In recent decades, the search for new ways to promote bone healing has increasingly turned to biology .In general, there are two routes through which the body forms bone: endochondral and intramembranous . The former process involves the local differentiation of progenitor cells into chondrocytes that lay down a cartilaginous matrix. The cartilage is then replaced by bone through a process known as endochondral ossification, during which the cartilaginous matrix is degraded, blood vessels invade the cartilage and chondrocytes are replaced by osteoblasts. The intramembranous formation of bone involves the differentiation of progenitor cells directly into osteoblasts without a cartilaginous intermediate. Both the endochondral and intramembranous routes lead to the formation of immature, woven bone that undergoes remodeling into mature, lamellar bone.
Study of osteogenesis has identified a number of growth factors with the ability to stimulate one or more of the steps involved in endochondral and intramembranous bone formation. The best characterised of these factors are the bone morphogenetic proteins (BMPs). Several members of this large family, including BMP-2, BMP-4, BMP-6, BMP-7 and BMP-9, are able to induce the formation of bone when implanted experimentally into the muscles of laboratory animals . Recombinant, human BMP-2 and BMP-7 are available clinically as the active components of the products Infuse® and OP-1® (osteogenic protein-1), respectively. Despite the powerful osteogenic properties of these proteins in laboratory animals, their clinical performance is mixed . For instance, they appear to be much more effective for spinal fusion than for long-bone healing, and need to be administered at extremely high doses of several milligrammes – many orders of magnitude greater than the levels at which they occur naturally in bone. The use of such high doses not only raises safety concerns, but also enormously increases the cost. In the USA, for example, a single dose of Infuse® can cost around $5000. There is considerable optimism that gene-therapy approaches can be harnessed to improve the effectiveness of osteogenic factors, such as BMP-2, while lowering both costs and the potential for side effects.
As its name suggests, gene therapy involves the transfer of genes, or more usually cDNA (complementary DNA), to patients for therapeutic purposes. Gene therapy was originally developed as a means of curing genetic diseases, but in recent years its potential use in treating nongenetic disorders has become increasingly appreciated and a range of nonmendelian diseases, including various cancers , arthritis and Parkinson disease have been the subject of clinical trials. In the context of bone healing, the aim is to deliver to the fracture site cDNAs encoding osteogenic proteins, such as BMPs. Successful gene delivery and expression leads to the continuous, local, focal synthesis of the osteogenic protein, which is likely to have undergone authentic post-translational modification and, unlike the recombinant product, to be uncontaminated by inactive, misfolded products that can trigger immune responses. Safety is further enhanced by gene transfer because the local production of osteogenic proteins at approximately physiological concentrations is less likely than the bolus application of a large amount of recombinant protein to lead to the systemic spread of these proteins to ectopic sites.
There are a large number of growth factors known to stimulate one or more of the processes involved in osteogenesis and bone healing. Most of these either stimulate the differentiation of progenitor cells into chondrocytes or osteoblasts, or enhance the bone-forming activities of mature osteoblasts. Because bone is highly vascularised, vascular endothelial growth factor (VEGF) and other angiogenic factors are also important, especially for intramembranous bone formation; chondrogenesis, the first step in endochondral ossification, does not require a blood supply. The importance of angiogenesis in bone healing has been nicely demonstrated by Peng and colleagues who used gene transfer to show that VEGF enhanced, and a VEGF antagonist (sFlt1) inhibited, repair of cranial defects in mice. Because individual growth factors act at different stages of osteogenesis, combinations of different factors should promote bone healing more potently than either factor alone. This has been confirmed in animal models using gene delivery of BMP-2 and BMP-7; BMP-4 and VEGF ; BMP-4 and transforming growth factor-β (TGF-β) .
Oncoretroviruses (usually referred to as retroviruses) and lentiviruses (also members of the retrovirus family) are RNA viruses that integrate their genetic material into the chromosomal DNA of the cells they infect, thus providing the potential for long-term transgene expression. Although this is highly desirable when treating a genetic disease for the lifetime of the patient, it is probably not necessary for healing a fracture, which might be expected to take 6–8 weeks. The continued expression of a transgene beyond this time would probably be counterproductive. Another disadvantage of integrating retrovirus and lentivirus vectors is their potential for causing insertional mutagenesis. This has led to leukaemia in children being treated for severe combined immunodeficiency disease (SCID). Retrovirus, but not lentivirus, has the additional disadvantage of requiring host-cell division for successful transduction . Lentivirus vectors are being engineered to prevent integration , but it still remains doubtful that such viruses could be used in humans to treat a non-life-threatening condition, such as an osseous nonunion, especially as the most commonly used lentiviral vectors come with the ‘psychological baggage’ of having been derived from HIV.
Adenovirus and recombinant (unlike wild-type) adenoassociated virus (AAV) are DNA viruses whose genomes do not integrate with high frequency into host-cell DNA. Nevertheless, they are capable of maintaining transgene expression for lengths of time compatible with the needs of bone healing. Among the advantages of adenovirus is the relative ease with which recombinant vector can be produced at high titre, and its high infectivity towards many cell types, regardless of whether they are dividing or not . they have been the most widely used vectors for studies of gene therapy for bone healing. A perceived disadvantage of adenovirus is its high antigenicity . Most of the population have circulating, neutralising antibodies to adenovirus serotype 5, the serotype most commonly used for gene transfer, which might reduce the effectiveness of adenovirus-based gene therapy. Moreover, the inflammatory response to adenovirus may be detrimental to healing.