Max von Pettenkofer-Institut, Department of Virology, Ludwig-Maximilians-Universität Munich, Munich, Germany.
Mol Ther. 2010 Nov;18(11):1896-906. doi: 10.1038/mt.2010.169. Epub 2010 Aug 17.
Sleeping Beauty (SB) transposase enables somatic integration of exogenous DNA in mammalian cells, but potency as a gene transfer vector especially in large mammals has been lacking. Herein, we show that hyperactive transposase system delivered by high-capacity adenoviral vectors (HC-AdVs) can result in somatic integration of a canine factor IX (cFIX) expression-cassette in canine liver, facilitating stabilized transgene expression and persistent haemostatic correction of canine hemophilia B with negligible toxicity. We observed stabilized cFIX expression levels during rapid cell cycling in mice and phenotypic correction of the bleeding diathesis in hemophilia B dogs for up to 960 days. In contrast, systemic administration of an inactive transposase system resulted in rapid loss of transgene expression and transient phenotypic correction. Notably, in dogs a higher viral dose of the active SB transposase system resulted into transient phenotypic correction accompanied by transient increase of liver enzymes. Molecular analysis of liver samples revealed SB-mediated integration and provide evidence that transgene expression was derived mainly from integrated vector forms. Demonstrating that a viral vector system can deliver clinically relevant levels of a therapeutic protein in a large animal model of human disease paves a new path toward the possible cure of genetic diseases.
睡美人（SB）转座酶能够使外源性 DNA 在哺乳动物细胞中进行体整合，但作为基因转移载体，特别是在大型哺乳动物中的效力一直不足。在此，我们展示了通过高容量腺病毒载体（HC-AdV）传递的 hyperactive 转座酶系统可导致犬因子 IX（cFIX）表达盒在犬肝脏中进行体整合，从而促进稳定的转基因表达和对犬血友病 B 的持续止血纠正，且毒性可忽略不计。我们观察到在快速细胞周期中，在小鼠中观察到稳定的 cFIX 表达水平，并且在血友病 B 犬中观察到出血倾向的表型纠正长达 960 天。相比之下，全身性给予无活性转座酶系统会导致转基因表达迅速丧失和表型纠正短暂。值得注意的是，在犬中，高剂量的活性 SB 转座酶系统会导致短暂的表型纠正，同时伴随着肝酶的短暂升高。对肝组织样本的分子分析揭示了 SB 介导的整合，并提供了证据表明转基因表达主要来源于整合的载体形式。证明病毒载体系统可以在人类疾病的大型动物模型中递送具有临床相关性的治疗蛋白水平，为治疗遗传疾病开辟了新的途径。
