Genetic retargeting of adenovirus: Novel strategy employing "deknobbing" of the fiber

For efficient and versatile use of adenovirus (Ad) as an in vivo gene thera py vector, modulation of the viral tropism is highly desirable. In this stu dy, a novel method to genetically alter the Ad fiber tropism is described. The knob and the last 15 shaft repeats of the fiber gene were deleted and r eplaced with an external trimerization motif and a new cell-binding ligand, in this case the integrin-binding motif RGD. The corresponding recombinant fiber retained the basic biological functions of the natural fiber, i.e., trimerization, nuclear import, penton formation, and ligand binding. The re combinant fiber bound to integrins but failed to react with antiknob antibo dy. For virus production, the recombinant fiber gene was rescued into the A d genome at the exact position of the wild-type (WT) fiber to make use of t he native regulation of fiber expression. The recombinant virus Ad5/FibR7-R GD yielded plaques on 293 cells, but the spread through the monolayer was t wo to three times delayed compared to WT, and the ratio of infectious to ph ysical particles was 20 times lower. Studies on virus tropism showed that A d5/FibR7-RGD was able to infect cells which did not express the coxsackie-a denovirus receptor (CAR), but did express integrins. Ad5/FibR7-RGD virus in fectivity was unchanged in the presence of antiknob antibody, which neutral ized the WT virus. Ad5/FibR7-RGD virus showed an expanded tropism, which is useful when gene transfer to cells not expressing CAR is needed. The descr ibed method should also make possible the construction of Ad genetically re targeted via ligands other than RGD.