Efficient adenoviral gene transfer to kidney cortical vasculature utilizing a fiber modified vector.
BACKGROUND
The alpha v integrin is present in vascular endothelium, including that of the kidney. It is also upregulated in the presence of inflammatory cytokines and in some neoplasms. In an effort to transduce vascular endothelial cells, we compare in vitro and in vivo adenoviral gene transfer of a vector with a high affinity peptide ligand to the alpha v integrin incorporated into the fiber coat protein AdZ.F(RGD) to an unmodified vector, AdZ.


METHODS
Cell transduction assays were performed on human umbilical vein endothelial cells (HuVEC) and human pulmonary epithelial cells (A549). In vitro competition assays were performed in the presence of either wild type (F5K) or chimeric (F5K(RGD)) soluble recombinant fiber protein. Transduction efficiency was determined by quantitative beta-galactosidase activity. In vivo gene transfer was compared infusing either AdZ or AdZ.F(RGD) into the left renal artery of the rat and assaying beta-galactosidase staining of the kidney. Gene transfer was also evaluated in the presence of a competitive RGD or control RGE peptide.


RESULTS
There was a marked increase in transgene expression in HuVEC cells with AdZ.F(RGD) as compared to AdZ. The increased expression with AdZ.F(RGD) was more prominent in the endothelial as opposed to the epithelial cell line. Furthermore pre-incubation of these cells with either F5K or F5K(RGD) soluble fiber protein markedly decreased beta-galactosidase activity of AdZ, whereas only the F5K(RGD) decreased beta-galactosidase activity of AdZ.F(RGD). AdZ.F(RGD) also resulted in significantly enhanced beta-galactosidase expression in the vascular endothelium of the kidney (for comparable amounts of virus injected) as well as significantly higher gene transfer to cortical vasculature. Coinfusion of an RGD peptide with AdZ.F(RGD) blocked gene transfer whereas a control RGD peptide did not.


CONCLUSIONS
We conclude that incorporating a high affinity peptide ligand into the adenoviral fiber protein can preferentially enhance in vitro and in vivo adenoviral transfection efficiency in endothelial cells. Enhancing transfection efficiency will not only broaden the scope of disease processes addressed with adenoviral vectors but also allow the use of lower titer, thereby limiting toxicity. This vector has additional potential to transduce endothelial cells within tumors or ischemic tissue where alpha v integrins are upregulated.
McDonald
G A
GA
Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA.
Zhu
G
G
Li
Y
Y
Kovesdi
I
I
Wickham
T J
TJ
Sukhatme
V P
VP
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D001667:Binding, Competitive; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007261:Infusions, Intra-Arterial; D039564:Integrin alphaV; D007672:Kidney Cortex / Q000098:blood supply*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D012077:Renal Artery; D001616:beta-Galactosidase / Q000235:genetics
D015703:Antigens, CD; D036022:Capsid Proteins; D039564:Integrin alphaV; C018315:hexon capsid protein, Adenovirus; D001616:beta-Galactosidase