EXPRESSION OF HUMAN BONE MORPHOGENIC PROTEIN-7 IN PRIMARY RABBIT PERIOSTEAL CELLS - POTENTIAL UTILITY IN GENE-THERAPY FOR OSTEOCHONDRAL REPAIR

A commonly encountered problem in orthopedics is bone and cartilage ti ssue injury which heals incompletely or without full structural integr ity. This necessitates development of improved methods for treatment o f injuries which are not amenable to treatment using current therapies . An already large and growing number of growth factors which play sig nificant roles in bone remodeling and repair have been identified in t he past few years. It is well established that bone morphogenic protei ns induce the production of new bone and cartilage. An efficient metho d of delivery of these growth factors by conventional pharmacological means has yet to be elucidated We wished to evaluate the use of retrov iral vector-mediated gene transfer to deliver genes of therapeutic rel evance for bone and cartilage repair. To determine the feasibility of using amphotropically packaged retroviral vectors to transduce primary rabbit mesenchymal stem cells of periosteal origin, primary periostea l cells were isolated from New Zealand white rabbits, transduced in vi tro with a retroviral vector bearing both the nuclear localized lacZ m arker gene and the neo(r) gene, and selected in G418. We used a conven ient model for analysis of in vivo stability of these cells which were seeded on to polymer scaffold grafts and implanted into rabbit femora l osteochondral defects. The nuclear localized beta-galactosidase prot ein was expressed in essentially 100% of selected cells in vitro and w as observed in the experimental explants from animals after both 4 and 8 weeks in vivo, while cells transduced with a retroviral vector bear ing only the neo(r) gene in negative control explants showed no blue s taining. We extended our study by delivering a gene of therapeutic rel evance, human bone morphogenic protein 7 (hBMP-7), to primary perioste al cells via retroviral vector. The hBMP-7 gene was cloned from human kidney 293 cell total RNA by RT-PCR into a retroviral vector under con trol of the CMV enhancer/promoter. Hydroxyapatite secretion, presumabl y caused by overexpression of hBMP-7, was observed on the surface of t he transduced and selected periosteal cells, however, this level of ex pression was toxic to both PA317 producer and primary periosteal cells . Subsequently, the strong CMV enhancer/promoter driving the hBMP-7 ge ne was replaced in the retroviral vector by a weaker enhancer/promoter from the rat beta-actin gene. Nontoxic levels of expression of hBMP-7 were confirmed at both the RNA and protein levels in PA317 producer a nd primary periosteal cell lines and cell supernatants. This work demo nstrates the feasibility of using a gene therapy approach in attempts to promote bone and cartilage tissue repair using gene-modified perios teal cells on grafts.