Enhanced Gene Delivery to Human Primary Endothelial Cells Using Tropism-Modified Adenovirus Vectors

Abstract

Endothelial cells have been noted to have relatively low expression of the native receptor for adenovirus serotype 5 (Ad5), coxsackie and adenovirus receptor (CAR), and are thus refractory to Ad5 infection. In this study, we hypothesize that increases in the infectivity of Ad5 in primary human pulmonary artery (HPAEC), coronary artery (HCAEC) and umbilical vein endothelial cells (HUVEC) can be achieved through genetic capsid modification of Ad5 to bypass CAR-dependent infection. The modifications tested in this study include incorporation of an integrin-binding RGD peptide motif (Ad5.RGD), a poly-lysine motif (Ad5.pK7), a combination of both of these peptide domains (Ad5.RGD.pK7), an adenovirus serotype 3 knob domain (Ad5/3Luc1) and canine adenovirus serotype 1 or 2 knob domains (Ad5Luc1-CK1 and Ad5Luc1-CK2). In HPAEC and HCAEC, the greatest infectivity enhancements were achieved using Ad5/3Luc1 (26-fold and 30-fold respectively). HUVEC was most readily infected by Ad5Luc1-CK1 (213-fold). These results demonstrate that gains in Ad5 infectivity in endothelial cells can be accomplished with genetic capsid modifications.

Fig. 1

Schematic representation of a generalized Ad vector (panel A), which can be genetically modified for enhanced infectivity by incorporation of peptide motifs or alternate fiber knob domains as shown in panel B. Wild type unmodified adenovirus serotype 5 fiber and knob domain is shown first (Ad5WT), then with increasingly complex incorporated peptide motifs (Ad5.pK7, Ad5.RGD, and Ad5.RGD.pK7) followed by incorporation of serotype 3 knob (Ad5/3Luc1) and replacement of the Ad5 knob domain with the knob domains from canine adenovirus serotypes 1 or 2 (Ad5Luc1-CK1 and Ad5Luc1-CK2 respectively).

Fig. 2

Luciferase activity expressed as relative light units (RLU) following infection with Ad5 wild type capsid control virus for 48h. Ad5Luc1 contains firefly luciferase as a reporter gene (panel A) and Ad5GL3 contains a dual reporter cassette with green fluorescent protein and luciferase (panel B).

Fig. 3

Fold increase in luciferase activity versus Ad5 vectors with native fibers for a panel of genetically capsid modified Ad vectors in human pulmonary artery (HPAEC), human coronary artery (HCAEC) and human umbilical vein (HUVEC) endothelial cells. Bars indicate the mean fold increase in luciferase compared to isogenic control (n=3, ± standard deviation).