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
Introduction
Species C adenovirus serotype 5 (Ad5) vectors achieve the highest level of transduction efficiency among existing vectors, and as such, have been widely used as a gene delivery vehicle for both gene therapy as well as for basic research. However, Ad5 vectors are associated with several problems, including low transduction efficiency in cells lacking the primary receptor, coxsackievirus-adenovirus receptor (CAR) (Li et al., 1999, Segerman et al., 2000), and the high hepatic tropism of Ad5 after intravenous administration (Kalyuzhniy et al., 2008, Waddington et al., 2008). To overcome such problems, several groups have developed fiber-substituted Ad5 vectors (Ad5F35) containing the fiber knob and shaft of Ad serotype 35 (Ad35) (Mizuguchi and Hayakawa, 2002, Shayakhmetov et al., 2000). Ad35 utilizes human CD46 as a cellular attachment receptor (Gaggar et al., 2003, Segerman et al., 2003). Ad5F35 vectors efficiently transduce a variety of cells because Ad5F35 vectors recognize human CD46 as an infection receptor. It is well-known that human CD46 is ubiquitously expressed on almost all human cells. Intravenous administration of Ad5F35 vectors results in a lower accumulation in the liver than that of Ad5 vectors, and Ad5F35 vectors do not show apparent tropism for certain organs (DiPaolo et al., 2006, Ni et al., 2005). Furthermore, recent studies have demonstrated that Ad5F35 vectors elicit less of an innate immune response than do conventional Ad5 vectors (DiPaolo et al., 2006, Ni et al., 2005). These properties indicate that Ad5F35 vectors would be suitable as a platform for targeted Ad vectors. To develop targeted Ad vectors based on Ad5F35 vectors, we previously developed a fiber-mutant Ad5F35-vector system in which foreign peptides can be incorporated into the FG or HI loop of the Ad35 fiber knob. Previous studies demonstrated that both FG and HI loops are crucial for binding of Ad35 fiber knob to human CD46 (Pache et al., 2008a, Wang et al., 2007). Fiber-mutant Ad5F35 vectors containing the integrin binding Arg-Gly-Asp (RGD) motif in the FG or HI loop transduced CD46-negative cells more efficiently in an RGD-dependent manner, compared with that of unmodified Ad5F35 vectors (Matsui et al., 2009). However, the transduction efficiency of these fiber-mutant vectors remained lower than had been expected; moreover, the transduction efficiency of the fiber-mutant Ad5F35 vectors was much lower than that of unmodified Ad5F35 vectors in CD46-positive cells, although these cells abundantly express αv-integrins. Therefore, to further improve upon the transduction efficiency achieved with fiber-mutant Ad5F35 vectors, we developed for the present study an Ad5F35-vector system in which foreign peptides could be simultaneously incorporated into both the FG and HI loops of the Ad35 fiber knob by means of in vitro ligation. In CD46-negative cells, the mutant Ad5F35 vectors containing the RGD peptides in both loops (Ad5F35-2xRGD-L2) yielded more than 12-fold and 3-fold greater transduction efficiency than that of the unmodified Ad5F35 vectors and mutant Ad5F35 vectors containing only one copy of the RGD peptide, respectively. Moreover, Ad5F35-2xRGD-L2 exhibited higher levels of cellular binding in CD46-negative cells, compared with that observed with the unmodified Ad5F35 vectors.
Section snippets
Cells
SK HEP-1 (an endothelial cell line derived from human liver), SF295 (a human glioblastoma multiforme cell line), and 293 cells were cultured with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). NIH3T3 (a mouse embryo fibroblast cell line) and B16 cells (B16BL6; a mouse melanoma cell line) were cultured with minimum essential medium (MEM) supplemented with 10% FCS.
Ad vectors
The vector plasmid for a fiber-mutant Ad5F35 vector, pAdHM75-Csp45I(FG)-XbaI(HI), was
Trimerization of the fibers containing RGD peptides into two distinct regions of the Ad35 fiber knob
We developed an Ad vector plasmid in which foreign peptides can be incorporated into both the FG and HI loops of the Ad35 fiber knob by means of in vitro ligation (Fig. 1A). We previously demonstrated that the FG and HI loops are suitable for the insertion of foreign peptides in the Ad35 fiber knob (Matsui et al., 2009). Using this Ad vector system, firefly luciferase-expressing Ad5F35 vectors containing the RGD peptide in both loops (Ad5F35-2xRGD-L2) were constructed. Table 1 shows the copy
Discussion
In this study, we developed novel mutant Ad5F35 vectors containing the RGD peptide in two distinct regions, i.e., in both the FG and HI loops, of the Ad35 fiber knob (Ad5F35-2xRGD-L2) to further improve the transduction efficiency of Ad5F35 vectors. In CD46-negative cells, Ad5F35-2xRGD-L2 showed greater transduction efficiency than that of the parent Ad5F35 vector and fiber-mutant Ad5F35 vectors containing only one copy of the RGD peptide in either the loop. Incorporation of two copies of the
Conclusions
In summary, fiber-mutant Ad5F35 vectors containing two copies of RGD peptide in two different loops of the Ad35 fiber knob are expected to be highly promising as a platform for targeted Ad vectors, as well as for use as a tool in functional analyses of genes. As a next step in the development of truly targeted Ad vectors, novel targeted molecules need to be identified that can be displayed on the Ad capsid; such work is currently underway in our facility.
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific Research (B) of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan. H. Matsui is the Research Fellow of the Japan Society for the Promotion of Science.
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