Use of quantitative TaqMan real-time PCR to track the time-dependent distribution of gene transfer vectors in vivo
Use of quantitative TaqMan real-time PCR to track the time-dependent distribution of gene transfer vectors in vivo
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DOI：
10.1006/mthe.2000.0203
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发表时间：
2000-12-01
影响因子：
12.4
通讯作者：
Crystal, RG
中科院分区：
文献类型：
--
作者：
Hackett, NR;El Sawy, T;Crystal, RG
为了评估基因转移载体的生物分布和药代动力学，使用荧光TaqMan化学的实时PCR来定量心肌给药后腺病毒基因转移载体（Ad）的组织水平。在优化表达人血管内皮生长因子（Ad（GV）VEGF 121.10）和大肠杆菌胞嘧啶脱氨酶（Ad（GV）CD. 10）的Ad载体的基因组检测之后，比较心肌内注射与冠状动脉内递送至猪左心室。心肌内给药后1小时，左心室Ad基因组水平为每个细胞基因组6.2拷贝，比冠状动脉内给药后每个细胞基因组0.24拷贝的水平高26倍。相对于1小时后的载体水平，心肌内和冠状动脉内给药后24小时的量分别下降了14倍和5.5倍。有趣的是，在冠状动脉内或心肌内给药猪后逃逸左心室的载体主要在肺内发现，这与Ad载体在啮齿动物中的生物分布存在显著差异。在这点上，在对猪静脉注射后，在肺中发现90%的回收载体，并且即使在肝内门静脉注射后，55%的回收载体也在肺中。这些数据具有重要的意义，关于使用实验动物的安全性研究管理的广告给人类。
To assess the biodistribution and pharmacokinetics of gene transfer vectors, real-time PCR with fluorescent TaqMan chemistry was used to quantify tissue levels of adenovirus gene transfer vectors (Ad) following myocardial administration. After optimizing the detection of the genome of Ad vectors expressing human vascular endothelial growth factor (Ad(GV)VEGF121.10) and Escherichia coli cytosine deaminase (Ad(GV)CD.10), a comparison was made of intramyocardial injection versus intracoronary delivery to the left ventricle of the pig. One hour post-intramyocardial administration, the left ventricular Ad genome level was 6.2 copies per cellular genome, 26-fold higher than the level of 0.24 copies per cellular genome following intracoronary administration. Relative to the vector levels after 1 h, the amount dropped 14- and 5.5-fold by 24 h following intramyocardial and intracoronary administration, respectively. Interestingly, the vector that escaped the left ventricle after intracoronary or intramyocardial administration to pigs was found primarily within the lung, an observation in marked variance to the biodistribution of Ad vector in rodents. In this regard, after intravenous injection to the pig, 90% of the recovered vector was found in the lung, and even after intrahepatic portal vein injection, 55% of the recovered vector was in the lung. These data have important implications regarding the use of experimental animals for safety studies on administration of Ad to humans.