RETROVIRAL-MEDIATED GENE-TRANSFER OF CSF-1 INTO OP OP STROMAL CELLS TO CORRECT DEFECTIVE IN-VITRO OSTEOCLASTOGENESIS/

Colony-stimulating factor-1 (CSF-1) released by stromal cells in the b one microenvironment is essential for the proliferation of osteoclast progenitors. in op/op mutant mice, a thymidine insertion in the coding sequence of the CSF-1 gene results in CSF-1 deficiency that in turn l eads to decreased osteoclast production and osteopetrosis. Because the osteopetrotic defect is due to the failure of stromal cells to produc e CSF-1, we determined if retroviral-mediated gene transfer of the wil d-type CSF-1 cDNA into op/op stromal cells would restore their ability to support osteoclast formation in vitro. A retroviral vector, L-CSF- 1-SN, was constructed by inserting 1,867 bp of the wild-type CSF-1 cDN A into pLXSN. After transduction with L-CSF-1-SN or LXSN constructs, a stable PA317 packaging cell line that produced a high viral titre was isolated. Viral supernatant from this line was used to infect op/op b one marrow stromal cells. Stable L-CSF-1-SN op/op stromal clones overe xpressed CSF-1 mRNA and released CSF-1 into conditioned medium, compar ed with no CSF-1 released by LXSN op/op stroma. The amount of CSF-1 pr oduced by two clones was similar to the physiologic level released by normal littermate stroma. Southern blot analysis confirmed the presenc e of intact proviral sequences in transduced cells. In coculture assay s, L-CSF-1-SN, but not LXSN, op/op stromal cells supported the formati on of TRAP-positive multinucleated cells in the absence of exogenous C SF-1. These findings indicate that genetically engineered stromal cell s may be used to improve defective osteoclastogenesis and suggest that targeting stromal cells to bone is a potentially useful therapeutic m odality for treating bone disorders. (C) 1998 Wiley-Liss, Inc.