CONSTITUTIVE AND INDUCIBLE CO-EXPRESSION SYSTEMS FOR NON-VIRAL OSTEOINDUCTIVE GENE THERAPY
CONSTITUTIVE AND INDUCIBLE CO-EXPRESSION SYSTEMS FOR NON-VIRAL OSTEOINDUCTIVE GENE THERAPY
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DOI：
10.22203/ecm.v027a13
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发表时间：
2014-01-01
影响因子：
3.1
通讯作者：
Redl, H.
中科院分区：
文献类型：
--
作者：
Feichtinger, G. A.;Hofmann, A. T.;Redl, H.
组织再生基因疗法需要递送治疗有效量的转基因的表达策略。由于生理表达模式比单一治疗基因的高水平表达更复杂，本研究旨在同时进行两种转基因的组成型或诱导型共表达，共表达人骨形态发生蛋白2和7（BMP 2/7）用骨钙素报告基因测定和成骨细胞标准测定在体外C2C12细胞中评价了来自组成型表达和多西环素诱导质粒的分化组成系统进行了额外的测试，在体内试点异位骨形成后，重复裸DNA注射到小鼠肌肉tissue.Inductor控制的分化在体外诱导共表达。两种共表达系统，诱导型和组成型，实现了显着更好的成骨分化比单因子表达。组成型共表达系统的效力取决于相对表达盒拓扑结构。在体内，异位骨形成在6/13只动物中得到证实（46%的骨形成效率）体外研究结果表明，所设计的单一载体BMP 2/7共表达策略介导上级骨诱导，可以以诱导物控制的方式应用，并且其效率取决于表达盒拓扑结构。体内结果表明，通过非病毒裸DNA基因转移应用的BMP 2/7的共表达有效地介导骨形成，而不应用生物材料、细胞或重组生长因子，为当前的治疗策略提供了一种有前景的替代方案，具有未来临床转化的潜力。
Tissue regenerative gene therapy requires expression strategies that deliver therapeutic effective amounts of transgenes. As physiological expression patterns are more complex than high-level expression of a singular therapeutic gene, we aimed at constitutive or inducible co-expression of 2 transgenes simultaneously.Co-expression of human bone morphogenetic protein 2 and 7 (BMP2/7) from constitutively expressing and doxycycline inducible plasmids was evaluated in vitro in C2C12 cells with osteocalcin reporter gene assays and standard assays for osteogenic differentiation. The constitutive systems were additionally tested in an in vivo pilot for ectopic bone formation after repeated naked DNA injection to murine muscle tissue.Inductor controlled differentiation was demonstrated in vitro for inducible co-expression. Both co-expression systems, inducible and constitutive, achieved significantly better osteogenic differentiation than single factor expression. The potency of the constitutive co-expression systems was dependent on relative expression cassette topology. In vivo, ectopic bone formation was demonstrated in 6/13 animals (46 % bone formation efficacy) at days 14 and 28 in hind limb muscles as proven by in vivo mu CT and histological evaluation.In vitro findings demonstrated that the devised single vector BMP2/7 co-expression strategy mediates superior osteoinduction, can be applied in an inductor controlled fashion and that its efficiency is dependent on expression cassette topology. In vivo results indicate that co-expression of BMP2/7 applied by non-viral naked DNA gene transfer effectively mediates bone formation without the application of biomaterials, cells or recombinant growth factors, offering a promising alternative to current treatment strategies with potential for clinical translation in the future.