Hepatic gene therapy: adenovirus enhancement of receptor-mediated gene delivery and expression in primary hepatocytes.
Hepatic gene therapy: adenovirus enhancement of receptor-mediated gene delivery and expression in primary hepatocytes.
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肝脏基因治疗：腺病毒增强原代肝细胞中受体介导的基因递送和表达。
DOI：
10.1073/pnas.90.6.2122
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发表时间：
1993
影响因子：
11.1
通讯作者：
Woo,SL
中科院分区：
文献类型：
--
作者：
Cristiano,RJ;Smith,LC;Woo,SL
我们将受体介导的DNA递送系统与腺病毒的内体裂解能力相结合，表明DNA可以被递送到原代肝细胞，导致基因的高水平表达。用去唾液酸偶联聚L-赖氨酸通过与肝去唾液酸糖蛋白受体结合将大肠杆菌β-半乳糖苷酶基因导入原代肝细胞时，只检测到低水平的β-半乳糖苷酶，转染率不到0.1%。这种活性水平可以通过DNA蛋白复合体与复制缺陷型腺病毒的共内化而大大提高，导致100%的肝细胞用5-溴-4-氯-3-吲哚-β-D-半乳糖苷染成蓝色。对β-半乳糖苷酶表达的定量分析也显示，活性提高了1000倍。为了测试这种DNA传递系统对纠正苯丙酮尿症的适用性，苯丙酮尿症是一种会导致儿童严重智力低下的代谢紊乱，我们将人苯丙氨酸羟基酶(PAH)基因传递给来自PAH缺陷小鼠品系的肝细胞，并展示了酶活性的完全重建。这种方法显示了将基因高效地输送到肝脏以纠正肝脏疾病的巨大希望。
We have combined a receptor-mediated DNA delivery system with the endosomal lysis ability of adenovirus and shown that DNA can be delivered into primary hepatocytes, resulting in a high level of gene expression. When asialoorosomucoid conjugated with poly(L-lysine) was used to deliver the Escherichia coli beta-galactosidase gene into primary hepatocytes through binding with the hepatic asialoglycoprotein receptor, only a low level of beta-galactosidase was detectable, with less than 0.1% of the hepatocytes being transfected. This level of activity can be greatly enhanced by the cointernalization of the DNA.protein complex with a replication-defective adenovirus, resulting in 100% of the hepatocytes staining blue with 5-bromo-4-chloro-3-indolyl beta-D-galactoside. Quantitative analysis of beta-galactosidase expression also showed a 1000-fold enhancement of activity. To test the applicability of this DNA delivery system for the correction of phenylketonuria, a metabolic disorder that causes severe mental retardation in children, we have delivered the human phenylalanine hydroxylase (PAH) gene to hepatocytes derived from a PAH-deficient mouse strain and demonstrated complete reconstitution of enzymatic activity. This method shows great promise for efficient gene delivery to the liver for correction of hepatic disorders.