Brooks Alan R, Harkins Richard N, Wang Peiyin, Qian Hu Sheng, Liu Pengxuan, Rubanyi Gabor M
Department of Gene Therapy, Berlex Biosciences, Richmond, CA 94804, USA.
J Gene Med. 2004 Apr;6(4):395-404. doi: 10.1002/jgm.516.
Although the transient nature of transgene expression using first-generation adenovirus (Ad) vectors is well known, the exact mechanisms responsible for this phenomenon are uncertain.
Rats were given intramuscular (i.m.) injections of a first-generation Ad containing the human fibroblast growth factor 4 (hFGF-4) gene driven by the cytomegalovirus (CMV) promoter and enhancer (CMV-PE). The copy number of hFGF-4 mRNA and viral DNA was measured in the same muscles by quantitative RT-PCR and quantitative PCR at times between 1 h and 84 days after virus injection. Quantitative Southern blot analysis for the intact hFGF-4 transcription unit DNA was also performed, and the methylation status of the CMV-PE DNA in the muscle was determined using bisulfite sequencing.
The copy number of hFGF-4 mRNA peaked at 6 h then decreased 56-fold by 24 h, and a further 240-fold between days 3 and 28. Although the viral DNA copy number also decreased 23-fold between 6 h and 28 days, the ratio of copies of hFGF-4 mRNA per copy of viral DNA decreased 385-fold over this period. Methylation of the CMV-PE DNA in the muscle at both CpG and non-CpG sites was observed 24 h after virus administration and had increased at day 7.
Decreased transcription associated with extensive methylation of the CMV-PE was the major mechanism responsible for the decrease in transgene mRNA levels. Strategies for preventing transcriptional silencing will be valuable for improving the duration of transgene expression from adenoviral vectors.
尽管使用第一代腺病毒（Ad）载体时转基因表达的短暂性是众所周知的，但导致这种现象的确切机制尚不确定。
给大鼠肌肉注射由巨细胞病毒（CMV）启动子和增强子（CMV-PE）驱动的含人成纤维细胞生长因子4（hFGF-4）基因的第一代腺病毒。在病毒注射后1小时至84天之间的不同时间，通过定量逆转录聚合酶链反应（RT-PCR）和定量聚合酶链反应（PCR）测量同一肌肉中hFGF-4 mRNA和病毒DNA的拷贝数。还对完整的hFGF-4转录单位DNA进行了定量Southern印迹分析，并使用亚硫酸氢盐测序法确定了肌肉中CMV-PE DNA的甲基化状态。
hFGF-4 mRNA的拷贝数在6小时达到峰值，然后在24小时时下降56倍，在第3天至28天之间进一步下降240倍。尽管病毒DNA拷贝数在6小时至28天之间也下降了23倍，但在此期间，每拷贝病毒DNA的hFGF-4 mRNA拷贝数的比率下降了385倍。在病毒给药后24小时观察到肌肉中CMV-PE DNA在CpG和非CpG位点的甲基化，并且在第7天有所增加。
与CMV-PE广泛甲基化相关的转录减少是转基因mRNA水平下降的主要机制。预防转录沉默的策略对于延长腺病毒载体转基因表达的持续时间将是有价值的。
