Engineering and In Vitro Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity.
Engineering and In Vitro Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity.
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工程和体外选择具有高肝细胞的新型AAV3B变体和降低的静脉反应性。
DOI：
10.1016/j.omtm.2020.09.019
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发表时间：
2020-12-11
期刊：
影响因子：
--
通讯作者：
Zolotukhin S
中科院分区：
文献类型：
--
作者：
Biswas M;Marsic D;Li N;Zou C;Gonzalez-Aseguinolaza G;Zolotukhin I;Kumar SRP;Rana J;Butterfield JSS;Kondratov O;de Jong YP;Herzog RW;Zolotukhin S
腺相关病毒（AAV）成功进行基因治疗的局限性可能包括预先存在的针对载体衣壳的中和抗体，其可阻止细胞进入，或靶细胞的低效转导，其可导致治疗性转基因的次优表达。重组血清型 3 AAV (AAV3) 是肝脏定向基因治疗的新兴候选药物。在这项研究中，我们通过使用源自 AAV3B 衣壳的组合文库，通过体外选择肝脏靶向 AAV 变体的定向进化，整合了合理设计。本文描述的 AAV3B-DE5 变体在原始病毒文库中无法检测到，但在人肝癌球体培养物中体外传代时获得了选择性优势。与AAV3B相比，AAV3B-DE5含有24个衣壳氨基酸取代，分布在所有五个可变区中，对VR-IV、VR-V和VR-VII具有很强的选择压力。在体内，AAV3B-DE5 在肝脏嵌合小鼠模型中表现出改善的人肝细胞向性。重要的是，该变体对人静脉注射免疫球蛋白 (i.v. Ig) 以及来自 100 名健康人类捐赠者的个体血清样本的血清反应性降低。因此，使用组合文库平台的分子进化产生了具有高肝细胞向性和增强的对预先存在的 AAV 中和抗体的逃避的病毒衣壳。 Biswas 及其同事描述了一种生成新型 AAV3B 变体的综合方法。通过将组合 AAV3B 衣壳库平台与肝细胞癌细胞系中的定向进化相结合，他们分离出了候选 AAV3B-DE5，该候选 AAV3B-DE5 表现出改善的人肝细胞趋向性，并降低了与人血清中预先存在的 AAV 中和抗体的血清反应性。
Limitations to successful gene therapy with adeno-associated virus (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene. Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy. In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by in vitro selection for liver-targeted AAV variants. The AAV3B-DE5 variant described herein was undetectable in the original viral library but gained a selective advantage upon in vitro passaging in human hepatocarcinoma spheroid cultures. AAV3B-DE5 contains 24 capsid amino acid substitutions compared with AAV3B, distributed among all five variable regions, with strong selective pressure on VR-IV, VR-V, and VR-VII. In vivo, AAV3B-DE5 demonstrated improved human hepatocyte tropism in a liver chimeric mouse model. Importantly, this variant exhibited reduced seroreactivity to human intravenous immunoglobulin (i.v. Ig), as well as individual serum samples from 100 healthy human donors. Therefore, molecular evolution using a combinatorial library platform generated a viral capsid with high hepatocyte tropism and enhanced evasion of pre-existing AAV neutralizing antibodies. Biswas and colleagues describe an integrated approach to generate novel AAV3B variants. By integrating a combinatorial AAV3B capsid library platform with directed evolution in a hepatocellular carcinoma cell line, they isolated a candidate AAV3B-DE5, which exhibits improved human hepatocyte tropism and reduced seroreactivity to pre-existing AAV neutralizing antibodies in human serum.
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影响因子：
20.3
作者：
Jiang, Haiyan;Couto, Linda B.;Pierce, Glenn F.
通讯作者：
Pierce, Glenn F.
影响因子：
5.4
作者：
Govindasamy, Lakshmanan;DiMattia, Michael A.;Agbandje-McKenna, Mavis
通讯作者：
Agbandje-McKenna, Mavis
影响因子：
12.4
作者：
Li, Shaoyong;Ling, Chen;Gao, Guangping
通讯作者：
Gao, Guangping
DOI：
10.1007/978-1-61779-370-7_3
发表时间：
2011-01-01
期刊：
ADENO-ASSOCIATED VIRUS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Agbandje-McKenna, Mavis;Kleinschmidt, Juergen
通讯作者：
Kleinschmidt, Juergen
影响因子：
--
作者：
Aurnhammer, Christine;Haase, Maren;Baiker, Armin
通讯作者：
Baiker, Armin