Jin Zhong Li

Osteogenic potential of five different recombinant human bone morphogenetic protein adenoviral vectors in the rat.

Bone morphogenetic protein (BMP) adenoviral vectors for the induction of osteogenesis are being developed for the treatment of bone pathology. However, it is still unknown which BMP adenoviral vector has the highest potential to stimulate bone formation in vivo. In this study, the osteogenic activities of recombinant human BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 adenoviruses were compared in vitro, in athymic nude rats, and in Sprague–Dawley rats. In vitro osteogenic activity was assessed by measuring the alkaline phosphatase activity in C2C12 cells transduced by the various BMP vectors. The alkaline phosphatase activity induced by 2 × 105 PFU/well of BMP viral vector was 4890 × 10−12 U/well for ADCMVBMP-9, 302 × 10−12 U/well for ADCMVBMP-4, 220 × 10−12 U/well for ADCMVBMP-6, 45 × 10−12 U/well for ADCMVBMP-2, and 0.43 × 10−12 U/well for ADCMVBMP-7. The average volume of new bone induced by 107 PFU of BMP vector in athymic nude rats was 0.37±0.03 cm3 for ADCMVBMP-2, 0.89±0.07 cm3 for ADCMVBMP-4, 1.02±0.07 cm3 for ADCMVBMP-6, 0.24±0.05 cm3 for ADCMVBMP-7, and 0.63±0.07 cm3 for ADCMVBMP-9. In immunocompetent Sprague–Dawley rats, no bone formation was demonstrated in the ADCMVBMP-2, ADCMVBMP-4, and ADCMVBMP-7 groups. ADCMVBMP-6 at a viral dose of 108 PFU induced 0.10±0.03 cm3 of new bone, whereas ADCMVBMP-9 at a lower viral dose of 107 PFU induced more bone, with an average volume of 0.29±0.01 cm3.

Ex vivo bone morphogenetic protein-9 gene therapy using human mesenchymal stem cells induces spinal fusion in rodents.

OBJECTIVE Ex vivo gene therapy with the use of human mesenchymal stem cells (hMSCs) and bone morphogenetic protein (BMP) genes provides a local supply of precursor cells and a supraphysiological dose of osteoinductive molecules that may promote bone formation in patients with inadequate hMSC populations because of age, osteoporosis, metastatic bone disease, iatrogenic depletion, or other metabolic derangements. This study was undertaken to evaluate the efficacy of ex vivo gene therapy with the use of hMSCs and the BMP-9 gene to promote spinal fusion in the rat. METHODS Sixteen athymic nude rats were treated with hMSCs transduced with recombinant, replication-defective Type 5 adenovirus containing the cytomegalovirus promoter and either the BMP-9 (Ad-BMP-9) or the &bgr;-galactosidase (Ad-&bgr;-gal) gene. Ad-&bgr;-gal served as the control. Each animal received a percutaneous, paraspinal injection of 106 hMSCs transduced with 50 plaque-forming units/cell adenovirus in the lumbar region, with Ad-BMP-9 on the left and Ad-&bgr;-gal on the right. At 8 weeks postinjection, computed tomographic scans of the lumbosacral spine were obtained, and the lumbosacral spine specimens were examined histologically. RESULTS Both computed tomographic studies and histological analysis clearly demonstrated large volumes of ectopic bone at the Ad-BMP-9-transduced hMSC injection sites, resulting in successful spinal fusion and no evidence of nerve root compression or local or systemic toxicity. The contralateral regions that were treated with Ad-&bgr;-gal-transduced hMSCs showed no evidence of osteogenesis. CONCLUSION The results of this study suggest that hMSC and BMP-9 ex vivo gene therapy may be useful in inducing spinal fusion as well as other related procedures and certainly warrants further clinical development.

Morphologic analysis of BMP-9 gene therapy-induced osteogenesis

The present study was performed to determine the histological, ultrastructural, and radiographic changes that occur over time at intramuscular BMP-9 gene therapy treatment sites. Several members of the bone morphogenetic protein (BMP) family have the potential to induce osteochondrogenesis when the protein is delivered to rodents, canines, rabbits, and nonhuman primates. Previous studies have also demonstrated that BMP gene therapy utilizing adenoviral vectors can also stimulate orthotopic and heterotopic bone formation in rodents and rabbits. Athymic nude and Sprague-Dawley rats were injected with Ad-BMP-9 or Ad-beta-Gal (3.75 x 10(9) particles) in their thigh musculature and light microscopic, electron microscopic, and computerized tomography analysis was performed 3, 6, 9, 12, 15, 18, 21, and 100 days later. To assess early mesenchymal cell proliferation, a bromodeoxyuridine (BrdU) immunohistochemical analysis was also performed 48, 60, and 72 hr postinjection in athymic nude rats. All animals demonstrated extensive endochondral bone formation at the Ad-BMP-9 treatment sites within 3 weeks. The Sprague-Dawley rats also exhibited a massive, acute inflammatory infiltrate during the first week. Proliferating mesenchymal stem cells were clearly evident as early as 2 days after treatment, which differentiated into small or hypertrophied chondrocytes during the next week. During the third week, the cartilaginous matrix mineralized and formed woven bone, which converted to lamellar bone by 3 months. No evidence of bone formation was demonstrated at the Ad-beta-Gal injection sites in the athymic nude or Sprague-Dawley rats. In addition, no cellular proliferation was seen at the Ad-beta-Gal treatment sites in the athymic nude animals as assessed by light microscopy and BrdU immunohistochemistry. The extensive bone formation induced by Ad-BMP-9 suggests that BMP gene therapy may have potential utility in the treatment of degenerative, rheumatic, or traumatic bone pathology.

Human Mesenchymal Stem Cells Transduced with Recombinant Bone Morphogenetic Protein-9 Adenovirus Promote Osteogenesis in Rodents

The present study was undertaken to determine whether ex vivo bone morphogenetic protein-9 (BMP-9) gene therapy using human mesenchymal stem cells (hMSCs) can induce endochondral bone formation in athymic nude rats. An in vitro study was initially performed on hMSCs to evaluate morphological changes and osteoblastic differentiation induced by replication-defective adenovirus type 5 with the cytomegalovirus promoter and either the BMP-9 (Ad-BMP-9) or beta-galactosidase (Ad-beta-gal) gene. In vivo, athymic nude rats received an injection (10(6) hMSCs transduced with recombinant adenovirus at 50 PFU/cell) into the anterior thigh musculature: Ad-BMP-9 on the left and Ad-beta-gal (control) on the right. Computed tomography scans and histological analysis were obtained 7, 14, 28, 42, 56, and 84 days postinjection. In vitro, human mesenchymal stem cells treated with Ad-BMP-9 (50 PFU/cell) showed signs of differentiation, whereas hMSCs treated with 250 and 1250 PFU/cell showed cytotoxicity. In vivo, computed tomography and histological analysis clearly demonstrated ectopic bone at hMSC/Ad-BMP-9 treatment sites, whereas the hMSC/Ad-beta-gal treatment sites showed no evidence of osteogenesis. None of the animals showed clinical evidence of toxicity. Ex vivo gene therapy with hMSC/BMP-9 may be efficacious for promoting bone formation for a variety of bone pathologies and certainly warrants further investigations.

Osteogenesis in rats induced by a novel recombinant helper-dependent bone morphogenetic protein-9 (BMP-9) adenovirus.

Although recombinant first‐generation BMP adenoviruses can induce ectopic bone formation in immune deficient animals, the osteoinductive activity of these BMP vectors is reduced in immune competent animals. Helper‐dependent adenoviral vectors have been developed to decrease the immune response and, therefore, increase gene expression in immune competent animals compared with first‐generation vectors. In the present study, the osteoinductive activity of a helper‐dependent GFP and BMP‐9 adenoviral vector (ADGBMP9) was evaluated in vitro and in vivo.

CT and radionuclide study of BMP-2 gene therapy-induced bone formation

RATIONALE AND OBJECTIVES Gene therapy techniques have the potential to treat numerous diseases, from cancer to diabetes. One promising application is the use of bone morphogenetic protein (BMP) gene transfer to induce bone formation. Previous studies have demonstrated that both direct and ex vivo BMP gene therapy have the capacity to initiate the normal endochondral pathway, leading to rapid mature bone formation. In the present study, computed tomography (CT) and radionuclide imaging was used to assess bone formation induced by BMP gene therapy accurately and noninvasively. MATERIALS AND METHODS Athymic nude rodents were treated with 1.25 x 10(10) particles of adenovirus-BMP-2 (Ad-BMP-2) (treatment group) or adenovirus-beta-gal (control group). At various intervals after treatment, the animals underwent CT, planar digital radiography, and planar radionuclide scintigraphic imaging. RESULTS Radionuclide scintigraphy clearly demonstrated active bone deposition that began as early as 15 days after treatment and peaked at approximately 36 days, only at the Ad-BMP-2 injection sites. CT clearly demonstrated ectopic bone induction over time at the Ad-BMP-2 treatment sites, in perfect correlation with the scintigraphic findings. CONCLUSION This study clearly illustrates that gene therapy-induced osteogenesis can be studied with multimodality imaging and supports the use of these approaches in future preclinical and clinical studies.

Identification of the deleted in liver cancer 1 gene, DLC1, as a candidate meningioma tumor suppressor.

OBJECTIVEMeningiomas are the second most common primary tumors of the central nervous system. Meningiomas at the cranial base pose technical challenges and result in increased morbidity. To investigate the molecular mechanisms of meningioma formation, the expression profiles of 12 000 genes from meningiomas and dural specimens were compared. METHODSRibonucleic acid from 6 meningiomas (World Health Organization Grade I) and 4 dural specimens was profiled using U95A GeneChips (Affymetrix, Inc., Santa Clara, CA). Expression profiles of the 2 groups were compared using dChip and Data Mining Tool software packages (Affymetrix, Inc.) to identify differentially expressed genes. Down-regulation of a differentially expressed tumor suppressor gene, deleted in liver cancer 1 (DLC1), was verified by quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemical staining. Function and methylation of DLC1 were assessed by ectopic expression in 5 primary cultures, demethylation assay using 5-aza-2′-deoxycytidine, and methylation-specific polymerase chain reaction in 4 meningioma samples. RESULTSGene expression profiling revealed up-regulation of 5 genes (fibroblast growth factor 9, gibbon leukemia virus receptor 2, cyclin D1, eukaryotic translation initiation factor 5A, and 28S ribosomal ribonucleic acid) and down-regulation of 35 genes, including DLC1, in meningiomas. The down-regulation of DLC1 in meningiomas was confirmed by quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemical staining. Transfection of DLC1 complementary deoxyribonucleic acid into primary cultures of 5 meningiomas resulted in decreased replication. Although demethylation decreased meningioma cell growth rates in vitro, methylation-specific polymerase chain reaction did not detect DLC1 promoter methylation. CONCLUSIONThe results suggest that DLC1 may function as a tumor suppressor gene in meningiomas. Furthermore, DLC1 promoter methylation does not appear to be responsible for the decreased DLC1 expression in these tumors.

Long-term tracing of adenoviral expression in rat and rabbit using luciferase imaging.

Luciferase optical imaging provides a novel method to monitor transgene expression in small living animals. As the genetic and immunological heritages of particular animals significantly affect the expression of adenovirus‐delivered transgenes, it is essential to know the expression patterns specific to athymic nude and Sprague‐Dawley rats, two strains commonly used in rodent models. In this study we set out to determine these patterns. At the same time, we tested luciferase optical imaging in a larger animal, the rabbit.

Local immunomodulation with CD4 and CD8 antibodies, but not cyclosporine A, improves osteogenesis induced by ADhBMP9 gene therapy

This study was designed to see if immunosuppression achieved using local application of cyclosporine A (Cs. A) or CD4 and CD8 antibodies would improve bone formation following intramuscular injections of human BMP-4 and BMP-9 adenoviral vectors (ADhBMP4 and ADhBMP9) in Sprague–Dawley rats. Cs. A was injected into the thigh muscle. After 2 days, ADhBMP4, ADhBMP9, and the antibodies were separately injected into the left and right rear legs. At this time, the number of CD4+/CD3+ cells was significantly lower and the number of CD8+/CD3+ cells higher in the Cs. A group than in the control group (P<0.01). The total number of white blood cells 3 days following injection of CD4 and CD8 antibodies was significantly lower than that before the injection (P<0.01). At 4 weeks after the viral and antibody injections, mean bone volumes at the ADhBMP9 treatment sites were 0.29±0.01 cm3 in the viral control group, 0.17±0.03 cm3 in the Cs. A-ADhBMPs group, and 0.59±0.07 cm3 in the antibodies-ADhBMPs group. ADhBMP4 did not induce new bone formation in any group. This study demonstrates that local immunomodulation may improve the osteogenic potential of bone morphogenetic protein gene therapy in the clinical setting.

Non‐invasive imaging of firefly luciferase reporter gene expression using bioluminescence imaging in human prostate cancer models

Monitoring the expression of therapeutic genes in targeted tissues in disease models is important to assess the effectiveness and safety of systems of gene therapy delivery. In the present study, we employed a CCD (charge‐coupled‐device) imaging system to monitor how a prostate‐specific adenovirus vector (AdPSA‐Luc) mediated the long‐term, sustained expression of firefly luciferase (Luc) in living human prostate cancer mouse models. The in vivo bioluminescence imaging revealed significantly high levels of luciferase expression up to 1 month, not only in prostate tumours, but also in lungs after intratumoural injection. Systemic tail vein injection of AdPSA‐Luc revealed significant luciferase expression in lungs of both human prostate cancer mouse models and naïve mice, but significantly higher in the former, while the control virus, AdCMV‐Luc, containing CMV (cytomegalovirus) promoter and luciferase gene, just restricted expression in the livers. Our findings demonstrate the ability of the cooled CCD camera to sensitively and non‐invasively track the location, magnitude and persistence of luciferase gene expression in human prostate cancer mouse models. Monitoring of gene therapy studies in small animals may be aided considerably with further extensions of this technique.