Site Specific Modification of Adeno-Associated Virus Enables Both Fluorescent Imaging of Viral Particles and Characterization of the Capsid Interactome.
Site Specific Modification of Adeno-Associated Virus Enables Both Fluorescent Imaging of Viral Particles and Characterization of the Capsid Interactome.
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DOI：
10.1038/s41598-017-15255-2
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发表时间：
2017-11-07
影响因子：
4.6
通讯作者：
Azzouz M
中科院分区：
文献类型：
--
作者：
Chandran JS;Sharp PS;Karyka E;Aves-Cruzeiro JMDC;Coldicott I;Castelli L;Hautbergue G;Collins MO;Azzouz M
腺相关病毒（aav）由于其低毒性、高稳定性和很少整合到宿主基因组中而成为有吸引力的基因治疗载体。在病毒衣壳上表达配体可以将aav重新定位到新的细胞类型，但在衣壳上发现的耐受肽插入的位点有限。在这里，我们将一个位点特异性的四胱氨酸序列整合到AAV血清型9 （AAV9）衣壳中，从而允许用荧光染料或生物素标记病毒颗粒。我们证明了荧光标记的颗粒在体外是可检测的，并探索了该方法在小鼠体内使用延时成像的实用性。我们利用生物素化的病毒颗粒生成了两种不同的AAV相互作用组，并鉴定了几种具有高度代表性的功能类别的蛋白质：肌动蛋白/细胞骨架蛋白结合、RNA结合、RNA剪接/加工、染色质修饰、细胞内运输和RNA转运蛋白。为了研究衣壳相互作用组的生物学相关性，我们在AAV转导之前调节了来自相互作用组的两种蛋白质的表达。阻断整合素αVβ6受体功能可降低AAV9转导，而降低组蛋白去乙酰化酶4 （HDAC4）表达可增强AAV转导。我们的方法展示了在不影响病毒滴度或感染性的情况下将基序插入AAV衣壳的策略。
Adeno-associated viruses (AAVs) are attractive gene therapy vectors due to their low toxicity, high stability, and rare integration into the host genome. Expressing ligands on the viral capsid can re-target AAVs to new cell types, but limited sites have been identified on the capsid that tolerate a peptide insertion. Here, we incorporated a site-specific tetracysteine sequence into the AAV serotype 9 (AAV9) capsid, to permit labelling of viral particles with either a fluorescent dye or biotin. We demonstrate that fluorescently labelled particles are detectable in vitro, and explore the utility of the method in vivo in mice with time-lapse imaging. We exploit the biotinylated viral particles to generate two distinct AAV interactomes, and identify several functional classes of proteins that are highly represented: actin/cytoskeletal protein binding, RNA binding, RNA splicing/processing, chromatin modifying, intracellular trafficking and RNA transport proteins. To examine the biological relevance of the capsid interactome, we modulated the expression of two proteins from the interactomes prior to AAV transduction. Blocking integrin αVβ6 receptor function reduced AAV9 transduction, while reducing histone deacetylase 4 (HDAC4) expression enhanced AAV transduction. Our method demonstrates a strategy for inserting motifs into the AAV capsid without compromising viral titer or infectivity.
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影响因子：
12.3
作者：
Akerman M;Fregoso OI;Das S;Ruse C;Jensen MA;Pappin DJ;Zhang MQ;Krainer AR
通讯作者：
Krainer AR
影响因子：
64.5
作者：
Li, XL;Manley, JL
通讯作者：
Manley, JL
DOI：
10.1038/sj.mt.6300053
发表时间：
2007-03
期刊：
Molecular therapy : the journal of the American Society of Gene Therapy
影响因子：
--
作者：
Akache B;Grimm D;Shen X;Fuess S;Yant SR;Glazer DS;Park J;Kay MA
通讯作者：
Kay MA
影响因子：
4.6
作者：
Dayton RD;Wang DB;Klein RL
通讯作者：
Klein RL
影响因子：
34.7
作者：
Lee, Edward B.;Lee, Virginia M-Y;Trojanowski, John Q.
通讯作者：
Trojanowski, John Q.