Topoisomerase inhibition accelerates gene expression after adeno-associated virus-mediated gene transfer to the mammalian heart
Topoisomerase inhibition accelerates gene expression after adeno-associated virus-mediated gene transfer to the mammalian heart
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DOI：
10.1038/sj.mt.6300071
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发表时间：
2007-04-01
影响因子：
12.4
通讯作者：
French, Brent A.
中科院分区：
文献类型：
--
作者：
Prasad, Konkal-Matt R.;Xu, Yaqin;French, Brent A.
腺相关病毒2型（AAV 2）载体用于心脏基因治疗的效用受到最大基因表达前延迟期延长的限制。拓扑异构酶抑制可以在体内诱导AAV 2介导的基因表达，但在不同组织中的成功率不同。在这项研究中，我们证明，拓扑异构酶抑制可以加速AAV 2介导的基因在小鼠心脏中的表达。我们使用表达萤火虫荧光素酶的AAV 2载体，并使用非侵入性生物发光成像监测表达动力学。在单独接受载体的组中，心脏荧光素酶活性从第2周开始明显，并逐渐增加，到注射后9周达到稳定的平台期。在接受载体和喜树碱（CPT）的组中，荧光素酶表达从第2天到第4天是明显的，并且在注射后3-4周快速增加以达到稳定的平台，比没有CPT时快近3倍（P < 0.05）。心脏组织中AAV 2基因组的Southern印迹分析显示，CPT处理的小鼠中AAV 2基因组从其单链形式快速转化为双链形式。荧光素酶表达的非侵入性测定与体外荧光素酶测定相关性良好。通过磁共振成像评估，直接注射AAV 2载体和长期荧光素酶基因表达对正常心脏功能没有可检测的影响。
Utility of adeno-associated virus 2 (AAV2) vectors for cardiac gene therapy is limited by the prolonged lag phase before maximal gene expression. Topoisomerase inhibition can induce AAV2-mediated gene expression in vivo, but with variable success in different tissues. In this study, we demonstrate that topoisomerase inhibition can accelerate AAV2-mediated gene expression in the mouse heart. We used an AAV2 vector expressing firefly luciferase and monitored expression kinetics using non-invasive bioluminescence imaging. In the group receiving vector alone, cardiac luciferase activity was evident from week 2 onward and increased progressively to reach a steady plateau by 9 weeks postinjection. In the group receiving vector and camptothecine (CPT), luciferase expression was evident from days 2 to 4 onward and increased rapidly to reach a steady plateau by 3-4 weeks postinjection, nearly three times faster than in the absence of CPT (P < 0.05). Southern blot analysis of AAV2 genomes in cardiac tissue showed rapid conversion of the AAV2 genome from its single-stranded to double-stranded form in CPT-treated mice. Noninvasive determinations of luciferase expression correlated well with in vitro luciferase assays. Direct injection of the AAV2 vector and long-term luciferase gene expression had no detectable effects on normal cardiac function as assessed by magnetic resonance imaging.