生物发光病毒粒子外壳：用于定量细胞和活体动物中腺相关病毒载体动力学的新工具。
Bioluminescent virion shells: new tools for quantitation of AAV vector dynamics in cells and live animals.
作者信息
Asokan A, Johnson J S, Li C, Samulski R J
机构信息
Department of Pharmacology, Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7352, USA.
出版信息
Gene Ther. 2008 Dec;15(24):1618-22. doi: 10.1038/gt.2008.127. Epub 2008 Jul 31.
Current technologies for visualizing infectious pathways of viruses rely on fluorescent labeling of capsid proteins by chemical conjugation or genetic manipulation. For noninvasive in vivo imaging of such agents in mammalian tissue, we engineered bioluminescent Gaussia luciferase-tagged Adeno-associated viral (gLuc/AAV) vectors. The enzyme was incorporated into recombinant AAV serotypes 1, 2 and 8 capsids by fusing to the N-terminus of the VP2 capsid subunit to yield bioluminescent virion shells. The gLuc/AAV vectors were used to quantify kinetics of cell-surface-binding by AAV2 capsids in vitro. Bioluminescent virion shells displayed an exponential decrease in luminescent signal following cellular uptake in vitro. A similar trend was observed following intramuscular injection in vivo, although the rate of decline in bioluminescent signal varied markedly between AAV serotypes. gLuc/AAV1 and gLuc/AAV8 vectors displayed rapid decrease in bioluminescent signal to background levels within 30 min, whereas the signal from gLuc/AAV2 vectors persisted for over 2 h. Bioluminescent virion shells might be particularly useful in quantifying dynamics of viral vector uptake in cells and peripheral tissues in live animals.
目前用于可视化病毒感染途径的技术依赖于通过化学偶联或基因操作对衣壳蛋白进行荧光标记。为了在哺乳动物组织中对这类病原体进行非侵入性体内成像，我们构建了生物发光的高斯荧光素酶标记的腺相关病毒（gLuc/AAV）载体。通过与衣壳亚基VP2的N端融合，将该酶整合到重组AAV血清型1、2和8的衣壳中，从而产生生物发光的病毒粒子外壳。gLuc/AAV载体用于在体外定量AAV2衣壳的细胞表面结合动力学。在体外细胞摄取后，生物发光的病毒粒子外壳的发光信号呈指数下降。在体内肌肉注射后也观察到类似趋势，尽管不同AAV血清型的生物发光信号下降速率有显著差异。gLuc/AAV1和gLuc/AAV8载体的生物发光信号在30分钟内迅速下降至背景水平，而gLuc/AAV2载体发出的信号持续超过2小时。生物发光的病毒粒子外壳在定量活体动物细胞和外周组织中病毒载体摄取动力学方面可能特别有用。
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