Preparation of Trojan horse liposomes (THLs) for gene transfer across the blood-brain barrier.
Preparation of Trojan horse liposomes (THLs) for gene transfer across the blood-brain barrier.
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DOI：
10.1101/pdb.prot5407
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发表时间：
2010-04
影响因子：
--
通讯作者：
W. Pardridge
中科院分区：
文献类型：
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作者：
W. Pardridge
非病毒质粒DNA在静脉内施用封装在特洛伊木马脂质体（THL）（也称为聚乙二醇化免疫脂质体（PIL））中的DNA后经由穿过血脑屏障（BBB）的经血管途径递送至脑。脂质体表面覆盖有数千股聚合物（例如，聚乙二醇[PEG]），并且1%-2%的聚合物链的尖端与充当分子特洛伊木马（MTH）的靶向单克隆抗体缀合。MTH结合受体（例如，对于转铁蛋白或胰岛素），在体内触发受体介导的THL穿过BBB的转胞吞作用，以及受体介导的内吞作用进入BBB以外的脑细胞。转基因表达在脑中的持续性与附加型质粒DNA的降解速率呈负相关。THL技术使外源基因能够在单次静脉给药的1天内在成年脑（或其他器官）的大多数细胞中广泛表达。THL的应用包括使用组织特异性启动子的组织特异性基因表达，在静脉内酪氨酸羟化酶基因治疗后实验性帕金森病中纹状体酪氨酸羟化酶的完全正常化，在使用THL的每周静脉内反义基因治疗后患有脑肿瘤的小鼠的存活时间增加100%，每周静脉注射RNA干扰（RNAi）基因治疗颅内脑肿瘤小鼠的生存时间增加90%。该方案描述了用于体外或体内基因转移的THL的制备。
Nonviral plasmid DNA is delivered to the brain via a transvascular route across the blood-brain barrier (BBB) following intravenous administration of DNA encapsulated within Trojan horse liposomes (THLs), also called PEGylated immunoliposomes (PILs). The liposome surface is covered with several thousand strands of polymer (e.g., polyethylene glycol [PEG]), and the tips of 1%-2% of the polymer strands are conjugated with a targeting monoclonal antibody that acts as a molecular Trojan horse (MTH). The MTH binds to a receptor (e.g., for transferrin or insulin) on the BBB and brain cell membrane, triggering receptor-mediated transcytosis of the THL across the BBB in vivo, and receptor-mediated endocytosis into brain cells beyond the BBB. The persistence of transgene expression in the brain is inversely related to the rate of degradation of the episomal plasmid DNA. THL technology enables an exogenous gene to be widely expressed in the majority of cells in adult brain (or other organs) within 1 d of a single intravenous administration. Applications of the THLs include tissue-specific gene expression with tissue-specific promoters, complete normalization of striatal tyrosine hydroxylase in experimental Parkinson's disease following intravenous tyrosine hydroxylase gene therapy, a 100% increase in survival time of mice with brain tumors following weekly intravenous antisense gene therapy using THLs, and a 90% increase in survival time with weekly intravenous RNA interference (RNAi) gene therapy in mice with intracranial brain tumors. This protocol describes the preparation of THLs for use in gene transfer in vitro or in vivo.