Making gene therapy for osteosarcoma a reality

Osteosarcoma is the most common primary malignancy of bone and the second most common malignancy in adolescents after lymphoma [1]. Prior to the advent of multiagent chemotherapy, long-term survival was a dismal 20% [2] and surgical resection most often meant amputation of the affected limb. Current treatment for high-grade osteosarcoma now combines surgery with neoadjuvant and adjuvant chemotherapy regimens [3]. Long-term survival has improved to 60–75% [4] and advances in diagnostic imaging, chemotherapy and surgical technique have made limb-salvage a reality [4]. However, despite these improvements, 30% of patients remain resistant to chemotherapy and most of these eventually succumb to metastases [2]. A great deal of current research is therefore focused on identifying and characterizing additional therapeutic targets for osteosarcoma. The aim of such targeted treatments is to reduce toxicity while maintaining or enhancing efficacy of current chemotherapeutic agents.

Gene therapy is one such targeted technique that is being investigated for application to osteosarcoma. It aims to reduce tumor proliferation and metastasis by specifically targeting the genetic aberrations responsible for osteosarcoma progression. There are a number of methods for achieving altered gene expression in targeted cells and these include the introduction of plasmid DNA, ribozymes, DNAzymes, antisense molecules, decoy oligodeoxynucleotides and siRNA [3]. Ribozymes, DNAzymes, antisense molecules and siRNA all interrupt gene expression at the level of RNA, while decoy oligodeoxynucleotides prevent interaction between transcription factors and the promoter region of a gene [5]. Gene therapy may be broadly classified according to the method used for transferring these nucleic acid constructs into diseased cells: viral versus nonviral gene therapy. The applicable viral vectors include retroviruses, adenoviruses, lentiviruses and herpes viruses, while nonviral methods have largely been centered on liposomal complexes and nanoparticles [3].

Understanding the molecular pathogenesis of osteosarcoma identifies potential targets for gene therapy

Osteosarcoma is an aggressive cancer thought to arise from mesenchymal stem cells [6]. Both environmental and genetic factors contribute to its pathogenesis. Exposure to ionizing radiation [7–9], methylcholanthrene [10], beryllium oxide [11] and zinc beryllium silicate [12] are all known to be associated with osteosarcoma development. Genetic damage by these agents leads to dysfunction of molecular signaling pathways and unchecked cell proliferation, invasion and metastasis. An understanding of these key molecular events is critical for the identification of novel therapeutic targets, and gene therapy aims to exploit one or more of these targets to reverse tumorigenesis.

As is the case for other cancers, mutations in tumor-suppressor genes contribute to osteosarcoma formation. The retinoblastoma (Rb) and p53 proteins have been well documented for their roles in osteosarcoma pathogenesis. Loss of the Rb gene underlies an increased familial risk of osteosarcoma [13], while also conferring resistance to methotrexate [14]. An inherited mutation to p53 underlies Li Fraumeni syndrome and the development of multiple cancers. Rb and p53 have been examined as possible targets for gene therapy in osteosarcoma. The wild-type Rb gene was introduced by way of adenoviral vector into MG-63, K-HOS, SJSA-1 and SaOS-2 cell lines, resulting in reduced osteosarcoma cell proliferation [3]. Ternovoi et al. reviewed the prospects for p53 gene therapy and highlighted both the insufficient efficacy of adenoviral vectors and the low levels of apoptosis induced by wild-type p53 when used as a sole agent [15]. Since then, Oshima et al. have successfully exploited p53 analogues, p73 and p63, in 11 osteosarcoma cell lines using adenoviral vectors [16]. p63γ caused the greatest effect on osteosarcoma apoptosis both in vitro and in vivo.

Dysfunctional transcription may contribute to osteosarcoma tumorigenesis. The activator protein-1 complex (AP-1) product is a regulator of transcription that controls proliferation, differentiation and bone cell metabolism. The subunits of AP-1, Fos and Jun, are upregulated in high-grade osteosarcomas compared with benign osteoblastic lesions and low-grade osteosarcomas [17,18]. Fos overexpression causes the malignant transformation of osteoclasts in transgenic mice [19], while Fos and Jun double-transgenic mice are found to develop osteosarcomas with a higher frequency than c-Fos-only transgenic mice [20]. Dz13 is a DNAzyme that cleaves c-Jun mRNA and is capable of inhibiting cancer cell growth in vitro. Dz13, when encapsulated in chitosan nanoparticles, is efficacious against osteosarcoma both in vitro and in vivo when combined with doxorubicin therapy [21]. Nonencapsulated Dz13 is not efficacious and, thus, highlights the importance of optimizing delivery vectors for gene therapy.

Our understanding of the metastatic cascade for osteosarcoma has also enabled gene therapy to also be applied to the mediators of cell adhesion, migration and invasion. The ezrin protein plays key roles in cell–cell interactions and signal transduction. It is also involved in linking actin filaments with the cell membrane receptor CD44 [22]. Overexpression of ezrin is associated with metastatic progression [23]. In a pediatric cohort of osteosarcoma patients, it was related to reduced disease-free intervals. The risk of relapse was 80% greater for children with high ezrin-expressing osteosarcomas compared with low ezrin-expressing tumors. As an applied target for gene therapy, downregulation of ezrin in a mouse model of osteosarcoma resulted in reduced rates of pulmonary metastasis [24]. This result is particularly encouraging as pulmonary metastatic disease represents the major cause of death and hence major challenge when developing strategies against osteosarcoma.

The urokinase plasminogen activator (uPA) system is also critical to the regulation of osteosarcoma invasion and metastasis. The uPA ligand binds to its receptor uPAR, leading to activation and cleavage of plasminogen to plasmin [25]. Plasmin degrades the extracellular matrix and further facilitates tumor invasion by activating matrix metalloproteinases [26]. There is an inverse relationship between uPA expression and survival for osteosarcoma [27], a relationship which makes uPA an attractive target for gene therapy. Using antisense clones with an in vivo model of osteosarcoma, our laboratory has been able to demonstrate a therapeutic effect for uPAR downregulation, which inhibited both osteosarcoma growth and pulmonary metastases [28]. More recently, a DNAzyme against uPAR has also shown similar effects in vitro and in vivo[29].

Finally we have extended a gene therapy approach to the novel anti-osteosarcoma agent pigment epithelium-derived factor (PEDF). PEDF has both direct and indirect effects on osteosarcoma progression. It induces apoptosis and inhibits cell cycling of osteosarcoma cells, promotes cell adhesion and limits invasion. PEDF’s indirect effects rely on its ability to specifically target tumor vasculature for destruction [30]. These effects have all been confirmed in vitro using SaOS-2 osteosarcoma cells transfected with a PEDF plasmid (pPEDF). pPEDF also inhibited both primary osteosarcomas and pulmonary metastases when applied to an in vivo orthotopic model. In addition, tumor microvessel density and osteolysis were also reduced [31]. Even in pre-established tumors, PEDF gene delivery has resulted in reduced tumor growth in combination with doxorubicin therapy [32].

Viral vector versus nonviral-mediated gene therapy

Gene therapy relies on either a viral vector or nonviral vehicle for the introduction of therapeutic genes or gene-interfering agents into diseased cells. Although a high transfection efficiency achieved with viral-vectors is beneficial for testing genes in the laboratory setting, alternative transfection methods have been sought for patient use, given the significant risk of side effects [33] and even death [34–36] when using viral vectors clinically. There is greater immunoreactivity associated with viral vectors and also the potential to exchange genetic material with other viruses that may be present in human tissues [37].

Retroviruses, adenoviruses, lentiviruses and herpes viruses have been used for gene therapy [3]. Retroviral vectors are potent in their ability to maintain long-term gene therapy; however, there is major risk for malignant transformation of the host cell genotype. Retrovirus transfection was used in the hope of curing severe combined immunodeficiency in a number of children; however, five children developed leukemia. This was due to incorporation of the transgene next to an oncogene [38,39].

Adenoviruses are being used increasingly for gene therapy work because they do not incorporate into the host genome and therefore pose less risk for transformation. Furthermore, adenovirus transfection acts as a form of virotherapy by replicating itself within and then lyzing the tumor cell [40]. One of the challenges for adenovirus tranfection is targeting the virus to the tumor cell. Normally the virus binds to cells expressing the Coxsackie adenovirus receptor. However, Coxsackie adenovirus receptor expression is very limited on tumor cells so bispecific molecules have been developed that bind to the virus with one end and a tumor-specific surface molecule via the other end. This is known as ‘transduction targeting’ [41]. Osteosarcoma has been targeted in this way by using the bispecific molecule 425-s11, which binds to EGF receptor – a frequently expressed receptor in osteosarcoma [42].

Another method of increasing tumor specificity is to employ ‘transcription targeting’. This involves incorporating a tumor-specific promoter sequence into the transgene so that only tumor cells will transcribe and overexpress the gene. The osteocalcin promoter, normally found in osteoblasts, has been used with effect in osteosarcoma. Higher tumor specificity can be achieved when both methods, transduction and transcription targeting, are employed simultaneously [43].

Virotherapy can also be focused on tumor cells in a similar fashion whereby tumor-specific promoters are linked to viral replication genes in a conditional manner. The result is that the virus can infect all cells but only tumor cells utilizing that particular promoter region will allow the virus to replicate and then lyse the cell. This clearly limits damage to healthy cells. The hTERT promoter associated with osteosarcoma telomerase expression may be used in this way [44].

The previously described limitations and safety issues related to viral gene therapy have led to a focus on nonviral vectors. These are divided into two main categories, liposomes and polymers. Such vectors are less immunoreactive than viruses and lack the potential for oncogenic transformation but do not self-replicate [45]. We have recently reviewed the use of nonviral methods for gene delivery [45] and published results for a chitosan hydrogel system for osteosarcoma therapy [32]. The chitosan hydrogel enabled the sustained local release of pPEDF to orthotopically induced osteosarcoma. Primary tumor growth was inhibited significantly and combining gene therapy with conventional chemotherapy further heightened this therapeutic effect.

Animal models of pulmonary metastatic osteosarcoma have also been used to test the efficacy of nonviral gene therapy in this difficult problem. These models are clinically relevant given that 25–50% of patients who present with initially nonmetastatic disease go on to develop metastases [46], primarily pulmonary metastases. Worth et al. initially delivered an adenoviral vector containing the IL-12 gene intranasally in a nude mouse model of metastatic osteosarcoma [47]. IL-12 is antitumorigenic via its effects on natural killer and T cells [47]. This unique drug delivery system caused significant inhibition of pulmonary metastases; however, it was later abandoned by the authors for a nonviral vehicle, namely polyethyleneimine (PEI). Densmore et al.[48] and Jia et al.[49,50] have used this polycationic DNA carrier to deliver plasmid p53 and IL-12, respectively, to pulmonary metastases as an aerosol. Significant reductions in size and number of pulmonary metastases were demonstrated with these treatments. Intranasal PEI–IL-12 has most recently been combined with intraperitoneal ifosfamide [51]. Increased efficacy was demonstrated with the combined therapy, highlighting a potential application for PEI-IL-12 as a chemotherapy dose-reducing agent.

Conclusion

Gene therapy for osteosarcoma is gaining additional momentum with the introduction of delivery vehicles such as hydrogels [52] and modified adenoviral vectors. With safer gene-delivery vehicles being produced, nucleic acid-based therapeutic constructs are becoming more amenable to clinical application. Targets such as ezrin [24], uPAR [28,29] and PEDF [30–32] have been shown to be efficacious for osteosarcoma in animal models. A combination of gene therapy and conventional therapies may yet achieve more complete disease control and warrants further evaluation for osteosarcoma.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.