Rundle, CH, Strong, DD, Chen, ST, Linkhart, TA, Sheng, MH, Wergedal, JE et al.. Retroviral-based gene therapy with cyclooxygenase-2 promotes the union of bony callus tissues and accelerates fracture healing in the rat. J Gene Med 10: 229-241

Department of Medicine, Loma Linda University, Loma Linda, CA, USA.
The Journal of Gene Medicine (Impact Factor: 2.47). 03/2008; 10(3):229-41. DOI: 10.1002/jgm.1148
Source: PubMed


An in vivo gene therapy strategy was developed to accelerate bone fracture repair.
Direct injection of a murine leukemia virus-based vector targeted transgene expression to the proliferating periosteal cells arising shortly after fracture. Cyclooxygenase-2 (Cox-2) was selected because the transgene for its prostaglandin products that promote angiogenesis, bone formation and bone resorption, are all required for fracture healing. The human (h) Cox-2 transgene was modified to remove AU-rich elements in the 3'-untranslated region and to improve protein translation.
In vitro studies revealed robust and sustained Cox-2 protein expression, prostaglandin E(2) and alkaline phosphatase production in rat bone marrow stromal cells and osteoblasts transgenic for the hCox-2 gene. In vivo studies in the rat femur fracture revealed that Cox-2 transgene expression produced bony union of the fracture by 21 days post-fracture, a time when cartilage persisted within the fracture tissues of control animals and approximately 1 week earlier than the healing normally observed in this model. None of the ectopic bone formation associated with bone morphogenetic protein gene therapy was observed.
This study represents the first demonstration that a single local application of a retroviral vector expressing a single osteoinductive transgene consistently accelerated fracture repair.

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Available from: K-H William Lau, Oct 23, 2014
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    • "We have previously demonstrated that Cox-2 gene therapy could indeed improve endochondral bone fracture repair.[12] However, the therapeutic effect was unexpectedly not seen until the endochondral stage of bone fracture repair, during fracture chondrogenesis, when the callus cartilage is being remodeled to bone and well after the inflammatory stage has subsided, during which time Cox-2 expression has been observed to initiate fracture healing.[13] "
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    ABSTRACT: Background Cyclo-oxygenase-2 (Cox-2) is an inflammatory mediator that is necessary for the tissue repair, including bone fracture healing. Although the application of Cox-2 gene therapy to a murine closed femoral fracture has accelerated bony union, but the beneficial effect was not observed until the endochondral stage of bone repair that is well after the inflammatory stage normally subsides. Methods To identify the molecular pathways through which Cox-2 regulates fracture healing, we examined gene expression profile in fracture tissues in response to Cox-2 gene therapy during the endochondral bone repair phase. Cox-2 gene therapy was applied to the closed murine femur fracture model. Microarray analysis was performed at 10 days post-fracture to examine global gene expression profile in the fracture tissues during the endochondral bone repair phase. The entire repertoire of significantly expressed genes was examined by gene set enrichment analysis, and the most up-regulated individual genes were evaluated further. Results The genes that normally promote inflammation were under-represented in the microarray analysis, and the expression of several inflammatory chemokines was significantly down-regulated. There was an up-regulation of two key transcription factor genes that regulate hematopoiesis and erythropoiesis. More surprisingly, there was no significant up-regulation in the genes that are normally involved in angiogenesis or bone formation. However, the expression of two tissue remodeling genes was up-regulated. Conclusions The down-regulation of the inflammatory genes in response to Cox-2 gene therapy was unexpected, given the pro-inflammatory role of prostaglandins. Cox-2 gene therapy could promote bony union through hematopoietic precursor proliferation during endochondral bone repair and thereby enhances subsequently fracture callus remodeling that leads to bony union of the fracture gap.
    Full-text · Article · Aug 2014
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    • "This was attended by increases in expression of receptors of VEGFs (Vegfr1 and Vegfr2 mRNA). Thus, these findings substantiate the previous reports that COX2 is associated with increased angiogenesis during fracture healing [29] [43]. In the case of fracture callus, this angiogenesis was specifically mediated by VEGF-A and VEGF-C through VEGFR-1 and -2. "
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    ABSTRACT: This study sought to determine the cellular and molecular mechanisms of cyclooxygenase-2 (COX2) gene therapy to accelerate fracture repair in a mouse multiple tibial fractures model. The lenti-COX2 (or lenti-gfp control vector) was injected into fractures on day 1 post-fracture. At days 3-7, the COX2 treatment increased Sdf1-, Cxcr4-, Nes-, and Podxl-expressing mesenchymal stem cells (MSCs) within fracture calluses, suggesting an enhanced MSC recruitment or expansion. The COX2-treated mice formed smaller cartilaginous calluses that had less cartilage tissues than control mice. The expression of Sox9 mRNA was 7-fold less in COX2-treated than in control calluses at day 14, implying that COX2 reduces chondrocytic differentiation of MSCs. The therapy also enhanced angiogenesis as reflected by increased immunostaining of CD31, vWF, and α-SMA over controls in the cartilaginous callus at day 14-21. At which time, the COX2 gene therapy promoted bony remodeling of the cartilaginous callus to bridge the fracture gap that was accompanied by 2-fold increase in osteoclasts along the surface of the woven bone and an onset of osteogenesis. Blocking angiogenesis with daily injection of endostatin from day 4 to day 10 into fracture sites blocked the COX2-mediated reduction of callus size that was associated with an increase in hypertrophic chondrocytes and concomitant reduction in osteoclasts. In conclusion, COX2 accelerates fracture healing in part through three biological actions: 1) increased recruitment/expansion of MSCs; 2) decreased cartilaginous callus formation; and 3) increased angiogenesis-dependent cartilage remodeling. These effects were associated with an earlier onset of bony bridging of the fracture gap.
    Full-text · Article · Jan 2013 · Bone
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    • "The COX-2 gene was expressed in osteoblasts and other cells at the fracture site. Unlike BMP-2 and FGF-2 that were expressed from retroviral vectors, application of the COX-2 retroviral vector accelerated bony union of the callus tissues and hence improved healing of the fractured bone, possibly through angiogenic mechanisms (Rundle et al., 2008). Similar results were observed in PTH treatments in the rat femur fracture, although unlike COX-2 gene therapy, this approach required repeated applications of PTH (Alkhiary et al., 2005). "

    Full-text · Chapter · Aug 2011
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