Ultrasound and microbubble-assisted gene delivery in Achilles tendons: Long lasting gene expression and restoration of fibromodulin KO phenotype
Ultrasound and microbubble-assisted gene delivery in Achilles tendons: Long lasting gene expression and restoration of fibromodulin KO phenotype
复制标题
DOI：
10.1016/j.jconrel.2011.08.020
复制
发表时间：
2011-12-10
影响因子：
10.8
通讯作者：
Pichon, Chantal
中科院分区：
文献类型：
--
作者：
Delalande, Anthony;Bouakaz, Ayache;Pichon, Chantal
本研究的目的是利用超声和微泡将基因递送至跟腱。其原理是将超声辅助递送与超声诱导的蛋白质表达刺激相结合。我们发现，在存在5×10⁵个BR14微泡的情况下，向小鼠跟腱注射10μg编码荧光素酶基因的质粒，然后在1MHz、200kPa、40%占空比的条件下进行10分钟的超声暴露，可实现高效转染且无毒性。荧光素酶的表达率比单独注射质粒时高100倍。值得注意的是，荧光素酶转基因可稳定表达长达108天。从这些经超声穿孔的跟腱中提取的DNA可有效转化感受态大肠杆菌，这表明持久完整的质粒DNA是这种长期基因表达的原因。我们利用这种方法恢复了纤维调节蛋白基因敲除小鼠中纤维调节蛋白的表达。注射后一周，通过定量PCR检测到显著的纤维调节蛋白表达。有趣的是，对这些跟腱的超微结构分析显示，胶原纤维的直径分布和圆形度与野生型小鼠相似。我们的研究结果表明，这种基因递送方法在旨在调节愈合或修复退变肌腱的临床应用中具有前景，并且由于与病毒方法相比具有安全性，在基因治疗方面也具有很大的潜力。（C）2011爱思唯尔B.V.保留所有权利。
The aim of this study is to deliver genes in Achilles tendons using ultrasound and microbubbles. The rationale is to combine ultrasound-assisted delivery and the stimulation of protein expression induced by US. We found that mice tendons injected with 10 mu g of plasmid encoding luciferase gene in the presence of 5x10(5) BR14 microbubbles, exposed to US at 1 MHz, 200 kPa, 40% duty cycle for 10 min were efficiently transfected without toxicity. The rate of luciferase expression was 100-fold higher than that obtained when plasmid alone was injected. Remarkably, the luciferase transgene was stably expressed for up to 108 days. DNA extracted from these sonoporated tendons was efficient in transforming competent E. coli bacteria, indicating that persistent intact pDNA was responsible for this long lasting gene expression. We used this approach to restore expression of the fibromodulin gene in fibromodulin KO mice. A significant fibromodulin expression was detected by quantitative PCR one week post-injection. Interestingly, ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar to that of wild type mice. Our results suggest that this gene delivery method is promising for clinical applications aimed at modulating healing or restoring a degenerative tendon while offering great promise for gene therapy due its safety compared to viral methods. (C) 2011 Elsevier B. V. All rights reserved.