Mechanism of bone formation by LIM mineralization protein–1 gene therapy

Abstract

Purpose of study: LIM mineralization protein-1 (LMP-1), an osteoinductive protein identified in 1998, is thought to induce secretion of soluble factors that convey its osteoinductive activity and capability of inducing spine fusion. LMP-1 has been successfully delivered using several ex vivo gene therapy techniques and has proven its ability to consistently induce spine fusion in experimental models. The purpose was to determine the mechanism of bone formation of LMP-1.Methods used: In Phase I, conditioned medium from rat osteoblast cultures overexpressing the LMP-1 cDNA was placed on nondifferentiating cultures. The overexpression cultures were cotreated with antisense oligonucleotides complimentary to bone morphogenic protein (BMP)-4, BMP-6, BMP-7 or a nonsense sequence to see if any of those BMPs were candidates for the LMP-1–induced differentiation factor. In Phase 2, A549 cells were used to test for the induction of candidate BMPs in vitro. The cells were infected in chamber slides for 2 days with adenovirus (MOI=10) containing the cDNA for LMP-1 (AdLMP-1) or B-galactosidase (AdBgal). Untreated cells were used as an additional negative control. Cells were analyzed by immunohistochemical methods for BMP family members. In Phase 3, 16 athymic rats each received four subcutaneous implants on the chest. Human buffy coat cells from peripheral blood were used to deliver the LMP-1 cDNA. One million cells were infected for 10 minutes (MOI = 4.0) with either AdLMP-1 or AdBgal (control) and then placed on a collagen disc and implanted. The animals were sacrificed at intervals, and explants were analyzed by histology and immunohistochemistry.of findings: In Phase 1 the antisense experiments suggested that BMP-4 and BMP-7, but not BMP-6, were required for LMP-1 activity. In Phase 2, the A549 cells treated by AdLMP-1 had strong intracellular reaction for BMP-2, BMP-4 and BMP-7. Some cells also stained slightly positive with the anti–BMP-6 antibody. There was no specific staining for any of these BMPs in the cells treated with AdBgal or the untreated control cells. In Phase 3 human leukocytes were implanted on collagen discs in rats and by Day 3 an increased number of cells were seen at the periphery of the AdLMP-1–treated implants. By Day 5, the number of cells surviving in the center of the implant was diminished, especially in the controls. Extracellular matrix could be seen deposited near the cells in the periphery by day 7 in the Ad-LMP implants. By Day 10, osteoblast-like cells were observed in the voids between the collagen fibers. Osteoid and some mineralized bone were consistently seen by Day 14 growing inward from the edge of the implant. No cartilage phase was observed. There was strong BMP-4 and BMP-7 staining on Day 3 and Day 5 in AdLMP-1 implants within cells, confirming the in vitro observations.Relationship between findings and existing knowledge: Little is known about the mechanism of action of LMP-1 or its relationship to BMPs. The osteoinductive activity of LMP-1 appears to involve synthesis of several BMPs, including BMP-4 and BMP-7. In addition, BMP-2, BMP-6 and TGF-b were also be induced by LMP-1.Overall significance of findings: The LMP-1 cDNA can be transferred into peripheral blood leukocytes in 10 minutes without special equipment. LMP-1 is able to induce the complete membranous bone formation cascade in vivo. Future studies will determine if there are advantages to induce local synthesis of multiple BMP family members, as with LMP-1, as an alternative to implanting larger doses of a single recombinant BMP.Disclosures: Device or drug: LMP-1. Status: not approved.Conflict of interest: Scott D. Boden, grant research support and consultant, Medtronic Sofamor Danek; Scott D. Boden, University licensed, LMP-1 to Medtronic Sofamor Danek.