DNA shuffling of adeno-associated virus yields functionally diverse viral progeny
DNA shuffling of adeno-associated virus yields functionally diverse viral progeny
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DOI：
10.1038/mt.2008.167
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发表时间：
2008-10-01
影响因子：
12.4
通讯作者：
Schaffer, David V.
中科院分区：
文献类型：
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作者：
Koerber, James T.;Jang, Jae-Hyung;Schaffer, David V.
腺相关病毒 (AAV) 载体是极其有效的基因递送载体，具有广泛的应用。然而，这些和其他载体的治疗功效目前受到安全、有效的基因传递障碍的限制，包括预先存在的抗病毒免疫和脱靶细胞的感染。最近，我们实现了 AAV 的定向进化，包括生成基于血清型 2 的随机诱变病毒库和高通量选择，以设计增强的病毒载体。在这里，我们通过进行高效体外重组显着扩展了这种能力，以创建大量亲本 AAV 血清型（AAV1、2、4-6、8 和 9）的大型（107）、多样化的随机嵌合体库。为了分析这种高度嵌合的病毒的存活程度，我们选择了用于高效病毒包装和感染的文库，并成功回收了许多新型嵌合体。这些新病毒在体外和体内表现出广泛的细胞趋向性，并且对人静脉注射免疫球蛋白（IVIG）具有增强的抵抗力，凸显了这些嵌合体与其亲本血清型之间的许多功能差异。因此，定向进化有可能产生无限数量的具有新颖基因传递特性的新 AAV 变体，并且对这些变体的后续分析可以进一步扩展 AAV 生物学的基础知识。
Adeno-associated virus (AAV) vectors are extremely effective gene-delivery vehicles for a broad range of applications. However, the therapeutic efficacy of these and other vectors is currently limited by barriers to safe, efficient gene delivery, including pre-existing antiviral immunity, and infection of off-target cells. Recently, we have implemented directed evolution of AAV, involving the generation of randomly mutagenized viral libraries based on serotype 2 and high-throughput selection, to engineer enhanced viral vectors. Here, we significantly extend this capability by performing high-efficiency in vitro recombination to create a large (107), diverse library of random chimeras of numerous parent AAV serotypes (AAV1, 2, 4-6, 8, and 9). In order to analyze the extent to which such highly chimeric viruses can be viable, we selected the library for efficient viral packaging and infection, and successfully recovered numerous novel chimeras. These new viruses exhibited a broad range of cell tropism both in vitro and in vivo and enhanced resistance to human intravenous immunoglobulin (IVIG), highlighting numerous functional differences between these chimeras and their parent serotypes. Thus, directed evolution can potentially yield unlimited numbers of new AAV variants with novel gene-delivery properties, and subsequent analysis of these variants can further extend basic knowledge of AAV biology.