122. Expression of hCD40 in the Pulmonary Endothelium Targeted Via a Fiber-Modified Adenovirus Improves Gene Delivery to the Lung

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The lung is an attractive target for gene therapy since many diseases such as cystic fibrosis, lung cancer, primary pulmonary hypertension and acute respiratory distress syndrome require novel therapeutic approaches. However, pulmonary gene transfer by adenovirus (Ad) vectors via the vascular route has proven to be limited with respect to efficiency due to low expression of human coxsackie adenovirus receptor (hCAR) in the lung. Conversely, the liver expresses high levels of hCAR leading to ectopic transduction and the risk of liver damage. Therefore, development of CAR-independent gene transfer strategy to the lung is needed. We recently constructed an Ad in which the fiber was replaced by human CD40 ligand (Ad5 Luc FF / CD40L) and showed the utility of this fiber replacement strategy for genetic targeting of the virus to human CD40 (Belousova et al. J. Virol 77:11367–77, 2003). In addition, we recently showed the promoter for vascular endothelial growth factor receptor 1 (fms-like tyrosine kinase-1; flt-1) has high activity in pulmonary endothelial cells, low activity in hepatocytes (Nicklin et al. Hypertension 38:65–70, 2001), and also provided transcriptional targeting to the lung vasculature (Reynolds et al. Nat. Biotechnol.19:838–42, 2001). We hypothesized that hCD40 expression, transcriptionally targeted to the vascular endothelium via the flt-1 promoter, could provide CD40-targeted gene delivery to the lung in a CAR-independent manner. We constructed an E1-deleted recombinant adenovirus expressing hCD40 driven by the flt-1 promoter (Ad5flt1-hCD40). We then analyzed hCD40 expression and flt-1 activity in several cell lines by luciferase assay and fluorescence-activated cell sorting (FACS) analysis. We selected NTERA-2 cells as low hCD40 and flt-1 positive cell line, and HeLa cells as low hCD40 and flt-1 negative cell line as control for further experiments. Forty-eight hours after the first infection with Adflt1-hCD40 and the control virus, we quantitated hCD40 expression on the cell surface by FACS analysis. Adflt1-hCD40 infected NTERA-2 cells had markedly increased hCD40 expression compared to either control virus infected cells or flt-1 negative HeLa cells. Further, we evaluated infectivity enhancement gained by increased hCD40 in the flt-1 positive cell line. Forty-eight hours after the first infection, cells were infected with Ad5 Luc FF / CD40L vectors at an MOI of 1000 viral particles / cell to determine infectivity enhancement accrued via CD40 targeting. Luciferase assays were performed 24 hours post second infection. Ad5 Luc FF / CD40L vector infectivity in Ad flt-1 hCD40 infected NTERA-2 cells showed 13-fold greater luciferase reporter gene activities versus negative control virus infected cells. These data suggest that expression of hCD40 in pulmonary endothelial cells using recombinant Ad vectors containing hCD40 gene under the control of the flt-1 promoter and targeted with a novel fiber modified Ad vector can increase the efficacy and specificity of adenoviral gene therapy for lung disease.