Background: Gene therapy approaches targeting vascular diseases require an efficient and specific gene transfer to the vascular wall. Previously applied vector systems fail to cover these requirements and induce strong host immune responses. Adeno-associated virus (AAV) vectors enable long-term gene transfer into the myocardium. However, they fail to efficiently transduce endothelial cells. We therefore sought out to develop a targeted AAV vector by modification of the capsid surface and use of a vascular promoter.
Methods: In order to restrict transgene expression to endothelium, we used the murine vascular cadherin-5 (mveCad-5) core promoter. For augmentation of expression levels, we generated additionally a chimeric promoter by fusing a CMV-enhancer (CMVenh) to mveCad-5. The constructs were tested in vitro to identify the most efficient promoter for further in vivo studies. For in vivo gene transfer, we compared naturally occurring AAV serotypes 8 and 9 to an AAV9 variant obtained by in vitro selection of a random AAV peptide display library. Vectors carrying a gene encoding the Cre recombinase under control of the CMV or CMVenh-mveCad5 promoters, respectively, were injected intravenously into adult ROSAmT/mG ("Tomato")-mice. The mouse model harbors a cell membrane-targeted, two-color fluorescent Cre-responsive reporter allele enabling expression of green fluorescence in Cre recombinase positive cells. After 3 weeks mice were sacrificed and representative tissues were analyzed by fluorescence microscopy for detection of green fluorescent cells.
Results: Our in vitro study showed that replacement of CMV by the mveCad-5 promoter restricted transgene expression to endothelial cells but overall expression was lower than that obtained with the CMV promoter. The CMVenh-mveCad5 promoter instead, resulted in high expression levels in endothelial cells with lower expression in other cells. While AAV8 and AAV9 failed to enable gene expression in endothelial cells in vivo, the peptide-targeted AAV9 vector enabled a highly efficient recombination in endothelial cells of the aorta. While both the CMV and CMVenh-mveCad5 promoter resulted in efficient EGFP expression in the aortic endothelium, unspecific extravascular expression was higher with the CMV-promoter.
Conclusion: Taken together, we could show that a targeted AAV-vector can be used for efficient gene transfer into endothelial cells of the aorta in vivo. Besides its use for validation of potentially therapeutic targets for vascular diseases this vector may also be suitable for conditionally knocking out genes in the vascular system.