A NEW RECOMBINANT ADENO-ASSOCIATED VIRUS (AAV)-BASED RANDOM PEPTIDE DISPLAY LIBRARY SYSTEM: INFECTION-DEFECTIVE AAV1.9-3 AS A NOVEL DETARGETED PLATFORM FOR VECTOR EVOLUTION.
A NEW RECOMBINANT ADENO-ASSOCIATED VIRUS (AAV)-BASED RANDOM PEPTIDE DISPLAY LIBRARY SYSTEM: INFECTION-DEFECTIVE AAV1.9-3 AS A NOVEL DETARGETED PLATFORM FOR VECTOR EVOLUTION.
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DOI：
10.1142/s1568558610000197
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发表时间：
2010-10-01
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影响因子：
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通讯作者：
Nakai, Hiroyuki
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文献类型：
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作者：
Adachi, Kei;Nakai, Hiroyuki
通过病毒衣壳的基因工程随后选择的定向进化已经成为产生具有所需向性和增强性质的新型重组腺相关病毒（rAAV）载体的有力手段。最有效的方法之一是使用基于rAAV的随机肽展示文库。在这里，我们报告了一个新的系统的基础上感染缺陷rAAV1.9-3作为一个平台，随机肽展示，并表明，生物淘选库在体外有效地识别恢复和增强rAAV转导的肽。rAAV1.9-3具有基因工程改造的AAV 1衣壳，氨基酸445-568被AAV 9的氨基酸取代，并且已被鉴定为当输注到小鼠中时表现出显著受损的感染性和延迟的血液清除的变体。在这项研究中，我们产生了rAAV1.9-3变体文库，其中7-或12-mer随机肽在衣壳氨基酸位置590处表达。对原代人皮肤成纤维细胞的三轮阳性选择成功地鉴定了新的rAAV-肽变体，其比原型rAAV 2更有效地使它们增殖。因此，我们的研究表明，感染缺陷的rAAV变体作为一种新的脱靶平台的随机肽展示库。我们还描述了一个简短的回顾rAAV为基础的随机肽展示库方法的最新进展。
Directed evolution through genetic engineering of viral capsids followed by selection has emerged as a powerful means to create novel recombinant adeno-associated virus (rAAV) vectors with desired tropism and enhanced properties. One of the most effective approaches uses rAAV-based random peptide display libraries. Here we report a novel system based on an infection-defective rAAV1.9-3 as a platform for random peptide display, and show that biopanning of the libraries in vitro effectively identifies the peptides that restore and enhance rAAV transduction. rAAV1.9-3 has a genetically engineered AAV1 capsid with amino acids 445-568 being replaced with those of AAV9, and has been identified as a variant exhibiting significantly impaired infectivity and delayed blood clearance when infused into mice. In this study, we generated rAAV1.9-3 variant libraries in which 7- or 12-mer random peptides were expressed at the capsid amino acid position 590. Three rounds of positive selection for primary human dermal fibroblasts successfully identified new rAAV-peptide variants that transduce them more efficiently than the prototype rAAV2. Thus our study demonstrates that an infection-defective rAAV variant serves as a novel detargeted platform for random peptide display libraries. We also describe a brief review of recent progress in rAAV-based random peptide display library approaches.