Faculty Bibliography

STUDY DESIGN: Rat spinal fusion model. OBJECTIVE: This study aimed to compare the efficacy of lentiviral gene therapy, and adenoviral gene therapy in inducing spinal fusion in an immune competent rat spinal fusion model. SUMMARY OF BACKGROUND DATA: Recombinant bone morphogenetic proteins (BMPs) have also been used for spinal fusion successfully in clinical trials. However, large doses of BMPs are required to induce adequate bone repair. Hence, regional gene therapy may be a more efficient method to deliver proteins to a specific anatomic site. Recently, lentiviral vectors based on human immunodeficiency virus have been developed for gene therapy. However, lentiviral gene therapy for spinal fusion has not been compared with adenoviral gene therapy. METHODS: Lewis rats were divided into 7 groups. group I, II, III, and IV rats were implanted with a collagen sponge containing rat bone marrow cells (RBMCs) transfected with Lenti-BMP-2, Adeno-BMP-2, Lenti-GFP, Adeno-LacZ, respectively. Group V, VI, and VII rats were implanted with a collagen sponge containing recombinant BMP-2, a collagen sponge containing untransfected RBMCs, and a collagen sponge alone, respectively. The rats were assessed at 4, 6, and 8 weeks after implantation. After sacrificing the rats, their spines were explanted and assessed by manual palpation, high-resolution microcomputed tomography, and histologic analysis. RESULTS: Spinal fusion was observed in all animals in group I, II, and V rats at 8 weeks. None of the rats in groups III, IV, VI, and VII showed spinal fusion. The volumes of the new bone in the area between the L4 and L5 transverse processes were greater in group I rats than in group II, and V rats with a significant difference. CONCLUSION: BMP-2-producing RBMCs developed using lentiviral gene transfer induced more abundant bone within the fusion mass than the RBMCs transduced via adenoviral gene transfer and recombinant protein therapy.

The objective of this study was to compare the efficacy of adenoviral and lentiviral regional gene therapy in a rat critical sized femoral defect model. The healing rates and quality of bone repair of femoral defects treated with syngeneic rat bone marrow cells (RBMCs) transduced with either lentiviral vector (Group I) or adenoviral vector (Group II) expressing bone morphogenetic protein-2 (BMP-2) gene were assessed. RBMCs transduced with the adenoviral vectors produced more than 3 times greater (p<0.001) BMP-2 when compared to RBMCs transduced with lentiviral vectors in an in vitro evaluation. Serial bioluminescent imaging demonstrated short duration luciferase expression (less than 3 weeks) in defects treated with RBMCs co-transduced with two adenoviral vectors (Group IV; adenovirus expressing BMP-2 and luciferase [Ad-BMP-2+Ad-Luc]). In contrast, the luciferase signal was present for 8 weeks in defects treated with RBMCs co-transduced with two lentiviral vectors (Group III; lentivirus expressing BMP-2 and luciferase gene [LV-BMP-2+LV-Luc]). There were no significant differences with respect to the radiological healing rates (p=0.12) in defects treated with lentiviral versus adenoviral mediated BMP-2 gene transfer. Biomechanical testing of healed Group I femoral specimens demonstrated significantly higher energy to failure (p<0.05) when compared to Group II defects. Micro CT analysis revealed higher bone volume/tissue volume fraction (p=0.04) in Group I defects when compared to Group II defects. In conclusion, prolonged BMP-2 expression associated with lentiviral mediated gene transfer demonstrated a trend towards superior quality of bone repair when compared to adenoviral mediated transfer of BMP-2. These results suggest that the bone repair associated with regional gene therapy is influenced not just by the amount of protein expression but also by duration of protein production. This observation needs validation in a more biologically challenging environment where differences in healing rates and quality of bone repair are more likely to be significantly different.

The combination of small-animal PET/CT scans and conventional imaging methods may enhance the evaluation of in vivo biologic interactions of murine models in the study of prostate cancer metastasis to bone. METHODS: Small-animal PET/CT scans using (18)F-fluoride ion and (18)F-FDG coregistered with high-resolution small-animal CT scans were used to longitudinally assess the formation of osteoblastic, osteolytic, and mixed lesions formed by human prostate cancer cell lines in a severe combined immunodeficient (SCID) mouse tibial injection model. These scans were correlated with plain radiographs, histomorphometry, and soft-tissue measurements. RESULTS: Small-animal PET/CT scans were able to detect biologic activity of cells that induced an osteoblastic lesion 2 wk earlier than on plain radiographs. Furthermore, both the size and the activity of the lesions detected on PET/CT images significantly increased at each successive time point (P < 0.05). (18)F-FDG lesions strongly correlated with soft-tissue measurements, whereas (18)F-fluoride ion activity correlated with bone volume measured on histomorphometric analysis (P < 0.005). Osteolytic lesions were successfully quantified using small-animal CT, whereas lesion sizes measured on (18)F-FDG PET scans also strongly correlated with soft-tissue tumor burden (P < 0.05). In contrast, for mixed lesions, (18)F-fluoride ion and (18)F-FDG PET/CT scans detected only minimal activity. CONCLUSION: (18)F-FDG and (18)F-fluoride ion PET/CT scans can be useful tools in characterizing pure osteolytic and osteoblastic lesions induced by human prostate cancer cell lines. The value of this technology needs further evaluation to determine whether these studies can be used effectively to detect more subtle responses to different treatment regimens in animal models.

Targeted gene transduction to organs and tissues of interest is the ultimate goal of therapeutic gene delivery. Lentiviral vectors (LVs) are powerful tools for stable gene delivery but their integration into undesired cell types poses a serious safety concern for their use in the clinic. Here we report the development of a new dual-targeted LV that can preferentially home to and express in prostate cancer bone metastases in vivo after systemic delivery. Transductional targeting is mediated by a modified Sindbis virus envelope that interacts with the prostate stem cell antigen (PSCA) expressed by prostate cancer cells, and transcriptional targeting is mediated by a prostate cell specific promoter. Homing to prostate tumors was achieved in 70% of the animals. Importantly, tumors could be detected in some cases by molecular imaging prior to X-ray detection. The dual-targeted vector presents enhanced specificity with respect to individual transcriptional or transductional targeted vectors. Transgene expression in the liver was 190 times lower than the expression associated with solely transductionally targeted vectors, and there was 12 times less vector DNA than the amount present with solely transcriptionally targeted vectors. The LV presented here is a powerful tool for obtaining stable and site-specific gene expression and can be easily modified for its use in other diseases.

Multicentric giant cell tumour (GCT) is extremely rare; no case has been previously reported where two lesions occurred in the same foot at different sites. We report a case involving the calcaneus and subsequently the 3(rd) toe of the same foot and review the reported literature. In established cases of multicentricity, the histopathology has to be properly reviewed and the patient has to be followed up for a longer time with serial whole body assessment to pick up any subsquent lesions. The treatment of the local disease does not differ from a standard GCT.

Establishment of skeletal metastasis involves bidirectional interactions between the tumor cell and the cellular elements in the bone microenvironment. A better understanding of the pathophysiology of bone metastasis will be critical in developing the means to prevent bone metastasis or inhibit its progression. The receptor activator of nuclear factor-kappaB (RANK)/RANK ligand pathway has emerged as the key pathway regulating osteolysis in skeletal metastasis. A number of candidate factors, including the Wnt (wingless int) proteins, endothelin-1, and bone morphogenetic proteins, have been implicated in the establishment of osteoblastic metastasis. The complex nature of tumor-bone microenvironment interactions and the presence of multiple pathways that lead to bone metastasis suggests that simultaneous targeting of these pathways in the metastatic cascade are required for effective treatment. This review discusses current understanding of the pathophysiologic mechanisms that underlie the establishment of bone metastasis and potential molecular therapeutic strategies for prevention and treatment of bone metastasis.