engleski

THE ABILITY OF RGD4C AND NGR4C TARGETING MOTIFS TO MEDIATE DIRECT INTERNALIZATION OF RETARGETED ADENOVIRUSES

Even though vectors based on adenovirus type 5 (Ad5) have been intensively investigated for tumor gene therapy their major limitation is nonspecific distribution in tissue after in vivo gene transfer. One strategy for genetic retargeting of Ad5 is based on the modification of tropism by genetic incorporation of a targeting ligand into the adenovirus fiber protein. We have previously shown that Ad5NGR4C containing NGR sequence (asparagine-glycine-arginine) flanked by 4 cysteines binds to APN expressed on RD cells, and we provided evidence that disulfide bond formation within Ad5NGR4C is required for targeting properties of NGR4C. In this study we wanted to answer whether NGR4C can mediate direct internalization of Ad5NGR4C, or in other words, whether APN functions as attachment and/or internalization receptor. The experiments were performed with 6 viruses: Ad5wt, Ad5NGR4C, Ad5RGD4C, Ad5FbΔ 639, Ad5FbΔ 639NGR4C and Ad5FbΔ 639RGD4C. The Ad5FbΔ 639 possess shortened fiber protein obtained by deletion of the middle part within the shaft domain. The infection by Ad5FbΔ 639 is independent on the presence of CAR on the cell surface. Due to the length of the fiber protein as well as its inability to bend, the retargeting motif incorporated in the HI-loop of Ad5FbΔ 639 fiber protein can mediate more efficient transduction only if it also mediates direct Ad5 vector internalization. As a proof of concept we incorporated RGD4C motif into Ad5FbΔ 639. Ad5RGD4C transduces RD cells 47-fold better than Ad5wt. The increase in transduction efficacy for Ad5FbΔ 639RGD4C in comparison to Ad5FbΔ 639 was 27-fold indicating that RGD4C incorporated in fiber protein, after binding to integrins, indeed mediates direct Ad5 internalization. In order to investigate whether NGR4C motif incorporated in fiber protein would allow for direct internalization we measured binding to RD cells. Ad5NGR4C and Ad5FbΔ 639NGR4C bind to RD cells more efficiently than Ad5wt and Ad5FbΔ 639, 2.8-fold and 6.6-fold respectively. The 2.5-fold increased transduction efficacy was observed for Ad5NGR4C in comparison to Ad5wt. However, the transduction efficacy of Ad5FbΔ 639NGR4C and Ad5FbΔ 639 was similar. We conclude that NGR4C incorporated into HI loop of short fiber protein, despite increased attachment to cell surface APN, does not possess the same capability as RGD4C, i.e. does not mediate direct internalization of Ad5 vector. As a conclusion we propose that retargeting motifs should be selected not only by ability to increase transduction, but also by ability to provoke direct internalization resulting in infection mechanism that might be less dependent on integrins present on the cell surface.

RGD4C; NGR4C; adenovirus; preusmjeravanje; direktna internalizacija

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