Hydroxyethyl Starch-Based Preservation Solutions Enhance Gene Therapy Vector Delivery Under Hypothermic Conditions
Hydroxyethyl Starch-Based Preservation Solutions Enhance Gene Therapy Vector Delivery Under Hypothermic Conditions
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DOI：
10.1002/lt.21623
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发表时间：
2008-12-01
影响因子：
4.6
通讯作者：
van der Laan, Luc J. W.
中科院分区：
文献类型：
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作者：
Henry, Scot D.;van der Wegen, Pascal;van der Laan, Luc J. W.
离体肝脏灌注为安全、有效的基因治疗提供了独特的前景，可以针对同种异体移植排斥或丙型肝炎病毒（HCV）再感染等复发性疾病。我们的目的是研究低温条件下器官保存溶液对基于载体的基因治疗的影响。威斯康星大学 (UW) 溶液、组氨酸色氨酸酮戊二酸 (HTK)、EloHaes、聚乙二醇钠-UW 溶液 [Institut Georges Lopez 1 溶液 (IGL-1)] 和 Dulbecco 改良 Eagle's 培养基 (DMEM) 培养基（对照）在 2°C 或 37°C 下测试慢病毒载体转导效率肝癌细胞系 Huh-7 和原代人或小鼠肝细胞。表达短发夹 RNA 的慢病毒载体用于靶向 HCV 复制。使用有效的短发夹 RNA 载体，转导与治疗效果直接相关，低转导产生低敲低，反之亦然。载体孵育时间短至 10 分钟即可观察到绿色荧光蛋白 (GFP) 报告基因表达。 37 摄氏度孵育 2 小时后，UW 的转导率最高 (62%+/- 6 SEM)；它们显着高于 HTK (21%+/- 7 SEM)。尽管添加羟乙基淀粉 (HES) 显着改善了转导，但 UW 中存在的腺苷和谷胱甘肽在补充 HTK 时均未增加转导。为了排除尺寸排阻作为 HES 机制的可能性，对 IGL-1 进行了测试，但并未产生比 HTK 或 DMEM 更好的转导效果。当补充 UW 时，阴离子化合物减少了转导，这表明 HES 的电荷相互作用机制。总之，本研究表明在低温肝脏灌注条件下可以实现有效的载体递送。与非淀粉溶液相比，华盛顿大学提供了更好的肝细胞转导能力。肝脏移植 14:1708-1717, 2008。(C) 2008 AASLD。
Isolated liver perfusion offers a unique prospect for safe, effective targeting of gene therapies that can be directed against allograft rejection or recurrent diseases such as reinfection by hepatitis C virus (HCV). We aimed to examine the effect of organ preservation solutions on vector-based gene therapy delivery under hypothermic conditions. University of Wisconsin (UW) solution, histidine tryptophan ketoglutarate (HTK), EloHaes, sodium-poly(ethylene glycol)-UW solution [Institut Georges Lopez 1 solution (IGL-1)], and Dulbecco's modified Eagle's medium (DMEM) culture medium (control) were tested at 2 degrees C or 37 degrees C for lentiviral vector transduction efficiencies to the hepatoma cell line Huh-7 and primary human or mouse hepatocytes. Lentiviral vectors expressing short hairpin RNA were used to target HCV replication. With a potent short hairpin RNA vector, transductions were directly correlated to the therapeutic effect, with low transduction yielding low knockdown and vice versa. Green fluorescent protein (GFP) reporter gene expression was observed with vector incubation times as short as 10 minutes. The highest transductions were seen, after 2-hour 37 degrees C incubation, in UW (62%+/- 6 SEM); they were significantly higher than those in HTK (21%+/- 7 SEM). Neither adenosine nor glutathione, present in UW, provided any increase in transduction when supplemented to HTK, although the addition of hydroxyethyl starch (HES) significantly improved transductions. To rule out size exclusion as a mechanism of HES, IGL-1 was tested but did not result in better transductions than HTK or DMEM. When supplemented to UW, anionic compounds reduced transduction, and this indicated a charge interaction mechanism of HES. In conclusion, this study demonstrates that effective vector delivery can be achieved under conditions of hypothermic liver perfusion. UW provides superior transduction to hepatocytes over nonstarch solutions. Liver Transpl 14:1708-1717, 2008. (C) 2008 AASLD.