Martin Karine, Brie Anne, Saulnier Patrick, Perricaudet Michel, Yeh Patrice, Vigne Emmanuelle
UMR1582 CNRS/IGR/Aventis-Gencell, Villejuif Cedex, France.
Mol Ther. 2003 Sep;8(3):485-94. doi: 10.1016/s1525-0016(03)00182-5.
Targeting adenovirus encoding therapeutic genes to specific cell types has become a major goal in gene therapy. Coxsackievirus and adenovirus receptor (CAR) and alpha(V) integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. Redirecting Ad5-based vectors requires abrogation of the natural interaction between the viral capsid and its cellular receptors and simultaneous introduction of a new binding specificity into the viral capsid. To abrogate native Ad5 tropism, fiber knob mutations Pro409Glu and Lys417Ala were each incorporated into adenoviral vectors, while the RGD motif was deleted from the penton base. In vitro transduction experiments showed that these capsid mutations eliminated Ad5 interactions with CAR and alpha(V) integrins. Moreover, incorporation in the fiber HI loop of a vitronectin-derived ligand (VN4) specific for the uPAR/CD87 receptor provided the Lys417Ala virus with an alternative entry pathway specific for uPAR-expressing cells, indicating a successful in vitro retargeting of the vector. Unexpectedly, however, simultaneous disruption of Ad5 binding to CAR and alpha(V) integrins had no effect on liver gene transfer following systemic administration in mice. This study highlights the need to understand better the molecular determinants involved in adenovirus uptake by the liver to control the fate of adenoviral vectors in vivo.
将携带治疗性基因的腺病毒靶向特定细胞类型已成为基因治疗的主要目标。柯萨奇病毒和腺病毒受体（CAR）以及α（V）整合素已被确定为在体外转导过程中与基于5型腺病毒（Ad5）的载体相互作用的主要细胞表面成分。重新定向基于Ad5的载体需要消除病毒衣壳与其细胞受体之间的天然相互作用，并同时在病毒衣壳中引入新的结合特异性。为了消除天然Ad5的嗜性，将纤维结突变Pro409Glu和Lys417Ala分别引入腺病毒载体中，同时从五聚体基部删除RGD基序。体外转导实验表明，这些衣壳突变消除了Ad5与CAR和α（V）整合素的相互作用。此外，在纤维HI环中引入对uPAR/CD87受体特异的玻连蛋白衍生配体（VN4），为Lys417Ala病毒提供了一条特异于表达uPAR细胞的替代进入途径，表明该载体在体外成功实现了重新靶向。然而，出乎意料的是，在小鼠全身给药后，Ad5与CAR和α（V）整合素结合的同时破坏对肝脏基因转移没有影响。这项研究强调需要更好地了解肝脏摄取腺病毒所涉及的分子决定因素，以控制腺病毒载体在体内的命运。
