Assessment of optimal transduction of primary human skin keratinocytes by viral vectors
Assessment of optimal transduction of primary human skin keratinocytes by viral vectors
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DOI：
10.1002/jgm.768
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发表时间：
2005-09-01
影响因子：
3.5
通讯作者：
Meneguzzi, G
中科院分区：
文献类型：
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作者：
Gagnoux-Palacios, L;Hervouet, C;Meneguzzi, G
遗传修饰的角质形成细胞产生适于递送治疗性多肽的可移植自我更新上皮细胞。然而，目前使用的各种病毒载体和实验条件使得关于人原代角质形成细胞转导效率的信息支离破碎或相互矛盾。为了比较目前使用的最有效的基因转移到人角质形成细胞的病毒载体的适用性，我们进行了比较研究，使用一组重组constructs.Methods对于每个载体，转导效率和转基因表达的持久性进行了定量荧光显微镜和流式细胞术分析的感染cells.Results我们表明：（1）犬和人腺病毒载体实现了原代和永生化角质形成细胞的高效但瞬时的转导;（2）腺病毒相关病毒（AAV）载体使永生化角质形成细胞增殖，尽管具有短暂的基因表达（< 4天），但不能感染原代角质形成细胞;（3）在适当的条件下，逆转录病毒和慢病毒载体可以永久性地覆盖100%的原代角质形成细胞，但慢病毒载体更有效地靶向高度克隆形成的角质形成细胞。AAV载体不适合于转染原代角质形成细胞，而人和犬腺病毒载体似乎适合于实现治疗产品的短期递送。重组逆转录病毒提供转基因的持续表达，但慢病毒载体是最适合于离体基因治疗，因为它们能够抑制原代角质形成细胞的克隆形成。版权所有（c）2005年约翰威利父子有限公司。
Background Genetically modified keratinocytes generate transplantable self-renewing epithelia suitable for delivery of therapeutic polypeptides. However, the variety of viral vectors and experimental conditions currently used make fragmented or contradictory the information on the transduction efficiency of the human primary keratinocytes. To compare the suitability of the most currently used viral vectors for efficient gene transfer to human keratinocytes, we have performed a comparative study using a panel of recombinant constructs.Methods For each vector, the transduction efficiency and the persistence of the transgene expression were quantified by fluorescence microscopy and flow cytometry analysis of the infected cells.Results We show that: (1) canine and human adenoviral vectors achieve a highly efficient but transient transduction of both primary and immortalized keratinocytes; (2) the adenovirus-associated virus (AAV) vectors transduce immortalized keratinocytes, albeit with a short-lived gene expression (< 4 days), but fail to infect primary keratinocytes; and (3) under appropriate conditions, the oncoretroviral and lentiviral vectors can permanently transduce up to 100% of primary keratinocytes, but the highly clonogenic keratinocytes are more efficiently targeted by lentiviral vectors.Conclusions Therefore, AAV vectors are unsuitable to transduce primary keratinocytes, while human and canine adenoviral vectors appears to be appropriate to achieve short-term delivery of therapeutic products. Recombinant retroviruses provide sustained expression of the transgene, but the lentiviral vectors are the most suitable for ex vivo gene therapy because of their ability to transduce clonogenic primary keratinocytes. Copyright (c) 2005 John Wiley & Sons, Ltd.