RESUMO
BACKGROUND: To date, the significant osteoinductive potential of bone morphogenetic protein 2 (BMP-2) non-viral gene therapy cannot be fully exploited therapeutically. This is mainly due to weak gene delivery and brief expression peaks restricting the therapeutic effect. OBJECTIVE: Our objective was to test the application of minicircle DNA, allowing prolonged expression potential. It offers notable advantages over conventional plasmid DNA. The lack of bacterial sequences and the resulting reduction in size, enables safe usage and improved performance for tissue regeneration. METHODS: We inserted an optimized BMP-2 gene cassette with minicircle plasmid technology. BMP-2 minicircle plasmids were produced in E. coli yielding plasmids lacking bacterial backbone elements. Comparative studies of these BMP-2 minicircles and conventional BMP-2 plasmids were performed in vitro in cell systems, including bone marrow derived stem cells. Tests performed included gene expression profiles and cell differentiation assays. RESULTS: A C2C12 cell line transfected with the BMP-2-Advanced minicircle showed significantly elevated expression of osteocalcin, alkaline phosphatase (ALP) activity, and BMP-2 protein amount when compared to cells transfected with conventional BMP-2-Advanced plasmid. Furthermore, the plasmids show suitability for stem cell approaches by showing significantly higher levels of ALP activity and mineralization when introduced into human bone marrow stem cells (BMSCs). CONCLUSION: We have designed a highly bioactive BMP-2 minicircle plasmid with the potential to fulfil clinical requirements for non-viral gene therapy in the field of bone regeneration.
Assuntos
Proteína Morfogenética Óssea 2/genética , Regeneração Óssea/genética , Terapia Genética , Osteogênese/genética , Fosfatase Alcalina/genética , Animais , Proteína Morfogenética Óssea 2/farmacologia , Diferenciação Celular/genética , Escherichia coli/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Humanos , Camundongos , Osteocalcina/genética , Plasmídeos/genética , Plasmídeos/farmacologia , TransfecçãoRESUMO
Monitoring of cell differentiation is a crucial aspect of cell-based therapeutic strategies depending on tissue maturation. In this study, we have developed a noninvasive reporter system to trace murine skeletal muscle differentiation. Either a secreted bioluminescent reporter (Metridia luciferase) or a fluorescent reporter (green fluorescent protein [GFP]) was placed under the control of the truncated muscle creatine kinase (MCK) basal promoter enhanced by variable numbers of upstream MCK E-boxes. The engineered pE3MCK vector, coding a triple tandem of E-Boxes and the truncated MCK promoter, showed twentyfold higher levels of luciferase activation compared with a Cytomegalovirus (CMV) promoter. This newly developed reporter system allowed noninvasive monitoring of myogenic differentiation in a straining bioreactor. Additionally, binding sequences of endogenous microRNAs (miRNAs; seed sequences) that are known to be downregulated in myogenesis were ligated as complementary seed sequences into the reporter vector to reduce nonspecific signal background. The insertion of seed sequences improved the signal-to-noise ratio up to 25% compared with pE3MCK. Due to the highly specific, fast, and convenient expression analysis for cells undergoing myogenic differentiation, this reporter system provides a powerful tool for application in skeletal muscle tissue engineering.
Assuntos
Diferenciação Celular , Creatina Quinase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência/métodos , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/citologia , Animais , Células Cultivadas , Creatina Quinase/genética , Elementos Facilitadores Genéticos , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Camundongos , Músculo Esquelético/metabolismo , Regiões Promotoras Genéticas , Razão Sinal-RuídoRESUMO
Detection of osteogenic differentiation is crucial for bone tissue engineering. Despite established standard end point assays, there is increasing demand for methods allowing noninvasive kinetic differentiation monitoring. Reporter gene assays employing tissue-specific promoters and suitable reporter genes fulfill these requirements. Many promoters, however, exhibit only weak cis-activating potential, thus limiting their application to generate sensitive reporter gene assays. Therefore, the aim of this study was to design a reporter gene assay employing elements of the murine osteocalcin promoter coupled to a viral enhancer for signal amplification. Additionally, the system's practicability was enhanced by introducing a secreted luciferase as a quantifiable reporter gene. The constructs were tested in C2C12 cells stimulated with recombinant human bone morphogenetic protein 2 for osteogenic differentiation in two-dimensional and three-dimensional culture. Osteogenic differentiation was confirmed by standard assays for osteogenesis. The reporter gene signal was detected through a secreted luciferase or fluorescence microscopy for enhanced yellow fluorescent protein. The constructs exhibited strong activation upon treatment with recombinant human bone morphogenetic protein 2. Weak background expression was observable in negative controls, attributed to the pan-active viral enhancer. In conclusion, a novel enhancer/tissue-specific promoter combination allows specific signal-amplified, kinetic monitoring of osteogenic differentiation in a nonsample-destructive manner.