LMP-1 retroviral gene therapy influences osteoblast differentiation and fracture repair: a preliminary study.
LIM mineralization protein-1 (LMP-1) is a novel intracellular osteogenic factor associated with bone development that has been implicated in the bone morphogenetic protein (BMP) pathway. This preliminary study evaluated the possibility of LMP-1-based retroviral gene therapy to stimulate osteoblast differentiation in vitro and fracture repair in vivo. A Moloney leukemia virus (MLV)-based retroviral vector to express LMP-1 with a hemagglutinin (HA) tag was developed, and its effects were evaluated on MC3T3-E1 cell differentiation and in the rat femur fracture model. MC3T3-E1 osteoblasts transduced with the MLV-HA-LMP-1 vector demonstrated significantly increased osteoblast marker gene expression (P < 0.05) and mineral deposition compared to control transduced cells. Femoral midshaft fractures were produced in Fischer 344 rats by the three-point bending technique. The MLV-HA-LMP-1 or control vector was applied at the fracture site through percutaneous injections 1 day postfracture. Analysis of fracture healing of 10 MLV-HA-LMP-1-treated and 10 control MLV-beta-galactosidase (beta-gal)-treated animals was completed at 3 weeks by X-ray, peripheral quantitative computed tomography, and histology. MLV-HA-LMP-1-treated animals had 63% more bone mineral content at the fracture site (P < 0.01), 34% greater total hard callus area (P < 0.05), and 45% less cartilage in the fracture callus (P < 0.05) compared to MLV-beta-gal-treated animals. There was no effect of LMP-1 treatment on the density of the hard callus. Immunohistochemistry revealed expression of the LMP-1 transgene in the fracture callus at 21 days postfracture. Immunohistochemistry also revealed that LMP-1 transgene expression did not result in an increase in BMP-4 expression in the fracture callus. Compared to MLV-BMP-4 gene therapy studies, MLV-HA-LMP-1 gene therapy improved bony union of the fracture gap to a greater extent and did not cause heterotopic bone formation. This suggests that LMP-1 may be a better potential candidate for gene therapy for fracture repair than BMP-4. These exciting, albeit preliminary, findings indicate that LMP-1-based gene therapy may potentially be a simple and effective means to enhance fracture repair that warrants further investigation.
Strohbach
Cassandra A
CA
Musculoskeletal Disease Center, Jerry L. Pettis Memorial VA Medical Center, 11201 Benton Street, Loma Linda, CA 92357, USA.
Rundle
Charles H
CH
Wergedal
Jon E
JE
Chen
Shin-Tai
ST
Linkhart
Thomas A
TA
Lau
K-H William
KH
Strong
Donna D
DD
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001861:Bone Regeneration / Q000502:physiology; D001842:Bone and Bones / Q000166:cytology / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D003598:Cytoskeletal Proteins; D004195:Disease Models, Animal; D050723:Fractures, Bone / Q000628:therapy*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D060588:LIM Domain Proteins; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D012190:Retroviridae; D014161:Transduction, Genetic
D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; C484380:PDLIM7 protein, human