Genetic Retargeting of Adenovirus: Novel Strategy Employing “Deknobbing” of the Fiber

Abstract

For efficient and versatile use of adenovirus (Ad) as an in vivo gene therapy vector, modulation of the viral tropism is highly desirable. In this study, 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 replaced 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 recombinant fiber bound to integrins but failed to react with antiknob antibody. For virus production, the recombinant fiber gene was rescued into the Ad genome at the exact position of the wild-type (WT) fiber to make use of the native regulation of fiber expression. The recombinant virus Ad5/FibR7-RGD yielded plaques on 293 cells, but the spread through the monolayer was two to three times delayed compared to WT, and the ratio of infectious to physical particles was 20 times lower. Studies on virus tropism showed that Ad5/FibR7-RGD was able to infect cells which did not express the coxsackie-adenovirus receptor (CAR), but did express integrins. Ad5/FibR7-RGD virus infectivity was unchanged in the presence of antiknob antibody, which neutralized 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 described method should also make possible the construction of Ad genetically retargeted via ligands other than RGD.

Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu

“…30 Generation of recombinant truncated knobless fibers Recombinant fibers containing the native fiber tail and the first seven shaft repeats, followed by an external trimerization motif from lung surfactant protein D (neck region peptide (NRP)), 39 a linker from SpA and monomeric or dimeric versions of ZH as a new cellular ligand, were constructed and evaluated as previously described. [10][11][12] A schematic representation of the constructed recombinant Ad5 fibers studied is shown in Figure 1. Recombinant fibers were designated R7, followed by the name of the new ligand/s (Figure 1b).…”

“…4 In order to be useful as an in vivo gene therapy vector, Ad requires re-targeting to new cellular receptors (transductional re-targeting) as well as de-targeting from normal tissue receptors. 5 De-targeting of Ad requires at least three steps: (i) ablation of the natural binding of the fiber knob to the CAR receptor, 6 which can be achieved by point mutations in the sequences responsible for CARbinding [7][8][9] or by generation of de-knobbed fibers; [10][11][12][13][14] (ii) removal of the KKTK motif in the third shaft repeat in the fiber that seems to be responsible for the HS-GAG binding 15 that mediates liver uptake and (iii) mutation of the integrin-binding motifs in the penton base to lower unwanted uptake in normal tissues. 16 Transductional retargeting can be achieved by the use of bispecific reagents, chemical coupling of ligands to the virion or by genetic fusion of a suitable ligand with a viral capsid protein.…”

“…Previous results have shown that viable re-targeted Ad5 can be constructed by genetically removing the knob, shortening the shaft and introducing the neck-region peptide from human lung surfactant D as a new trimerization motif, followed by a new cell-binding ligand. 10 However, it has been demonstrated that recombinant adenovirus with modified capsid proteins are difficult to produce to wild-type titers, and exhibits poor growth characteristics. 10,12 The poor performance of viruses with de-knobbed fibers is due to the fact that the knob has other important functions in the life cycle of the virus than just binding to the cellular receptor.…”

“…10 However, it has been demonstrated that recombinant adenovirus with modified capsid proteins are difficult to produce to wild-type titers, and exhibits poor growth characteristics. 10,12 The poor performance of viruses with de-knobbed fibers is due to the fact that the knob has other important functions in the life cycle of the virus than just binding to the cellular receptor. [20][21][22][23][24][25] These facts make such viruses difficult to use for gene therapeutic purposes.…”

Transductional targeting of adenovirus vectors for gene therapy

“…This novel Ad variant lacks the ability to interact with CAR and demonstrated up to a 100-fold increase in reporter gene expression to cells presenting an artificial 6-His-binding receptor. A similar 'de-knobbing' strategy was employed by Magnussen et al, 111 wherein an integrin-binding RGD motif was utilized, resulting in selective infection of integrin-expressing cell lines in vitro, as well as human glandular cells. 112 Based on the feasibility of fiber replacement with T4 fibritin, an elegant system was devised wherein the trimeric CD40 ligand was fused to the C-terminus of this artificial fiber.…”