Enhanced transduction efficiency of fiber-substituted adenovirus vectors by the incorporation of RGD peptides in two distinct regions of the adenovirus serotype 35 fiber knob.

摘要

Fiber-substituted Ad serotype 5 vectors containing the fiber protein from Ad serotype 35 (Ad5F35) exhibit properties that render them suitable as a platform for targeted Ad vectors. Ad5F35 vectors do not show apparent tropism in certain organs, including the liver, and they elicit less innate immunity than other vectors after intravenous administration. In order to develop a targeted Ad vector, we previously developed fiber-mutant Ad5F35 vectors containing the integrin binding Arg-Gly-Asn (RGD) motif in the FG or HI loop of the Ad35 fiber knob. Mutant Ad5F35 vectors containing the RGD peptide in the FG or HI loop transduced CD46-negative cells more efficiently in an RGD-dependent manner, as compared to the efficiency achieved with unmodified Ad5F35 vectors (Matsui et al., 2009. Gene Therapy 16, 1050-1057). However, the transduction efficiency of the mutant Ad5F35 vectors in CD46-negative cells remained lower than had been expected. Ad5F35 vectors containing the RGD peptide in the HI or FG loop enabled a 6-fold higher transduction efficiency than that achieved with unmodified Ad5F35 vectors in CD46-negative cells, although this cell type abundantly expresses alpha(v)-integrins. In the present study, we aimed to enhance the transduction efficiency of fiber-mutant Ad5F35 vectors. To this end, we developed an Ad5F35-vector system in which foreign peptides could be incorporated into regions of FG and HI loops of the Ad35 fiber knob by means of in vitro ligation. Using this Ad5F35-vector system, firefly luciferase-expressing mutant Ad5F35 vectors containing the RGD peptides in both loops (Ad5F35-2xRGD-L2) were constructed. In CD46-negative cells, Ad5F35-2xRGD-L2 showed 12-fold and 3-fold greater transduction efficiency than unmodified Ad5F35 vectors and mutant Ad5F35 vectors containing only one copy of the RGD peptide in the FG or the HI loop. In addition, transduction with Ad5F35-2xRGD-L2 in CD46-negative cells was RGD peptide-dependent. These results indicate that fiber-mutant Ad5F35 vectors, by which foreign peptides can be simultaneously incorporated into both the FG and the HI loops of the Ad35 fiber knob, could be a promising gene delivery vehicle for various gene therapies, and could facilitate basic research efforts such as analyses of gene function.