In conclusion, transplantation of genetically modified cells is a promising tool for the treatment of articular cartilage defects such as resulting from trauma or osteoarthritis. In the past decades, considerable progress has been made to lay the scientific foundation for the use of modified cells. In particular, articular chondrocytes and, more recently, MSCs have been genetically modified using nonviral or viral methods. When used in model systems of cell transplantation, structural improvements occurred in the new repair tissue. Moreover, in small animal models, transplantation of genetically modified cells has yielded significant structural improvements of the repair tissue. Lately, preclinical animal models have been applied to study the effects of overexpression of therapeutic genes (118
). However, to realize the full potential of these approaches in the clinics, the following issues have to be addressed:
1. the proof of the safety of this approach in preclinical and clinical trials
2. the choice of the ideal gene vector system
3. the choice of the optimal therapeutic gene(s)
4. the delivery without (eg, for large osteoarthritic lesions) or with a supportive biomaterial (for focal lesions),
5. the establishment of the optimal cell dose.
For traumatic cartilage defects, single or combined overexpression of therapeutic genes, including growth and transcription factors, is feasible and results in structural improvements. Significant data have been collected in animal models of chondral and osteochondral defects. For osteoarthritis, transplantation approaches using genetically engineered cells have been less developed. Ex vivo models have already convincingly shown that genetically modified chondrocytes adhere to human osteoarthritic cartilage explants, and that inhibition of matrix degradation is possible. Animal studies using genetically modified cells for the treatment of osteoarthritis have mainly relied on the intra-articular injection of these cells, making a specific targeting of osteoarthritic areas difficult. Moreover, it remains to be seen whether areas of cartilage loss may be repopulated by such approaches, considering that the nature of the osteoarthritic loss of articular cartilage is unknown and since a simple injection of cells alone does not address other factors involved in the pathogenesis of osteoarthritis such as mechanical overload induced by axial malalignment.
Several gene transfer systems are capable of successfully modifying articular chondrocytes and MSCs, the two most promising cell types for transplantation approaches. However, each of the individual vector system has intrinsic limitations with regard to their efficacy (nonviral systems) and safety (eg, adenoviral systems) for human clinical applications. Most experts predict that initial human gene therapy trials based on the transplantation of genetically modified cells for articular cartilage repair will be conducted using rAAV vectors. The development of novel (third generation) adenoviral vectors may be another option, although significantly less immunogenic responses might be elicited when using rAAV. Vectors showing a capacity for site-specific integration have been designed for the purpose of achieving long-term gene expression, such constructs have not been tested yet in articular cartilage repair settings.
Supportive biomaterials may be used to deliver the genetically modified cells to focal articular cartilage defects, similar to the current clinical practice of treating cartilage lesions with matrix-assisted autologous chondrocyte transplantation, a technique in which the articular chondrocytes are attached to different types of scaffolds, eg, based on collagen types I/III, hyaluronic acid, polyglactin/poly-p-dioxanon, and fibrin-hyaluronan (141
). Such a delivery of genetically modified chondrocytes in conjunction with biomaterials may be advantageous compared to cell delivery alone, as it allows for a spatially controlled application of the modified cells to enhance chondrogenesis. An overexpression of therapeutic genes in such tissue-engineered constructs offers the important advantage of a complete filling of the defect with a construct actively supporting chondrogenesis. Although possible side-effects intrinsic to the biomaterial on transgene expression have to be ruled out in experimental studies, the current clinical consensus on the broad advantages of matrix-assisted autologous chondrocyte transplantation suggest that genetically modified cells may rather be delivered in supportive biomaterials to focal cartilage defects. For the treatment of osteoarthritis, however, no conclusions can be drawn at the moment because of the paucity of experimental preclinical and clinical in vivo data.
Possible side-effects have also to be considered. While intra-articular injection of an BMP-2 adenoviral vector resulted in the formation of osteophytes and spread of the vector DNA to the liver, lung, and spleen, no such effects were observed when an ex vivo approach was selected injecting modified cells overexpressing BMP-2 instead (31
). In fact, it appears that the therapeutic protein may exert its role in close proximity to the defect, as judged by the absence of elevated protein levels in the synovial fluid and peripheral blood and by the detection of only a few days of marker gene activity in cells adjacent to the site of the surgical approach.
The selection of therapeutic genes to specifically address individual parameters of chondrogenesis will continue to be a challenge. Progress may come from more insights into the regulation of chondrogenesis, eg, using genomic and proteomic profiles.
Open questions remain also in terms of finding the optimal cells dose. In the clinical protocols of autologous chondrocyte transplantation, chondrocytes are usually transplanted at densities of about 1
. When genetically modified articular chondrocytes overexpressing IGF-I were applied in a lapine model, about 75
were applied, a 75-fold higher dose (10
). In the clinical gene therapy study by Chris Evans, 1
(low dose), 1.5-5
(intermediate dose), and 6.5-10
(high dose) autologous synovial fibroblasts transduced with a retroviral vector encoding for an IL-1Ra cDNA were injected into metacarpophalangeal joints (2
). Because of its similarity with the protocol currently used for autologous chondrocyte transplantation, it is likely that a dose of 1
may be elected as a starting point of evaluation when genetically modified chondrocytes will be transplanted to focal cartilage defects in patients.
Nevertheless, nearly three decades of experimental and clinical research to advance the principles of articular chondrocyte transplantation have yielded significant technical improvements of clinical articular chondrocyte transplantation, such as chondrocyte application in highly specialized scaffolds for optimal cell attachment and distribution in vivo (141
). Many of the procedures required to constitute such ex vivo gene transfer approaches are already available for the orthopaedic surgeon. For example, cell (chondrocyte) isolation and in vitro cell culture – essential steps of such ex vivo protocols – are already in place. Moreover, the problem of the loss of the chondrocytic phenotype – as observed after prolonged monolayer culture (142
), has been sufficiently addressed. These autologous chondrocytes could easily be genetically modified ex vivo – as shown, for example, for hematopoietic stem cells (143
) – and could be implanted at the site of the cartilage defect, where a high concentration of the chondrogenic agent is needed. For the transplantation of MSC it remains to be seen whether the terminal differentiation of chondrocytes when they become hypertrophic – as observed in vitro – represents indeed a problem when implanted into articular cartilage defects in vivo (144
Despite these encouraging data, application of genetically modified cells to treat cartilage defects is still not on the horizon. Issues that need to be addressed include the duration of transgene expression, extended studies with both nonviral and viral transfer systems in preclinical models of focal and osteoarthritic cartilage defects, the continuing elucidation of the benefit of using ex vivo genetically modified cells vs direct gene transfer approaches, and the ongoing identification of optimal therapeutic factors. Future studies will also have to prove the safety of such approaches, since both traumatic defects and osteoarthritis are non-lethal diseases.
A key challenge in combining both experimental (in the case of genetic modification) and clinical knowledge (in the case of articular chondrocytes transplantation) and, as a result, to translate these progresses into clinical medicine is finally to establish a safe and efficient production line, including vector manufacturing, cell isolation, and genetic modification that meets all regulatory requirements.
Ultimately, the clinical potential for genetically modified articular chondrocytes and MSCs for the treatment of articular cartilage defect is likely to be realized by advances in the following areas: 1) development of a safe, highly efficient gene delivery system with sustained duration of transgene expression, 2) identification of optimal therapeutic gene(s), 3) combination of genetically modified articular chondrocytes and/or MSCs with scaffolds that better support chondrogenesis in vivo. After safe techniques for generating these genetically engineered cells are available, such cells may eventually provide new avenues for improved cell-based therapies for articular cartilage repair. This, in turn, may provide a crucial step toward the unanswered question of articular cartilage regeneration.