Chowdhury N R, Hays R M, Bommineni V R, Franki N, Chowdhury J R, Wu C H, Wu G Y
Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Biol Chem. 1996 Jan 26;271(4):2341-6. doi: 10.1074/jbc.271.4.2341.
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 (pSVK3-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 immunofluorescent 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.
通过去唾液酸糖蛋白受体介导的内吞作用递送至肝脏的DNA在48小时内在溶酶体中被降解。为了验证微管破坏应通过中断内体向溶酶体的转运来促进转基因持久性的假设，将含有细菌氯霉素乙酰转移酶（pSV2-CAT）或人胆红素-UDP-葡萄糖醛酸基转移酶-1（pSVK3-hBUGT1）基因的质粒与去唾液酸糖蛋白-聚赖氨酸缀合物复合，然后将1mg复合DNA静脉注射到胆红素-UDP-葡萄糖醛酸基转移酶缺陷的Gunn大鼠体内。在DNA注射前30分钟，一组大鼠腹腔注射0.75mg秋水仙碱/千克体重，免疫荧光共聚焦显微镜显示这会破坏微管网络。对照大鼠注射生理盐水。在接受pSV2-CAT的秋水仙碱预处理大鼠中，肝脏氯霉素乙酰转移酶活性持续9至14周，而在生理盐水预处理组中，该活性仅在48小时内可检测到。在接受pSVK3-hBUGT1的秋水仙碱预处理的Gunn大鼠中，DNA在肝脏中持续存在10周，胆红素葡萄糖醛酸酯在胆汁中排泄，血清胆红素水平在2至4周内下降25 - 35%，并在8周内保持降低。未经秋水仙碱预处理时，DNA仅在肝脏中可检测2天，血清胆红素水平未降低。因此，微管破坏提供了一种延长肝脏靶向基因治疗效果的非侵入性方法。
