Engineering and selection of shuffled AAV genomes: A new strategy for producing targeted biological nanoparticles
Engineering and selection of shuffled AAV genomes: A new strategy for producing targeted biological nanoparticles
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DOI：
10.1038/mt.2008.100
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发表时间：
2008-07-01
影响因子：
12.4
通讯作者：
Samulski, Richard J.
中科院分区：
文献类型：
--
作者：
Li, Wuping;Asokan, Aravind;Samulski, Richard J.
我们报告了一种基于DNA改组的方法，通过定向进化开发细胞类型特异性载体。将腺相关病毒（AAV）血清型1-9的衣壳基因组随机片段化并使用PCR重新组装以产生嵌合衣壳文库。含有来自AAV 1、2、8和9的基因组片段的单个感染性克隆（嵌合-1829）从先前显示对AAV具有低容许性的整联蛋白缺失仓鼠黑素瘤细胞系中分离。分子模拟研究表明，AAV 2有助于在二十面体三重对称轴的表面环，而AAV 1和9分别有助于两重和五重对称相互作用。通过合理的诱变，C-末端结构域（AAV 9）被鉴定为黑色素瘤嗜性的关键结构决定因子。嵌合-1829利用硫酸乙酰肝素作为主要受体，比所有血清型更有效地转导黑素瘤细胞。此外，嵌合-1829在啮齿类动物（包括非人灵长类动物）的骨骼肌、肝脏和脑中显示出改变的向性。我们根据中和抗体（NAb）滴度和交叉反应性研究确定了一种独特的免疫学特征，这强烈支持分离合成实验室来源的衣壳变体。使用AAV或其他病毒衣壳序列将该技术应用于替代细胞/组织类型可能会产生一类新的生物纳米颗粒作为人类基因转移的载体。
We report a DNA shuffling-based approach for developing cell type-specific vectors through directed evolution. Capsid genomes of adeno-associated virus (AAV) sero-types 1-9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two- and fivefold symmetry interactions, respectively. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer.