Efficient bone formation by gene transfer of human LIM mineralization protein-3

Abstract

LIM mineralization protein (LMP) is a novel positive regulator of the osteoblast differentiation program. In humans, three different LMP splice variants have been identified: LMP-1, LMP-2, and LMP-3. Gene transfer of human LMP-1 (hLMP-1) induces expression of genes involved in bone formation, including certain bone morphogenetic proteins (BMPs), promotes bone nodule formation in vitro, ectopic bone formation in vivo, and is therapeutic in animal models of posterior thoracic and lumbar spine fusion. To examine the osteoinductive properties of the LMP-3 in vitro and in vivo, we have generated plasmid and adenoviral vectors expressing codon-optimized hLMP-3. Here we demonstrate that gene transfer of hLMP-3 induces expression of the bonespecific genes osteocalcin, osteopontin, and bone sialoprotein and induced bone mineralization in preosteoblastic and fibroblastic cells. We also demonstrate that hLMP-3 is able to induce bone mineralization and the expression of the bonespecific genes, BMP-2, OSX, RunX2, and alkaline phosphatase in human mesenchymal stem cells in a dose-dependent manner. Finally, we demonstrate that direct gene transfer of hLMP-3 into murine skeletal muscle results in ectopic bone formation more efficiently than BMP-2. These results demonstrate that hLMP-3 gene transfer can be used to promote bone formation in cell culture and in vivo as or more efficiently than BMP-2, thus establishing feasibility and efficacy of direct gene delivery of hLMP-3 to produce bone in vivo. These results suggest that gene transfer of hLMP-3 could be developed as a bone-inductive therapeutic agent for clinical applications.

Ex vivo gene therapy using autologous dermal fibroblasts expressing hLMP3 for rat mandibular bone regeneration

“…E1-and E3-deleted adenoviral vector expressing human LMP-3 (Ad-hLMP3) was constructed as previously described. 18 Briefly, the codon-optimized LMP-3 cDNA was subcloned into pcDNA3.1 TOPO T/A cloning plasmid (Invitrogen, Carlsbad, CA). A Sal I/Not I fragment containing hLMP3, derived from the plasmid pcDNA3.1/hLMP3, was inserted in a modified version of the shuttle vector pAdlox (GenBank U62024).…”

“…In particular, LMP-3 has been recently demonstrated to promote efficient bone formation in distinct animal models along with bone mineralization in cultured mesodermal cells. 18,19 Based on the transcriptional modulation of selected bone-specific genes, including BMP-2 and RunX2/CbfA1, LMP-3 seemed to act as an upstream modulator of the osteogenic lineage differentiation. 18 In this study, we used an LMP-3-based ex vivo procedure to promote the healing of a rat critical-size mandibular defect.…”

“…18,19 Based on the transcriptional modulation of selected bone-specific genes, including BMP-2 and RunX2/CbfA1, LMP-3 seemed to act as an upstream modulator of the osteogenic lineage differentiation. 18 In this study, we used an LMP-3-based ex vivo procedure to promote the healing of a rat critical-size mandibular defect. For this purpose we used rat autologous skin fibroblasts transduced with an adenoviral vector expressing the human LMP-3 gene adsorbed on a osteoconductive scaffold.…”

Genes and Molecular Pathways of the Osteogenic Process

“…The effectiveness of LMPs in inducing bone formation in vivo has been demonstrated, using different strategy to deliver the gene/peptide at the site where the bone healing was required (H.S. Lattanzi et al, 2008;Parrilla et al, 2010;Pola et al, 2004;Sangadala et al, 2009;Strohbach et al, 2008b;X. Wang et al, 2011;Yoon et al, 2004).…”

“…Liu et al, 2002;Viggeswarapu et al, 2001). Despite the truncation of nearly two third of the full-length isoform, LMP3 retains efficient osteogenic properties, demonstrated in vitro and in different animal models (Lattanzi et al, 2008;Parrilla et al, 2010;Pola et al, 2004). In vitro, both LMP1 and LMP3 induce osteogenic differentiation of mesenchymal progenitors, fibroblasts and pre-osteoblasts, through the transcriptional activation of BMP-family members (mainly BMP2, BMP4 and BMP7) and TGFβ1 protein (Bernardini et al, 2010 ;Minamide et al, 2003).…”