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首页> 外文OA文献 >Adenovirus Type 5 Viral Particles Pseudotyped with Mutagenized Fiber Proteins Show Diminished Infectivity of Coxsackie B-Adenovirus Receptor-Bearing Cells
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Adenovirus Type 5 Viral Particles Pseudotyped with Mutagenized Fiber Proteins Show Diminished Infectivity of Coxsackie B-Adenovirus Receptor-Bearing Cells

机译：用诱变的纤维蛋白假型化的5型腺病毒病毒颗粒显示了柯萨奇B型腺病毒受体细胞的感染力降低。

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A major limitation of adenovirus type 5 (Ad5)-based gene therapy, the inability to target therapeutic genes to selected cell types, is attributable to the natural tropism of the virus for the widely expressed coxsackievirus-adenovirus receptor (CAR) protein. Modifications of the Ad5 fiber knob domain have been shown to alter the tropism of the virus. We have developed a novel system to rapidly evaluate the function of modified fiber proteins in their most relevant context, the adenoviral capsid. This transient transfection/infection system combines transfection of cells with plasmids that express high levels of the modified fiber protein and infection with Ad5.βgal.ΔF, an E1-, E3-, and fiber-deleted adenoviral vector encoding β-galactosidase. We have used this system to test the adenoviral transduction efficiency mediated by a panel of fiber protein mutants that were proposed to influence CAR interaction. A series of amino acid modifications were incorporated via mutagenesis into the fiber expression plasmid, and the resulting fiber proteins were subsequently incorporated onto adenoviral particles. Mutations located in the fiber knob AB and CD loops demonstrated the greatest reduction in fiber-mediated gene transfer in HeLa cells. We also observed effects on transduction efficiency with mutations in the FG loop, indicating that the binding site may extend to the adjacent monomer in the fiber trimer and in the HI loop. These studies support the concept that modification of the fiber knob domain to diminish or ablate CAR interaction should result in a detargeted adenoviral vector that can be combined simultaneously with novel ligands for the development of a systemically administered, targeted adenoviral vector.
机译：基于腺病毒5型（Ad5）的基因治疗的主要局限性是无法将治疗基因靶向所选的细胞类型，这归因于广泛表达的柯萨奇病毒-腺病毒受体（CAR）蛋白的病毒自然趋向性。已经显示出对Ad5纤维瘤结构域的修饰可以改变病毒的向性。我们已经开发了一种新型系统，可以在最相关的环境中快速评估修饰的纤维蛋白的功能，即腺病毒衣壳。该瞬时转染/感染系统将细胞与表达高水平修饰纤维蛋白的质粒的转染结合，并感染了Ad5.βgal.ΔF，E1，E3-，E3和纤维缺失的编码β-半乳糖苷酶的腺病毒载体。我们已经使用该系统来测试由提议影响CAR相互作用的一组纤维蛋白突变体介导的腺病毒转导效率。通过诱变将一系列氨基酸修饰掺入到纤维表达质粒中，然后将所得的纤维蛋白掺入腺病毒颗粒中。位于纤维瘤AB和CD环中的突变显示HeLa细胞中纤维介导的基因转移的最大减少。我们还观察到了FG环中突变对转导效率的影响，表明结合位点可能延伸至纤维三聚体和HI环中的相邻单体。这些研究支持这样的概念，即修饰纤维瘤结构域以减少或消除CAR相互作用应导致脱靶的腺病毒载体，该载体可与新型配体同时结合以开发全身给药的靶向腺病毒载体。

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