Targeting transgene to the heart and liver with AAV9 by different promoters
Targeting transgene to the heart and liver with AAV9 by different promoters
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通过不同的启动子将 AAV9 转基因靶向心脏和肝脏
DOI：
10.1111/1440-1681.12453
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发表时间：
2015-10-01
影响因子：
2.9
通讯作者：
Gao, Xiao-Ming
中科院分区：
文献类型：
--
作者：
Chen, Bang-Dang;He, Chun-Hui;Gao, Xiao-Ming
腺相关病毒（Adeno-associated virus，AAV）已成为基因治疗中最有前途的基因转移工具之一。本研究旨在评价重组杆状病毒（rBac）系统生产的AAV 9载体的嗜性、基因转移效率和安全性。使用rBac系统产生AAV 9-CMV-GFP和AAV 9-CBA-GFP，静脉内（i. v.）并在给药后1、2、3、4、5和8周处死动物。通过荧光成像和Western blot分析GFP在不同器官中的表达。使用qPCR测量病毒基因组数量。通过流式细胞术测定AAV 9载体在原代心肌细胞和肝细胞中的体外转导效率。通过体内测定某些心脏和肝脏损伤生物标志物和肾功能测试以及体外TUNEL分析来评估AAV 9载体的毒性。数据显示，由rBac系统包装的AAV 9病毒颗粒在体内和体外均具有完全功能。CMV启动子主要诱导较高的心脏GFP转基因表达和DNA拷贝数，而CBA启动子导致小鼠肝脏中稳健的GFP表达和高载体DNA拷贝数，两者均以时间依赖性增加的方式。早在合并感染后3天和5天，在心脏和肝脏中也观察到这种明显的优先效应。AAV 9-CMV和AAV 9-CBA病毒包装均不诱导心、肝和肾损伤和细胞凋亡。这些结果表明，AAV 9-CMV可以有效和安全地指导心脏基因转移，而AAV 9-CBA优先用于肝脏基因转移。
Adeno-associated virus (AAV) has become one of the most promising gene transfer tools for gene therapy. This work aims to evaluate tropism, gene transfer efficiency and safety of AAV9 vectors produced with recombinant baculovirus (rBac)-based system. AAV9-CMV-GFP and AAV9-CBA-GFP were produced using a rBac system, 1 3 1011 particles of each vectors were administered intravenously (i.v.) into mice and animals were killed at 1, 2, 3, 4, 5 and 8 weeks after administration. The GFP expression in different organs was analyzed by fluorescence imaging and Western blot. Viral genomic quantities were measured using qPCR. In vitro transduction efficiency of AAV9 vectors in primary cardiomyocytes and hepatocytes was determined by flow cytometry. Toxicity of AAV9 vectors was evaluated by determining certain cardiac and liver injury biomarkers and renal function test in vivo and TUNEL analysis in vitro. The data showed that AAV9 viral particles packaged by the rBac system were fully functional in vivo and in vitro. The CMV promoter predominantly induced higher cardiac GFP transgene expression and DNA copy numbers while the CBA promoter resulted in robust GFP expression and high vector DNA copy numbers in mouse liver, both in a time-dependent increased manner. Such distinct preferential effects were also observed in the heart and liver as early as 3 and 5 days after co-infection. Both the AAV9-CMV and AAV9-CBA viral packages did not induce heart, liver and renal damage and cell apoptosis. These results indicated that AAV9-CMV can efficiently and safely direct cardiac gene transfer, whereas AAV9-CBA is preferential for liver gene delivery.