Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
Private practice, Visalia, CA, USA.
Gene Ther. 2018 Jul;25(4):260-268. doi: 10.1038/s41434-018-0024-9. Epub 2018 Jun 15.
In this study, we developed a lentiviral two-step transcriptional amplification (TSTA) system expressing bone morphogenetic protein-2 (BMP-2) under the control of a GAL4FF transactivator to enhance gene expression and limit toxicity for bone repair applications. To this end human MSCs, isolated from bone marrow or adipose tissue, were transduced overnight with a LV-TSTA system (GAL4FF or GAL4vp16) expressing BMP-2 or GFP and evaluated in vitro for transduction efficiency, mean fluorescence intensity, cell viability, and BMP-2 production. FACS analysis of GFP-transduced MSCs confirmed successful transduction with the GAL4FF+GFP vector. Moreover, ELISA demonstrated abundant BMP-2 production by GAL4FF+BMP2-transduced human MSCs over a period of 8 weeks, with minimal cytotoxicity at all time points. Compared to GAL4vp16, GAL4FF was superior with respect to BMP production at 1, 2, 4, 6, and 8 weeks in BMSCs. In ASCs, GAL4FF was still associated with higher BMP-2 production at weeks 2-8, but this difference was not as prominent as in BMSCs. To our knowledge, this is the first report of GAL4FF-mediated BMP-2 production by human BMSCs and ASCs. Compared to the standard GAL4vp16TSTA vector, GAL4FF was associated with lower cytotoxicity and higher in vitro gene expression in both BMSCs and ASCs.
在这项研究中，我们开发了一种基于慢病毒的两步转录扩增（TSTA）系统，该系统在 GAL4FF 转录激活子的控制下表达骨形态发生蛋白-2（BMP-2），以增强基因表达并限制其毒性，从而应用于骨修复。为此，我们从骨髓或脂肪组织中分离出人骨髓间充质干细胞（MSCs），用表达 BMP-2 或 GFP 的 LV-TSTA 系统（GAL4FF 或 GAL4vp16）转导过夜，然后在体外评估转导效率、平均荧光强度、细胞活力和 BMP-2 的产生。对 GFP 转导的 MSCs 的 FACS 分析证实了 GAL4FF+GFP 载体的成功转导。此外，ELISA 表明，GAL4FF+BMP2 转导的人 MSCs 在 8 周内大量产生 BMP-2，且在所有时间点均具有最小的细胞毒性。与 GAL4vp16 相比，GAL4FF 在 BMSCs 中 1、2、4、6 和 8 周时在 BMP 产生方面具有优势。在 ASCs 中，GAL4FF 仍与 2-8 周时的 BMP-2 产生量较高相关，但与 BMSCs 相比，这种差异并不明显。据我们所知，这是首次报道 GAL4FF 介导的人 BMSCs 和 ASCs 中 BMP-2 的产生。与标准的 GAL4vp16TSTA 载体相比，GAL4FF 在 BMSCs 和 ASCs 中均与较低的细胞毒性和更高的体外基因表达相关。
