Tracking Expression of Virally Mediated BMP-2 in Gene Therapy for Bone Repair

Abstract

Ex vivo gene therapy using stem cells transduced with viral vectors is a useful method for delivering a therapeutic protein to augment bone repair in animal models. However, the duration of cell-mediated protein production and the fate of the transduced cells are unknown. We constructed an adenoviral vector encoding Myc epitope tagged bone morphogenetic protein (BMP)-2 gene (AdBMP-2 Myc ). Rat bone marrow cells transduced with this vector produced biologically active BMP-2 protein, which was confirmed by Western blot analysis and alkaline phosphatase assay. Implantation of bone marrow cells infected ex vivo with AdBMP-2 Myc caused orthotopic bone formation in mouse hindlimbs and bony union of critical-sized mouse radial defects. Immunohistochemical analysis revealed that rBMCs expressed Myc epitope-tagged BMP-2 protein for 14 days in vivo and became incorporated in the endochondral fracture callus. This novel adenovirus encoding for epitope-tagged BMP-2 can be used for immunohistochemical tracking of transduced cells in ex vivo gene therapy for bone repair.Treatment of bone defects secondary to trauma, tumor, or infection often requires bone grafting. Bone grafting can be accomplished with autogenous bone graft, allograft bone from cadaveric donors, demineralized bone matrices, calcium-phosphate ceramics, or recombinant proteins. Autogenous bone graft from the iliac crest often is considered the first choice of bone-graft materials, but the supply is limited. In addition, harvesting autogenous bone graft can be associated with morbidity such as donor-site pain, nerve damage, and bleeding. 1,5,18 Increased attention has focused on using recombinant proteins such as BMPs to induce bone repair. 14,19 However, supraphysiologic doses of recombinant BMPs are required to induce bone formation in clinical cases. 7,14,34 There is also concern that one dose of exogenous protein will not be sufficient to induce an adequate osteoinductive response in patients with large bone defects or with significant soft tissue injury. 10 One potential alternative to direct recombinant protein delivery is to develop a biologic cellular delivery vehicle via gene therapy to enhance bone formation. 8 Ex vivo gene therapy has been used successfully in various preclinical animal models to promote bone repair. Critical-sized femoral and calvarial defects have healed successfully using ex vivo gene therapy strategies with adenoviral and retroviral vectors. 9,16,20,21 The duration of in vivo protein production and the fate of the infected cells in vivo still need to be defined. Do the transduced cells just act as cellular delivery vehicles or do they actively participate in the bone repair process? Bone marrow cells, adipose-derived stem cells, and muscle-derived stem cells, all of which can differentiate into bone under the appropriate stimuli, have been used as cellular delivery vehicles in ex vivo gene therapy strategies. 12,15,17,21,28,30 A potential advantage of using osteoprogenitor cells is that these transduced cells not only pr...

The Synergistic Effect of Autograft and BMP-7 in the Treatment of Atrophic Nonunions

“…A statistical validation of the interrater agreement between the radiograph reviewers was performed to attain a consensus on the time to nonunion healing with positive results (j = 0.76). The inclusion of three different anatomic sites (humerus, tibia, and femur) and the small number of the patients per anatomic site (7,19, and 19, respectively) do not allow comprehensive statistical analysis for further evaluation of our results ( Table 2). The absence of comparative functional outcome results during the period of followup of the 45 enrolled patients, as a result of the inadequacy of the retrospective part of our data, did not allow complete assessment of the progress of symptoms of the studied patients.…”

“…Since then, over 20 different BMP-homologous molecules have been described, 15 of which were identified in humans [43]. BMPs belong to the transforming growth factor-b superfamily, a large family of growth and differentiation factors, which are involved in several stages of embryonic organogenesis and in various stages of intramembranous and endochondral ossification during fracture healing [19,36]. They have been tested in various experimental animal nonunion models [32,39].…”

Efficient and Rapid Osteoinduction in an Immune-Competent Host

“…Perhaps the lack of viral transduction of these cells aided in their ability to undergo osteogenesis and contribute to bone. Gamradt et al (2006) suggested that mesenchymal stem cells transduced with first-generation adenoviral vectors could also contribute to newly forming bone as functional osteoblasts. Interestingly, in these studies, the authors tagged the BMP2 with a Myc epitope and thus the authors tracked secreted BMP2 rather than the delivery cells themselves.…”

Role of Mesenchymal Stem Cells in Bone Regenerative Medicine: What Is the Evidence?

“…Among the available viral methods, some viral vectors have been used widely by gene therapists, including oncogenic retroviral vectors, ade- [Santos et al, 2011]. BMP family genes are the most commonly used in genetic manipulation of MSCs due to their better regeneration properties [Gamradt et al, 2006;Zhang et al, 2012]. He et al [2013] evaluated the role of BMP-2 genetically engineered MSCs on the bone regeneration process in a critical sized calvarial bone defect model in rats.…”