title: Adenovirus-mediated gene therapy of osteoblasts in vitro and in vivo.
abstract: Modulation of biological pathways governing osteogenesis may accelerate osseous regeneration and reduce the incidence of complications associated with fracture healing. Transforming growth factor beta1 (TGF-beta1) is a potent growth factor implicated in the regulation of osteogenesis and fracture repair. The use of recombinant proteins, however, has significant disadvantages and has limited the clinical utility of these molecules. Targeted gene therapy using adenovirus vectors is a technique that may circumvent difficulties associated with growth factor delivery. In this study, we investigate the efficacy of replication-deficient adenoviruses containing the human TGF-beta1 and the bacterial lacZ genes in transfecting osteoblasts in vitro and osseous tissues in vivo. We demonstrate that adenovirus-mediated gene therapy efficiently transfects osteoblasts in vitro with the TGF-beta1 virus causing a marked up-regulation in TGF-beta1 mRNA expression even 7 days after transfection. Increased TGF-beta1 mRNA expression was efficiently translated into protein production and resulted in approximately a 46-fold increase in TGF-beta1 synthesis as compared with control cells (vehicle- or B-galactosidase-transfected). Moreover, virally produced TGF-beta1 was functionally active and regulated the expression of collagen IalphaI (5-fold increase) and the vascular endothelial growth factor (2.5-fold increase). Using an adenovirus vector encoding the Escherichia coli LacZ gene, we demonstrated that adenovirus-mediated gene transfer efficiently transfects osteoblasts and osteocytes in vivo and that transfection can be performed by a simple percutaneous injection. Finally, we show that delivery of the hTGF-beta1 gene to osseous tissues in vivo results in significant changes in the epiphyseal plate primarily as a result of increased thickness of the provisional calcification zone.
authors.affiliation: The Laboratory of Developmental Biology and Repair, The Institute of Reconstructive Plastic Surgery, and The Department of Surgery, New York University Medical Center, New York, New York 10016, USA.
authors.forename: B J
authors.initials: BJ
authors.lastname: Mehrara
authors.forename: P B
authors.initials: PB
authors.lastname: Saadeh
authors.forename: D S
authors.initials: DS
authors.lastname: Steinbrech
authors.forename: M
authors.initials: M
authors.lastname: Dudziak
authors.forename: J A
authors.initials: JA
authors.lastname: Spector
authors.forename: J A
authors.initials: JA
authors.lastname: Greenwald
authors.forename: G K
authors.initials: GK
authors.lastname: Gittes
authors.forename: M T
authors.initials: MT
authors.lastname: Longaker
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D001861:Bone Regeneration / Q000502:physiology; D002478:Cells, Cultured; D016228:Endothelial Growth Factors / Q000096:biosynthesis; D017102:Fracture Healing; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007763:Lac Operon; D008222:Lymphokines / Q000096:biosynthesis; D010006:Osteoblasts / Q000502:physiology*; D010012:Osteogenesis / Q000502:physiology; D016133:Polymerase Chain Reaction; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000627:therapeutic use*; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D016228:Endothelial Growth Factors; D008222:Lymphokines; D016212:Transforming Growth Factor beta; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D001616:beta-Galactosidase