Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
Hum Gene Ther. 2010 Jun;21(6):739-49. doi: 10.1089/hum.2009.190.
Abstract It has been shown that blood clotting factors, including factor X (FX), bind to the adenovirus serotype 5 (Ad5) hexon protein and target the virus to liver hepatocytes after intravenous injection. These factors bind to hexon via their conserved vitamin K-dependent gamma-carboxyglutamic acid (GLA) domains with subnanomolar affinity. In this work, we have used this strong interaction to retarget Ad to new receptors, using the GLA domain of FX fused to single-chain antibody variable fragment (ScFv). We demonstrate that fusion of the GLA domain of human FX to receptor-specific ScFvs will target Ad5 vectors to cells expressing these receptors. Fusion of an alphaHer2 ScFv to GLA increased in vitro transduction of Her2-positive versus Her2-negative cells when compared with untargeted virus. Similar results were obtained with ScFvs against the epidermal growth factor receptor (EGFR) and against the stem cell marker ATP-binding cassette protein G2 (ABCG2). Direct expression of GLA fusion protein from replication-defective or replication-competent Ad increased infection and killing of cancer cells in vitro and in vivo. These data demonstrate the potential of using GLA domains to bridge secreted ligands with intracellularly produced Ad5 vectors for vector targeting.
摘要 已有研究表明，凝血因子（包括因子 X，FX）可与血清 5 型腺病毒（Ad5）六邻体蛋白结合，在静脉注射后将病毒靶向肝脏肝细胞。这些因子通过其保守的维生素 K 依赖性γ-羧基谷氨酸（GLA）结构域与六邻体以纳摩尔级亲和力结合。在这项工作中，我们利用这种强相互作用，通过 FX 的 GLA 结构域融合单链抗体可变片段（ScFv），将 Ad 重新靶向新的受体。我们证明，将人 FX 的 GLA 结构域与受体特异性 ScFv 融合，可将 Ad5 载体靶向表达这些受体的细胞。与未靶向病毒相比，将 αHer2 ScFv 融合到 GLA 上，可增加体外转导 Her2 阳性细胞与 Her2 阴性细胞的比率。针对表皮生长因子受体（EGFR）和干细胞标记物三磷酸腺苷结合盒蛋白 G2（ABCG2）的 ScFv 也得到了类似的结果。从复制缺陷型或复制型 Ad 直接表达 GLA 融合蛋白可增加体外和体内癌细胞的感染和杀伤。这些数据表明，利用 GLA 结构域将分泌配体与细胞内产生的 Ad5 载体桥接，可用于载体靶向。
