Adenovirus-mediated gene transfer to liver grafts - An improved method to maximize infectivity
Adenovirus-mediated gene transfer to liver grafts - An improved method to maximize infectivity
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DOI：
10.1097/00007890-199812150-00020
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发表时间：
1998-12-15
期刊：
影响因子：
6.2
通讯作者：
Murase, N
中科院分区：
文献类型：
--
作者：
Chia, SH;Geller, DA;Murase, N
背景腺病毒基因治疗在肝移植中具有许多潜在的应用，但目前的移植载体递送方法缺乏效率并且需要高滴度。在本研究中，我们尝试使用编码 LacZ 标记基因 (AdLacZ) 的腺病毒载体，使用三种不同的肝移植递送方法来提高基因递送效率。 AdLacZ 通过以下方式递送至冷保存的大鼠肝移植物：(1) 通过门静脉连续灌注（门静脉灌注），(2) 通过门静脉和肝动脉连续灌注（双灌注），以及 (3) 通过夹紧流出物（夹紧技术）将病毒灌注物捕获在肝血管系统中。结果。使用 1x10(9) 斑块形成单位的 AdLacZ（感染复数为 0.4），移植后 48 小时通过 5-溴-4-色-3-吲哚基-β-D-半乳糖苷染色和 β-半乳糖苷酶测定确定 3 小时保存的肝移植物中的转导率，采用钳夹技术是最佳的（21.5+/-2.7%） 5-溴-4-色-3-吲哚基-β-D-吡喃半乳糖苷阳性细胞和81.1+/-3.6 U/g β-半乳糖苷酶），然后进行双重灌注（18.5+/-1.8%、66.6+/-19.4 U/g）和门静脉灌注（8.8+/-2.5%、19.7+/-15.4 U/g）。使用钳夹技术的进一步研究表明，在感染复数为 0.4 且冷缺血时间延长至 18 小时时，基因转移率接近最大 30%。转基因表达稳定2周，并在第28天缓慢下降至7.8+/-12.1%。通过组织病理学检查和肝酶证实缺乏炎症反应。在肝细胞中选择性诱导转导，而在肺和脾中几乎没有肝外转基因表达。结论。钳夹技术为冷保存的肝移植物提供了一种高效的病毒基因递送方法。该方法以最少的技术操作提供了腺病毒载体的最大感染性。
Background Adenoviral gene therapy in liver transplantation has many potential applications, but current vector delivery methods to grafts lack efficiency and require high titers. In this study, we attempted to improve gene delivery efficacy using three different delivery methods to liver grafts with adenoviral vector encoding the LacZ marker gene (AdLacZ).Methods. AdLacZ was delivered to cold preserved rat liver grafts by: (1) continuous perfusion via the portal vein (portal perfusion), (2) continuous perfusion via both the portal vein and hepatic artery (dual perfusion), and (3) trapping viral perfusate in the liver vasculature by clamping outflow (clamp technique).Results. Using 1x10(9) plaque-forming units of AdLacZ (multiplicity of infection of 0.4), transduction rate in 3-hr preserved liver grafts, determined by 5-bromo-4-chromo-3-indolyl-beta-D-galactopyranoside staining and beta-galactosidase assay 48 hr after transplantation, was best with clamp technique (21.5+/-2.7% 5-bromo-4-chromo-3-indolyl-beta-D-galactopyranoside- positive cells and 81.1+/-3.6 U/g beta-galactosidase), followed by dual perfusion (18.5+/-1.8%, 66.6+/-19.4 U/g) and portal perfusion (8.8+/-2.5%, 19.7+/-15.4 U/g). Further studies using clamp technique demonstrated a near-maximal gene transfer rate of 30% at multiplicity of infection of 0.4 with prolonged cold ischemia to 18 hr. Transgene expression was stable for 2 weeks and slowly declined to 7.8+/-12.1% at day 28. Lack of inflammatory response was confirmed by histopathological examination and liver enzymes. Transduction was selectively induced in hepatocytes with nearly no extrahepatic transgene expression in the lung and spleen.Conclusions. The clamp technique provides a highly efficient viral gene delivery method to cold preserved liver grafts. This method offers maximal infectivity of adenoviral vector with minimal technical manipulation.