Suicide gene approach using a dual-expression lentiviral vector to enhance the safety of ex vivo gene therapy for bone repair
Suicide gene approach using a dual-expression lentiviral vector to enhance the safety of ex vivo gene therapy for bone repair
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DOI：
10.1038/gt.2013.66
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发表时间：
2014-02-01
期刊：
影响因子：
5.1
通讯作者：
Lieberman, J. R.
中科院分区：
文献类型：
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作者：
Alaee, F.;Sugiyama, O.;Lieberman, J. R.
使用病毒载体过度表达BMP-2的“离体”基因治疗显示出在实验动物中治愈临界尺寸的骨缺损。为了提高其安全性，我们构建了一个双表达慢病毒载体，以过表达BMP-2或荧光素酶和HSV 1-tk类似物Delta tk（LV-Delta tk-T2 A-BMP-2/Luc）。我们假设给予更昔洛韦（GCV）将消除植入部位的转导细胞。与单基因载体相比，载体诱导的小鼠基质细胞系（W-20-17细胞）和小鼠骨髓细胞（MBMCs）中BMP-2和荧光素酶的表达减少了50%。与MBMCs相比，W-20-17细胞对GCV更敏感（MBMCs中GCV为1 μ g/ml时，12天时90 - 95%的细胞死亡，而W-20-17细胞中GCV为0.1 μ g/ml时，5天时90- 95%的细胞死亡）。LV-Δ tk-T2 A-BMP-2转导的MBMC的植入在4周时愈合了2 mm的股骨缺损。术后早期GCV治疗（0-14天）阻断了骨形成，证实了生物学反应。从第14天开始的延迟GCV处理2或4周减少了来自LV-Δ tk-T2 A-Luc转导的MBMC的荧光素酶信号，但信号没有完全消除。这些数据表明，这种自杀基因策略在未来具有临床应用的潜力，但需要进行优化以提高效率。
'Ex vivo' gene therapy using viral vectors to overexpress BMP-2 is shown to heal critical-sized bone defects in experimental animals. To increase its safety, we constructed a dual-expression lentiviral vector to overexpress BMP-2 or luciferase and an HSV1-tk analog, Delta tk (LV-Delta tk-T2A-BMP-2/Luc). We hypothesized that administering ganciclovir (GCV) will eliminate the transduced cells at the site of implantation. The vector-induced expression of BMP-2 and luciferase in a mouse stromal cell line (W-20-17 cells) and mouse bone marrow cells (MBMCs) was reduced by 50% compared with the single-gene vector. W-20-17 cells were more sensitive to GCV compared with MBMCs (90-95% cell death at 12 days with GCV at 1 mu g ml(-1) in MBMCs vs 90-95% cell death at 5 days by 0.1 mu g ml(-1) of GCV in W-20-17 cells). Implantation of LV-Delta tk-T2A-BMP-2 transduced MBMCs healed a 2mm femoral defect at 4 weeks. Early GCV treatment (days 0-14) postoperatively blocked bone formation confirming a biologic response. Delayed GCV treatment starting at day 14 for 2 or 4 weeks reduced the luciferase signal from LV-Delta tk-T2A-Luc-transduced MBMCs, but the signal was not completely eliminated. These data suggest that this suicide gene strategy has potential for clinical use in the future, but will need to be optimized for increased efficiency.