Hydrodynamic plasmid DNA gene therapy model in liver transplantation.
Hydrodynamic plasmid DNA gene therapy model in liver transplantation.
复制标题
肝移植中的流体动力学质粒DNA基因治疗模型。
DOI：
10.1016/j.jss.2006.04.020
复制
发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Geller,DavidA
中科院分区：
文献类型：
--
作者：
Tsoulfas,George;Takahashi,Yoshihito;Liu,Dexi;Yagnik,Gautam;Wu,Tong;Murase,Noriko;Geller,DavidA
背景通过对移植物进行基因修饰以减少保存损伤或同种异体排斥反应，在移植领域引起了极大的兴趣。尽管腺病毒基因转移在实验性肝移植中有效，但病毒毒性和安全性问题限制了其在临床试验中的潜在应用。因此，本研究的目的是建立一种在肝移植环境中进行非病毒基因转移的模型，以允许高效的转基因表达。材料和方法采用威斯康星大学(UW)保存的冷缺血3h的同种异体大鼠原位肝移植。建立了一种流体动力基因转移技术，通过阻断其他血管，将DNA通过体外静脉快速注入UW内，将质粒DNA输送到移植肝。使用标记基因荧光素酶和分泌型人α1-抗胰蛋白酶(α1-AT)的表达载体。通过移植肝组织学和肝脏转氨酶来评估肝损伤。用肝荧光素酶相对光单位活性和血清α1-AT蛋白水平检测转基因表达。研究的变量包括：(A)注射量对移植肝静脉压力的影响；(B)肝脏的损伤，通过肝酶和组织病理学测量；(C)叶之间的可变表达；(D)载体注射的UW体积；(E)DNA质粒量；(F)所用启动子的类型；(G)夹闭时间；以及(H)标记基因表达的时间进程。结果对照组大鼠接受了标准的同种原位大鼠肝移植，没有检测到肝脏荧光素酶活性或血清中人α1-AT。由于荧光素酶的表达不随剂量的增加而增加，因此最适剂量为400μg/只肝移植。此外，巨细胞病毒启动子比Rous肉瘤病毒有更高的表达。较高的注射压力梯度可以更有效地表达转基因，但也会造成更大的肝脏损伤，表现为转氨酶升高和小叶中心坏死。将注射量从移植物重量的75%降至50%，可将肝脏损伤减少4.5倍。虽然较高的UW注射量与表达增加相关，但仅50%的UW注射量导致荧光素酶的表达高达10,000,000 RLU/mg；这种表达在不同的肝叶中是均匀的。人α1-AT在受体血中早在6h即可检测到，24 h达高峰，并持续高达5d。结论我们开发了一种非病毒基因转移技术，该技术可以在冷保存的肝血管床上施加静压，从而实现有效的质粒传递。这一简单的策略应该被证明对在移植环境中对肝移植进行基因改造很有用。
BACKGROUNDThere is great interest in the field of transplantation to genetically modify grafts to decrease preservation injury or allograft rejection. Although adenoviral gene transfer has been effective in experimental liver transplantation, viral toxicity and safety concerns limit potential use in clinical trials. Therefore, the purpose of this study was to develop a model of nonviral gene transfer in the liver transplant setting, allowing for efficient transgene expression.MATERIALS AND METHODSOrthotopic syngeneic rat liver transplantation was performed with 3 h cold ischemia using University of Wisconsin (UW) preservation. A hydrodynamic gene transfer technique was developed where plasmid DNA was delivered to the liver graft by ex vivo rapid infusion of DNA in UW via the IVC with other vessels clamped. Expression plasmids for the marker genes luciferase and secreted human α1-antitrypsin (α1-AT) were used. Hepatic injury was assessed by graft histology and liver transaminases. Transgene expression was determined by hepatic luciferase relative light units activity (RLU) and serum α1-AT protein levels. Variables examined included the effect of (a) volume injected on the intravenous pressure in the liver graft; (b) injury to the liver, as measured by hepatic enzymes and histopathology; (c) variable expression between lobes; (d) volume of UW that the plasmid is administered in; (e) amount of DNA plasmid; (f) type of the promoter used; (g) clamp time; as well as (h) the time course of the marker gene expression.RESULTSControl rats underwent standard orthotopic syngeneic rat liver transplantation and had no detectable hepatic luciferase activity or serum human α1-AT. The optimal DNA plasmid dose was found to be 400 μg/liver graft, as there was no increase in the luciferase expression by increasing the dose. Furthermore, cytomegalovirus promoter yielded greater expression than Rous sarcoma virus. A high injection pressure gradient allowed for more efficient transgene expression, but produced greater liver injury shown by elevated transaminases and centrilobular necrosis. Lowering injection volume from 75 to 50% of graft weight decreased liver injury by 4.5-fold. Although higher UW injection volumes were associated with increased expression, volumes of only 50% led to luciferase expression up to 10,000,000 RLU/mg; this expression was homogeneous between the different liver lobes. Human α1-AT was detected in recipient blood as early as 6 h, peaked at 24 h, and remained high for 5 days.CONCLUSIONSWe have developed a nonviral gene transfer technique where hydrostatic pressure across the cold-preserved liver vascular bed allows for efficient plasmid DNA delivery. This simple strategy should prove useful to genetically modify liver grafts in the transplantation setting.