: 1103-1114Crossref PubMed Scopus (198) Google Scholar, 2Evans C.H. Using gene therapy to protect and restore cartilage.Clin. Orthop. 2000; 379: S214-s219Crossref Scopus (89) Google Scholar, 3Oakes D.A. Lieberman J.R. Osteoinductive applications of regional gene therapy: ex vivo gene transfer.Clin. Orthop. 2000; 379: S101-s112Crossref PubMed Scopus (61) Google Scholar]. These injuries frequently present enormous clinical challenges to both orthopedic surgeons and rheumatologists, and can result in considerable human suffering and economic loss. Various gene products, particularly growth factors, show remarkable promise as agents that can improve the healing of bone, cartilage, and other connective tissues. Their clinical utility, however, is limited by delivery problems. The attraction of gene transfer approaches is the unique ability to deliver authentically processed gene products to precise anatomical locations at therapeutic levels for sustained periods of time. However, unlike the treatment of chronic disease, it is neither necessary nor desirable for transgene expression to persist beyond the few weeks or months needed to achieve healing. It is also unlikely that the level of transgene expression will need to be closely regulated. Moreover, achieving targeted delivery is not an issue, because the same orthopedic procedures that are already used when operating on bones and joints can be adapted for the purposes of targeted gene transfer. Thus, the applications proposed in this review represent several of the few examples in which gene therapy has a good chance of clinical success using existing technology. There is great interest in developing gene therapy to enhance bone repair because the potential for sustained protein production may enable the host to respond to an osteoinductive stimulus in a more robust fashion. Regional gene therapy is an attractive option to enhance bone formation and repair because the genes can be delivered to a specific anatomic site, and the duration of protein production can be determined by selection of a particular vector. In fact, gene therapy for bone repair may be easier to develop for human use than for other diseases because the requirements for protein production are more variable. In many cases, protein production will only be required until the bone has healed [3Oakes D.A. Lieberman J.R. Osteoinductive applications of regional gene therapy: ex vivo gene transfer.Clin. Orthop. 2000; 379: S101-s112Crossref PubMed Scopus (61) Google Scholar, 4Scaduto A.A. Lieberman J.R. Gene therapy for osteoinduction.Orthop. Clin. North Am. 1999; 30: 625-633Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Lieberman J.R. The effect of regional gene therapy with bone Morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats.J. Bone Joint Surg. Am. 1999; 81: 905-917Crossref PubMed Scopus (494) Google Scholar]. One exception to this would be the treatment of osteoporosis, which may require the development of systemic gene therapy. In the future, gene therapy may be one option in a comprehensive strategy for the tissue engineering of bone. Gene therapy will not be for all patients, as some may be best treated with an autologous bone graft and others, with recombinant proteins and/or bone graft substitutes. However, in some patients gene therapy may offer the best potential for healing [3Oakes D.A. Lieberman J.R. Osteoinductive applications of regional gene therapy: ex vivo gene transfer.Clin. Orthop. 2000; 379: S101-s112Crossref PubMed Scopus (61) Google Scholar, 4Scaduto A.A. Lieberman J.R. Gene therapy for osteoinduction.Orthop. Clin. North Am. 1999; 30: 625-633Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Lieberman J.R. The effect of regional gene therapy with bone Morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats.J. Bone Joint Surg. Am. 1999; 81: 905-917Crossref PubMed Scopus (494) Google Scholar]. Enhanced bone formation is often required to treat bone loss associated with trauma, revision total joint arthroplasty, pseudarthrosis of the spine, and tumor resection. Autologous bone grafts harvested from the pelvis are the current gold standard for treating bone loss problems. However, only a limited amount of autogenous bone graft is available, and there are problems with donor site morbidity [6Colterjohn N.R. Bednar D.A. Procurement of bone graft from the iliac crest. An operative approach with decreased morbidity.J. Bone Joint Surg. Am. 1997; 79: 756-759Crossref PubMed Scopus (72) Google Scholar]. In a large retrospective study, a 10% incidence of minor complications and a 5.8% incidence of major complications were associated with bone graft harvest [7Arrington E.D. Complications of iliac crest bone graft harvesting.Clin. Orthop. 1996; 329: 300-309Crossref PubMed Scopus (1261) Google Scholar]. These limitations have prompted increased interest in the development of alternative bone grafts, such as allografts and demineralized bone matrices. Allograft bone has limited osteoinductive potential, and there is also the possibility of transmission of viral disease. At this time, demineralized bone matrices seem to be most appropriately used as bone graft extenders rather than bone graft substitutes. These materials are osteoconductive and provide a scaffold to promote bone formation, but they have limited osteoinductive activity. Several osteoinductive growth factors that can enhance bone repair have been identified. In particular, the bone morphogenetic proteins (BMPs) have been shown to induce bone formation in a variety of different animal models. Both BMP-2 and BMP-7 (osteogenic protein-1) have been used to heal critical-sized defects in sheep, dogs, and nonhuman primates [8Gerhart T.N. Healing segmental femoral defects in sheep using recombinant human bone morphogenetic protein.Clin. Orthop. 1993; 293: 317-326PubMed Google Scholar, 9Cook S.D. Recombinant human bone morphogenetic protein-7 Induces healing in a canine long-bone segmental defect model.Clin. Orthop. 1994; 301: 302-312PubMed Google Scholar, 10Cook S.D. Effect of recombinant human osteogenic protein-1 on Healing of segmental defects in non-human primates.J. Bone Joint Surg. Am. 1995; 77: 734-750Crossref PubMed Scopus (376) Google Scholar] and to induce the fusion of the spine in canines and nonhuman primates [11Sandhu H.S. Evaluation of rhBMP-2 with an OPLA carrier in a canine posterolateral (transverse process) spinal fusion model.Spine. 1995; 20: 2669-2682Crossref PubMed Scopus (155) Google Scholar, 12Boden S.D. Laparoscopic anterior spinal arthrodesis with rhBMP-2 in a titanium interbody threaded cage.J. Spinal Disord. 1998; 11: 95-101Crossref PubMed Scopus (192) Google Scholar]. Although these preclinical models demonstrate the potential efficacy of different BMPs, these animal models do not truly simulate the clinical situations often associated with bone loss problems. These studies were carried out using young animals with well-vascularized bone and intact soft tissue. In contrast, the healing potential of the bone in a clinical setting may be quite limited because of compromised vascularity, limited bone stock, and abundant fibrous tissue. In addition, bone repair may be inhibited in diabetic, elderly, or nicotine-addicted patients [4Scaduto A.A. Lieberman J.R. Gene therapy for osteoinduction.Orthop. Clin. North Am. 1999; 30: 625-633Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar]. Although recombinant BMP has been used successfully in a variety of animal studies, the success rate in humans is variable. In two clinical trials, large doses of BMP were required to induce adequate bone repair, suggesting that the mode of BMP delivery still requires further optimization [13Friedlaender G.E. Osteogenic protein-1 (bone morphogenetic protein-7) in the treatment of tibial nonunions.J. Bone Joint Surg. Am. 2001; 83-A: S151-S158Google Scholar, 14Boden S.D. The use of rhBMP-2 in interbody fusion cages. Definitive evidence of osteoinduction in humans: a preliminary report.Spine. 2000; 25: 376-381Crossref PubMed Scopus (566) Google Scholar]. It is not known what side effects are associated with such high doses of BMP. Therefore, there is concern that a single exposure to an exogenous growth factor may not induce an adequate osteogenic signal in many clinical situations. Gene therapy has the potential to provide more sustained protein release when necessary and to deliver protein in a more physiologic manner than recombinant proteins [3Oakes D.A. Lieberman J.R. Osteoinductive applications of regional gene therapy: ex vivo gene transfer.Clin. Orthop. 2000; 379: S101-s112Crossref PubMed Scopus (61) Google Scholar, 4Scaduto A.A. Lieberman J.R. Gene therapy for osteoinduction.Orthop. Clin. North Am. 1999; 30: 625-633Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar]. In vivo gene delivery involves directly delivering the gene into a specific anatomic site by transducing local cells. The advantages of this strategy are that it is a relatively simple technique and has the potential for lower cost. The disadvantages are the difficulty in targeting specific cells for transduction and in achieving high transduction efficiency. When using an adenovirus vector there is the additional concern about the development of an immune response as a result of the viral particles injected directly into the anatomic site, which can inhibit transgene expression [15Crystal R.G. Transfer of genes to humans: early lessons and obstacles to success.Science. 1995; 270: 404-410Crossref PubMed Scopus (988) Google Scholar, 16Anderson W.F. Human gene therapy.Nature. 1998; 392: 25-30Crossref PubMed Scopus (66) Google Scholar, 17Wilson J.M. Adenoviruses as gene-delivery vehicles.N. Engl. J. Med. 1996; 334: 1185-1187Crossref PubMed Scopus (433) Google Scholar]. Both viral and nonviral delivery methods have successfully induced bone formation in several animal models. Baltzer et al. used a first-generation adenoviral vector containing the cDNA for BMP-2 to heal critical-sized femoral defects in New Zealand white rabbits [18Baltzer A.W. Genetic enhancement of fracture repair: healing of an experimental segmental defect by adenoviral transfer of the BMP-2 gene.Gene Ther. 2000; 7: 734-739Crossref PubMed Scopus (272) Google Scholar]. In this experiment, healing of the bone defects was noted 7 weeks after viral injection. Ectopic bone formation has also been induced by the injection of a BMP-2-containing adenovirus into the thigh muscles of nude rats. However, bone formation was inhibited in immune competent Sprague-Dawley rats. In addition, an injection of the BMP-2 adenovirus produced less bone when injected into the triceps of immune competent mice compared with the same adenovirus injected into the triceps of nude mice [19Musgrave D.S. Adenovirus-mediated direct gene therapy with bone morphogenetic protein-2 produces bone.Bone. 1999; 24: 541-547Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar]. Okubu et al. noted increased bone formation after the direct injection of the BMP-2-containing adenovirus when the rats were also given cyclophosphamide. Although these experiments resulted in bone formation, they also suggest that the presence of an immune response against the adenoviral vectors limits the biological activity of the secreted protein [20Okubo Y. Osteoinduction by bone morphogenetic protein-2 via adenoviral vector under transient immunosuppression.Biochem. Biophys. Res. Commun. 2000; 267: 382-387Crossref PubMed Scopus (77) Google Scholar]. To avoid potential problems with the adenoviral vectors, nonviral delivery methods are being developed. Plasmid DNA encoding parathyroid hormone (PTH) 1-34 or BMP-4 placed in a gene-activated matrix (GAM) is one example of a nonviral gene therapy strategy that has been successfully used to promote osteogenesis in a rat bone-defect model. In this strategy, the plasmid PTH 1-34 or BMP-4 is incorporated into a collagen matrix and then implanted directly into a bone defect. Local fibroblasts in the area of the defect can then acquire the DNA and become local bioreactors for synthesis of the PTH or BMP-4 protein [21Fang J. Stimulation of new bone formation by direct transfer of osteogenic plasmid genes.Proc. Natl. Acad. Sci. USA. 1996; 93: 5753-5758Crossref PubMed Scopus (446) Google Scholar]. This strategy was successfully used to heal 5-mm defects in rats. A combination of both PTH 1-34 and BMP-4 showed enhanced healing (4 weeks versus 9 weeks) when compared with cDNA for either BMP-4 or PTH 1-34 alone. Bonadio et al. noted 6 weeks of in vivo protein production using a GAM containing PTH 1-34 cDNA to treat a canine tibial defect [22Bonadio J. Localized, direct plasmid gene delivery in vivo: Prolonged therapy results in reproducible tissue regeneration.Nat. Med. 1999; 5: 753-759Crossref PubMed Scopus (592) Google Scholar]. However, insufficient bone was produced to heal a critical-sized tibial defect in this canine model. These results suggest that either the local transfection efficiency is low or that PTH 1-34 has limited osteoinductive potential. Overall, the concept of using a GAM is attractive, and further study is necessary to determine the appropriate cDNA to use in this matrix. In addition to providing an osteoinductive gene to a desired site, the advantage of an ex vivo approach is that it enables surgeons to select specific cells (that is, bone marrow cells, muscle cells, or stem cells) that can participate in osteoinduction. This may enhance the bone repair process as both autocrine and paracrine responses can be elicited with secretion of the desired gene product. In addition, ex vivo methods may be safer than in vivo techniques when working with an adenovirus because no viral particles or DNA complexes are injected into the body. In general, ex vivo techniques have a high efficiency of cell transduction. The major disadvantage of this strategy is the requirement of an extra harvesting step and the increased time and cost of the process. However, it is theoretically feasible to harvest cells from the patient, have a short period of infection and then reimplant the transduced cells at the appropriate anatomic site [23Viggeswarapu M. Adenoviral delivery of LIM mineralization protein-1 induces new-bone formation in vitro and in vivo.J. Bone Joint Surg. Am. 2001; 83-A: 364-376Crossref PubMed Scopus (139) Google Scholar]. Because they are osteogenic, bone marrow cells are obvious candidates for ex vivo gene transfer in bone healing. Furthermore, bone marrow cell harvest is a well-established procedure and methods have been developed to produce a purified population of mesenchymal cells. These progenitor cells can be expanded over 1 billion-fold without losing their potency, and they have demonstrated the ability to induce sufficient bone formation to heal critical-sized defects in both rat and canine models [24Bruder S.P. Jaiswal N. Haynesworth S.E. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation.J. Cell Biochem. 1997; 64: 278-294Crossref PubMed Scopus (1292) Google Scholar, 25Bruder S.P. Bone regeneration by implantation of purified, Culture-expanded human mesenchymal stem cells.J. Orthop. Res. 1998; 16: 155-162Crossref PubMed Scopus (685) Google Scholar, 26Bruder S.P. The effect of implants loaded with autologous Mesenchymal stem cells on the healing of canine segmental bone defects.J. Bone Joint Surg. Am. 1998; 80: 985-996Crossref PubMed Scopus (765) Google Scholar]. Although these results are promising and have the possible advantage of avoiding the use of gene transfer techniques, it remains to be determined whether these cells alone would be able to heal a bone defect in a clinical situation. However, genetically manipulating these cells to produce an osteoinductive protein would further enhance the bone repair response. Lieberman et al. [5Lieberman J.R. The effect of regional gene therapy with bone Morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats.J. Bone Joint Surg. Am. 1999; 81: 905-917Crossref PubMed Scopus (494) Google Scholar] used an ex vivo gene transfer approach to heal critical-size femoral defects in a rat model. Rat bone marrow cells were cultured, infected with a BMP-2-containing adenovirus, and then implanted in the femoral defect site. Histomorphometric analysis revealed a more robust pattern of bone formation in femoral defects treated with BMP-2-producing bone marrow cells as compared with defects treated with recombinant BMP-2 protein placed on a collagen sponge [5Lieberman J.R. The effect of regional gene therapy with bone Morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats.J. Bone Joint Surg. Am. 1999; 81: 905-917Crossref PubMed Scopus (494) Google Scholar] (Fig. 1). Although biomechanical testing of the healed femurs did not show any differences between these groups with respect to energy-to-failure or torque-to-failure, this may be due to a lack of sensitivity of the rat model. The more robust bone formed by cells transduced with BMP-2 may be the result of a more continuous or physiological release of BMP-2 protein compared with the kinetics of BMP release from a collagen sponge. It is hypothesized that a more vigorous osteogenic activity is seen because both paracrine and autocrine responses occur, that is, the transduced bone marrow cells can respond to the BMP-2 protein that they are secreting. A variety of other cell types have been used to induce bone formation. BMP-7-transfected rat skin fibroblasts have been shown to heal critically-sized calvarial defects in Lewis rats. Skin fibroblasts are an attractive cellular delivery vehicle because they are easy to harvest and are readily available in all patients [27Krebsbach P.H. Gene therapy-directed osteogenesis: BMP-7-transduced human fibroblasts form bone in vivo.Hum. Gene Ther. 2000; 11: 1201-1210Crossref PubMed Scopus (218) Google Scholar]. There is one caveat when using fibroblasts to deliver osteoinductive signals: fibrous tissue can actually inhibit bone formation and is found in fracture non-union sites. Genetically engineered muscle-derived cells also have osteogenic potential [28Lee J.Y. Effect of bone morphogenetic protein-2-expressing Musclederived cells on healing of critical-sized bone defects in mice.J. Bone Joint Surg. Am. 2001; 83-A: 1032-1039Crossref PubMed Scopus (196) Google Scholar]. Lee et al. demonstrated that muscle cells transduced with a BMP-2-containing adenovirus were able to heal calvarial defects in SCID mice. These investigators also noted that a small percentage of muscle-derived cells implanted in calvarial defects actually differentiated into osteoblasts in vivo. Both muscle cells and bone marrow cells have a similar advantage in that they not only deliver an osteoinductive signal, but also contain osteogenic precursor cells. Stem cells harvested from fat also have potential for use in tissue engineering. Zuk et al. demonstrated that human adipose tissue obtained at the time of liposuction contains a fibroblast-like population of cells that can behave like mesenchymal stem cells [29Zuk P.A. Multilineage cells from human adipose tissue: implications for cell-based therapies.Tissue Eng. 2001; 7: 211-228Crossref PubMed Scopus (6524) Google Scholar]. When these lipoaspirate cells are grown in the appropriate medium, they can differentiate into bone, cartilage, fat, or muscle. Although the full osteogenic potential of these cells is still being analyzed, it is known that these stem cells can form bone in vivo when they are transduced with a BMP-2-containing adenovirus and implanted into a SCID mouse muscle pouch [30Dragoo J. Bone induction by genetically manipulated stem cells derived from fat.J. Bone Min. Res. 2001; 16: 5500Google Scholar]. Further work with these types of cells is being carried out (J.R.L.'s laboratory). Spinal fusion is one of the most commonly performed orthopedic surgical procedures in the United States, with over 980,000 performed every year. Approximately one-third of these procedures requires a bone graft [31Deutsche, Banc, A. B. 2001, Estimates and Company Information.Google Scholar]. In general, autogenous bone graft is a successful method for enhancing spine fusion, but non-union rates range from 5% to 35%. A variety of factors can influence the success of the spine fusion including mechanical instability of the spine, the stability of fixation, the quality of bone, the health of surrounding soft tissue, the type of bone graft used, and the concurrent use of medications and drugs such as nicotine. Concerns regarding the osteoinductive potential of various exogenous growth factors have lead investigators to explore the potential of gene therapy to induce fusion of the spine. Bone marrow cells transduced with a DNA plasmid containing the cDNA for LMP-1 (LIM mineralization protein-1) were assessed in a single intertransverse process lumbar spine fusion model [32Boden S.D. Lumbar spine fusion by local gene therapy with a CDNA encoding a novel osteoinductive protein (LMP-1).Spine. 1998; 23: 2486-2492Crossref PubMed Scopus (210) Google Scholar]. LMP-1 is a transcription factor that can promote endogenous expression of BMPs. Control animals were treated with a reverse copy of the cDNA that does not express LMP-1. Successful spine fusion was noted in all 11 sites treated with the LMP-1 transduced bone marrow cells. The spine fusion sites treated with control bone marrow cells alone did not fuse. Buffy-coat cells have also been engineered to promote bone formation using an LMP-1-containing adenovirus. Rabbits treated with these transduced peripheral blood-derived cells develop solid spine fusions [23Viggeswarapu M. Adenoviral delivery of LIM mineralization protein-1 induces new-bone formation in vitro and in vivo.J. Bone Joint Surg. Am. 2001; 83-A: 364-376Crossref PubMed Scopus (139) Google Scholar]. Peripheral blood cells are appealing as a cellular gene delivery vehicle because these cells are easier to harvest than bone marrow cells. However, it is unlikely that cells derived from peripheral blood have the same autocrine effects that are seen with transduced bone marrow cells. The results with LMP-1 in these preclinical models are impressive and could be adapted to treat other bone repair problems. However, no mice mutant or transgenic for LMP-1 have been reported and little is known of the possible side effects of LMP-1 overexpression. Wang et al. used a recombinant adenovirus containing the BMP-2 cDNA to induce an intertransverse lumbar spinal fusion in rats [33Wang J. Intertransverse process fusion in Lewis rats using bone marrow cells infected with a BMP-2 containing adenovirus.Trans. Orthop. Res. Soc. 2002; 25: 327Google Scholar]. Autologous bone marrow cells were harvested from Lewis rats, expanded in tissue culture, and then transduced with the BMP-2-containing adenovirus. These transduced cells were then loaded onto either guanidine-extracted demineralized bone matrix or collagen sponges and then implanted into the appropriate fusion site. The BMP-2-producing bone marrow cells induced spine fusion in all 15 spines that were treated. Bone marrow cells alone did not induce spine fusion. The results of these gene transfer preclinical studies are quite exciting and clearly demonstrate the adaptability of gene therapy for human use. The ability to deliver growth factors via gene therapy may lead to the development of less invasive operative techniques such as laparoscopic spine fusion. Such a development carries the potential for reduced operative morbidity, shortened time to wound healing, and diminished costs. The goal is to develop gene therapy as a part of an overall comprehensive tissue engineering strategy to enhance bone repair. Clearly gene therapy is not necessary for all patients, but it does have the potential to enable orthopedic surgeons to treat difficult bone repair problems that can not be handled successfully with our present technology. Cartilage is an avascular, aneural, alymphatic tissue that overlies the ends of the long bones and helps ensure the smooth, almost frictionless movement of joints. Approximately 95% of the tissue consists of a highly hydrated extracellular matrix with a unique composition and microarchitecture, enabling it to bear load effectively. This matrix is synthesized and maintained by the sparse population of articular chondrocytes embedded within it. The poor ability of cartilage to heal has been recognized since the time of Hippocrates. Indeed, it mounts almost no reparative response to traumatic injuries that fail to penetrate the subchondral bone underlying the cartilage. Such lesions are often painful and debilitating. They may also progress to osteoarthritis, a disease characterized by focal erosion of the articular cartilage. Deeper injuries that engage the subchondral bone are associated with the entry of blood and marrow contents into the lesion. Progenitor cells within these infiltrates form fibrocartilagenous repair tissue which, although inferior to native articular cartilage, can provide symptomatic improvement for extended periods of time before deteriorating because of defective load-bearing properties (Fig. 2). Cartilage injuries are common, symptomatic, and difficult to repair by surgical means alone. Therefore, there is great interest in improving outcomes through harnessing the biology of the system [34Athanasiou K.A. Basic science of articular cartilage repair.Clin. Sports Med. 2001; 20: 223-247Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar]. Among the more commonly used procedures is abrasion arthroplasty, in which the subchondral plate is deliberately violated to encourage the influx of marrow elements. Less commonly, autologous cartilage, harvested from the margins of the joint that do not bear load, is used as a source of autologous chondrocytes in partial-thickness defects. After expansion in culture, the cells are surgically transplanted to the injured site, where they effect a repair. This procedure is largely restricted to knee joints and remains controversial. Problems common to all methods of attempted cartilage repair are fusion of the repair cartilage with the surrounding endogenous undamaged cartilage and the formation of a uniform cartilaginous surface. The repair of full-thickness lesions is also challenged by the need for reconstitution of the underlying subchondral bone. Biological repair is further challenged by the need to recapitulate the exquisite molecular microarchitecture of cartilage. The mechanically demanding environment of articular cartilage is a harsh discriminator of inadequate repair tissue. Cartilage healing presents far greater challenges than bone healing to gene therapy. Unlike bone, cartilage lacks a robust, native healing response that could serve as the basis of a gene therapy. Furthermore, there is no clinical experience of cartilage repair using recombinant proteins upon which to draw. Nevertheless, gene therapy approaches to cartilage repair are encouraged by the ability of various gene products to enhance chondrogenesis [2Evans C.H. Using gene therapy to protect and restore cartilage.Clin. Orthop. 2000; 379: S214-s219Crossref Scopus (89) Google Scholar]. Examples include growth factors [35O'Connor W.J. The use of growth factors in cartilage repair.Orthop. Clin. North Am. 2000; 31: 399-410Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar] such as insulin-like growth factor-1 (IGF-1), transforming growth factor-β (TGF-β), fibroblast growth factors, and various members of the BMP family, as well as transcription factors such as SOX-9 [36Uusitalo H. Accelerated up-regulation of L-Sox5, Sox6, and Sox9 by BMP-2 gene transfer during murine fracture healing.J. Bone Miner. Res. 2001; 16: 1837-1845Crossref PubMed Scopus (64) Google Scholar], certain signaling molecules such as SMADs [37Fujii M. Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation.Mol. Biol. Cell. 1999; 10: 3801-3813Crossref PubMed Scopus (370) Google Scholar], and molecules that inhibit apoptosis such as BCL-2 [38Feng L. Evidence of a direct role for Bcl-2 in the regulation of articular chondrocyte apoptosis under the conditions of serum withdrawal and retinoic acid treatment.J. Cell. Biochem. 1998; 71: 302-309Crossref PubMed Scopus (43) Google Scholar]. Growth factors, however, are difficult to administer exogenously to sites of cartilage injury in a sustained and therapeutically useful fashion, whereas the intracellular mediators have the additional problem of needing to enter responsive cells. Gene delivery strategies promise to overcome these limitations. One issue to be addressed in implementing such strategies is the choice of target tissue. Synovium is the most accessible tissue in the joint. It is readily transduced by a wide variety of different vectors and has already served as the site of gene transfer in a clinical study related to the gene therapy of arthritis [39Evans C.H. Clinical trial to assess the safety, feasibility, and efficacy of transferring a potentially anti-arthritic cytokine gene to human joints with rheu