190. Strategies for Achieving High-Level, Stable, Endothelium-Specific Transgene Expression Using Helper-Dependent Adenovirus
Authors: Nagadhara Dronadula, Brad Wacker, Reginald van der Kwast, Jingwan Zhang, David A. Dichek
Journal: Molecular Therapy (2015)

Endothelial cells (EC) are attractive targets for gene therapy because they can deliver therapeutic proteins both locally and systemically. Helper-dependent adenoviral (HDAd) vectors are useful for EC-targeted gene therapy because they efficiently transduce EC and express transgenes persistently in vivo. However, obtaining high-level and stable in vivo expression from HDAd is a challenge. Our current goal of transducing EC with HDAd to treat atherosclerosis will likely require high-level, stable expression of atheroprotective genes such as apolipoprotein A-I (apoA-I) and interleukin-10 (IL-10). We previously showed: a) use of a CMV promoter to drive expression of apoA-I resulted in substantial early transgene expression followed by a steep decline; b) expression of IL-10 from the CMV promoter did not show this decline; c) a mammalian promoter/enhancer including elements from the mouse endothelin gene (4X-ETE) expressed IL-10 at least as high as the CMV promoter and showed more EC-specificity; and d) IL-10 expression from both promoters was low. We used codon optimization, the 4xETE promoter/enhancer, removal of the endogenous IL-10 3'UTR, and co-infusion of IL-10 and apoA-I vectors to attempt to achieve high-level, stable transgene expression. HDAd reagents were from AdVec. Expression cassettes were screened in vitro by transfection into bovine aortic EC, bovine aortic smooth muscle cells, and rabbit fibroblasts. A subset of cassettes was incorporated into HDAd vectors and tested in vivo in rabbit carotids. Gene expression was measured by quantitative RT-PCR of cell or artery extracts and by western blotting of EC-conditioned medium. Surprisingly, codon optimization did not increase mRNA or protein expression from either IL-10- or apoA-I-expressing vectors. In contrast to our previous results with IL-10-expressing vectors (in which the 4XETE promoter/enhancer expressed as highly as CMV in vivo), the 4XETE promoter/enhancer expressed apoA-I at only ~25% of levels obtained with the CMV promoter and did not show EC-specific expression. However, unlike the CMV-apoA-I vector, expression of apoA-I from the 4XETE vector did not decline from 3–28 days (expression was 18% higher at 28 vs 3 days; P=NS). Removal of the endogenous IL-10 3' UTR from 4XETE-IL-10 increased in vitro IL-10 mRNA by 4-fold and IL-10 protein by ~15-fold while preserving EC-selective expression. Co-infusion of an IL-10 vector along with the apoA-I vector significantly decreased early loss of transgene expression from CMV-apoA-I (37% decrease; P=0.2 for HDAdIL-10 co-infusion vs. 81% decrease; P<0.001 for HDAdNull co-infusion). Early loss of HDAd-mediated transgene expression can be ameliorated by either use of a mammalian promoter/enhancer (4XETE) or by co-infusion of a vector expressing an immunomodulatory cytokine (IL-10). Cell-type specificity of expression is influenced both by promoter/enhancer and transgene. Elimination of non-coding sequences that may mediate mRNA decay can dramatically increase transgene expression in EC, while preserving cell-type specificity.