Wiseman J W, Goddard C A, McLelland D, Colledge W H
1Department of Physiology, University of Cambridge, Cambridge, UK.
Gene Ther. 2003 Sep;10(19):1654-62. doi: 10.1038/sj.gt.3302050.
Polyethylenimine (PEI), a polycation with high ionic charge density, has recently been used as a gene therapy delivery agent. We have defined the optimal conditions for PEI-based transfection of airway epithelial cells in vitro and in vivo and used these conditions to restore Cl(-) channel activity in a CF mouse model. Three forms of PEI, a linear 22 kDa (ExGen 500) form and branched 25 or 50 kDa forms were evaluated. All forms of PEI significantly increased luciferase reporter gene expression compared to the liposome DCChol/DOPE in a human bronchial epithelial cell line (16HBE) irrespective of the extent of cell confluency. With subconfluent cells, gene expression was around 1000-, 200- and 25-fold higher than liposomes using linear 22, 25 and 50 kDa PEI, respectively. The transfection efficiency was reduced in confluent and polarized epithelial cells but linear 22 kDa PEI showed the smallest decrease and gave 8000-fold better transfection in polarized cells compared to liposomes. A comparison of linear 22 or 25 kDa PEI with DCChol/DOPE for airway delivery in vivo via intranasal instillation was also performed. Linear 22 kDa PEI gave significantly better luciferase reporter gene expression of 350-fold in the lung, 180-fold in the nose and 85-fold in the trachea compared to liposome. In contrast, the 25 kDa form of PEI was no better than DCChol/DOPE. Repeat dosing with linear 22 kDa PEI failed to give reporter gene delivery comparable to the initial dose. To establish that PEI can be used to deliver a physiologically relevent gene in vivo, we used it to restore Cl(-) secretion by CFTR gene delivery in the airways of a CF mouse model.
聚乙烯亚胺（PEI）是一种具有高离子电荷密度的聚阳离子，最近被用作基因治疗递送剂。我们已经确定了基于PEI的气道上皮细胞体外和体内转染的最佳条件，并利用这些条件在囊性纤维化（CF）小鼠模型中恢复Cl⁻通道活性。评估了三种形式的PEI，即线性22 kDa（ExGen 500）形式和分支的25 kDa或50 kDa形式。在人支气管上皮细胞系（16HBE）中，与脂质体DCChol/DOPE相比，所有形式的PEI均显著增加了荧光素酶报告基因的表达，且与细胞汇合程度无关。对于亚汇合细胞，使用线性22 kDa、25 kDa和50 kDa PEI时，基因表达分别比脂质体高约1000倍、200倍和25倍。在汇合和极化的上皮细胞中，转染效率降低，但线性22 kDa PEI的降低幅度最小，与脂质体相比，在极化细胞中的转染效果要好8000倍。还比较了线性22 kDa或25 kDa PEI与DCChol/DOPE通过鼻内滴注在体内进行气道递送的情况。与脂质体相比，线性22 kDa PEI在肺中的荧光素酶报告基因表达显著更好，为350倍，在鼻中为180倍，在气管中为85倍。相比之下，25 kDa形式的PEI并不比DCChol/DOPE好。用线性22 kDa PEI重复给药未能实现与初始剂量相当的报告基因递送。为了确定PEI可用于在体内递送生理相关基因，我们利用它通过在CF小鼠模型的气道中递送CFTR基因来恢复Cl⁻分泌。
