Microtubular disruption prolongs the expression of human bilirubin-uridinediphosphoglucuronate-glucuronosyltransferase-1 gene transferred into gunn rat livers

Abstract

DNA delivered to the liver by asialoglycoprotein receptor-mediated endocytosis is degraded in lysosomes within 48 h. To test the hypothesis that microtubular disruption should promote transgene persistence by interrupting endosomal translocation to lysosomes, plasmids containing bacterial chloramphenicol acetyltransferase (pSV2-CAT) or human bilirubin-UDP-glucuronosyltransferase-1 (pSVKS-hBUGT1) genes were complexed with asialoglycoprotein-polylysine conjugates, and 1 mg of the complexed DNA was injected intravenously into bilirubin-UDP-glucuronosyltransferase-deficient Gunn rats. 30 min before DNA injection, one group received 0.75 mg of colchicine/kg of body weight intraperitoneally, which was shown by immununofluoreseent confocal microscopy to disrupt the microtubular network. Control rats received normal saline. In colchicine-pretreated rats receiving pSV2-CAT, hepatic chloramphenicol acetyltransferase activity persisted for 9-14 weeks, whereas in the saline-pretreated group the activity was detectable for 48 h only. In colchicine-pretreated Gunn rats receiving pSVK3-hBUGT1, the DNA persisted in liver for 10 weeks, bilirubin glucuronides were excreted in bile, and serum bilirubin levels declined by 25-35% in 2-4 weeks and remained reduced for 8 weeks. Without colchicine pretreatment, the DNA was detectable in liver for 2 days only, and serum bilirubin levels were not reduced. Thus, microtubular disruption provides a noninvasive method for prolonging the effect of liver-targeted gene therapy.