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Gene Therapy: Identification of coagulation factor (F)X binding sites on the adenovirus serotype 5 hexon: effect of mutagenesis on FX interactions and gene transfer

机译:基因治疗:鉴定腺病毒5型六邻体上凝血因子(F)X结合位点:诱变对FX相互作用和基因转移的影响

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摘要

Recent studies have demonstrated the importance of coagulation factor X (FX) in adenovirus (Ad) serotype 5–mediated liver transduction in vivo. FX binds to the adenovirus hexon hypervariable regions (HVRs). Here, we perform a systematic analysis of FX binding to Ad5 HVRs 5 and 7, identifying domains and amino acids critical for this interaction. We constructed a model of the Ad5-FX interaction using crystallographic and cryo-electron microscopic data to identify contact points. Exchanging Ad5 HVR5 or HVR7 from Ad5 to Ad26 (which does not bind FX) diminished FX binding as analyzed by surface plasmon resonance, gene delivery in vitro, and liver transduction in vivo. Exchanging Ad5-HVR5 for Ad26-HVR5 produced deficient virus maturation. Importantly, defined mutagenesis of just 2 amino acids in Ad5-HVR5 circumvented this and was sufficient to block liver gene transfer. In addition, mutation of 4 amino acids in Ad5-HVR7 or a single mutation at position 451 also blocked FX-mediated effects in vitro and in vivo. We therefore define the regions and amino acids on the Ad5 hexon that bind with high affinity to FX thereby better defining adenovirus infectivity pathways. These vectors may be useful for gene therapy applications where evasion of liver transduction is a prerequisite.
机译:最近的研究表明,凝血因子X(FX)在腺病毒(Ad)血清型5介导的体内肝脏转导中的重要性。 FX绑定到腺病毒六邻体高变区(HVR)。在这里,我们对FX与Ad5 HVR 5和7的结合进行了系统的分析,确定了对该相互作用至关重要的域和氨基酸。我们使用晶体学和低温电子显微镜数据构建了Ad5-FX相互作用的模型,以识别接触点。通过表面等离振子共振,体外基因递送和体内肝脏转导分析,将Ad5 HVR5或HVR7从Ad5交换至Ad26(不结合FX)会减少FX结合。用Ad5-HVR5交换Ad26-HVR5会导致病毒成熟不足。重要的是,在Ad5-HVR5中仅2个氨基酸的明确诱变就避免了这种情况,并且足以阻止肝脏基因转移。另外,在Ad5-HVR7中4个氨基酸的突变或在451位的单个突变在体外和体内也阻断了FX介导的作用。因此,我们定义了Ad5六邻体上与FX具有高亲和力结合的区域和氨基酸,从而更好地定义了腺病毒感染性途径。这些载体可用于以肝转移为先决条件的基因治疗应用。

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