Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing
Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing
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DOI：
10.1038/ncomms4075
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发表时间：
2014-01-01
影响因子：
16.6
通讯作者：
Nakai, Hiroyuki
中科院分区：
文献类型：
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作者：
Adachi, Kei;Enoki, Tatsuji;Nakai, Hiroyuki
腺相关病毒（AAV）衣壳工程是一种新兴的方法，以推进基因治疗。然而，系统分析每个衣壳氨基酸如何有助于多种功能仍然具有挑战性。在这里，我们展示了一种名为AAV Barcode-Seq的新方法的原理证明和成功应用，该方法使我们能够以高通量方式表征数百种不同AAV菌株的表型，从而克服了系统分析中的技术困难。在这种方法中，我们生成DNA条形码标记的AAV文库，并通过Illumina条形码测序确定每个AAV株的表型谱。通过将这种方法应用于用DNA条形码标记的AAV衣壳突变体文库，我们可以绘制对结构完整性和功能（包括受体结合、向性、中和和血液清除）重要的AAV衣壳氨基酸的高分辨率图谱。因此，Barcode-Seq为病毒和基因治疗研究提供了一种新的工具来产生有价值的资源。
Adeno-associated virus (AAV) capsid engineering is an emerging approach to advance gene therapy. However, a systematic analysis on how each capsid amino acid contributes to multiple functions remains challenging. Here we show proof-of-principle and successful application of a novel approach, termed AAV Barcode-Seq, that allows us to characterize phenotypes of hundreds of different AAV strains in a high-throughput manner and therefore overcomes technical difficulties in the systematic analysis. In this approach, we generate DNA barcode-tagged AAV libraries and determine a spectrum of phenotypes of each AAV strain by Illumina barcode sequencing. By applying this method to AAV capsid mutant libraries tagged with DNA barcodes, we can draw a high-resolution map of AAV capsid amino acids important for the structural integrity and functions including receptor binding, tropism, neutralization and blood clearance. Thus, Barcode-Seq provides a new tool to generate a valuable resource for virus and gene therapy research.