E4ORF3 requirement for achieving long-term transgene expression from the cytomegalovirus promoter in adenovirus vectors

Abstract

Analysis of transgene expression under the control of the cytomegalovirus (CMV) promoter from adenovirus vectors in which the E4 region was modified indicated that E4ORF3 is required for long-term expression in the murine lung. CMV promoter truncation led to the persistence of expression in the absence of E4, thus eliminating the ORF3 requirement.

FIG. 1

Genomic structure of E4-modified adenovirus vectors. Expression cassettes replacing the E1 region are depicted on the left and variations of the E4 region are depicted on the right. (A) Vectors contain a CMV promoter element to drive β-galactosidase expression. Ad2/βgal-4 contains a wild-type (WT) E4 region, and Ad2/βgal-7 and -8 have deletions of E4 but retain ORF4 and ORF6 and -6/7, respectively. Ad2/βgal-9, -10, -11, -12, and -13 contain insertions or deletions in ORF1, ORF2, ORF3, ORF4, and ORF6/7, respectively. (B) Vectors contain a CMV promoter element to drive CFTR expression. Ad2/CFTR-16 contains a wild-type E4 region. Ad2/CFTR-5 and -18 have deletions of E4 but retain ORF6 and ORF3 and -4, respectively. BGH, bovine growth hormone. (C) ΔCMVβgal-1 contains a truncated CMV promoter element to drive β-galactosidase expression. The E4 region of this vector is completely deleted. SV40, simian virus 40.

FIG. 2

β-Galactosidase expression in the lungs of immunodeficient mice that received vectors in the Ad2/βgal series. BALB/c nude mice were given intranasal instillations of 3 × 10⁹ i.u. of each virus. Mice were sacrificed on days 3 and 14 postinstillation, and the β-galactosidase activity in the lung tissue was measured. Expression on day 14 is depicted as percentages of activity (relative light units [RLU]) detected on day 3. Each bar represents the average of data for four mice, except Ad2/βgal-4 (n = 12) and Ad2/βgal-11 (n = 8). Error bars indicate the standard deviation. ∗, significant difference (P < 0.05) from Ad2/βgal-4.

FIG. 3

Persistence of hCFTR expression in the lungs of immunocompetent mice. BALB/c mice were given intranasal instillations of 3 × 10⁹ i.u. of Ad2/CFTR-5, -16, or -18 and were sacrificed on days 3, 21, and 42 postinstillation. RNA samples from lungs were pooled at each time point (n = 4) with each vector, and hCFTR mRNA was measured by quantitative reverse transcription-PCR. □, Ad2/CFTR-16; ▵, Ad2/CFTR-18; ○, Ad2/CFTR-5.

FIG. 4

β-Galactosidase expression in the lungs of immunodeficient mice. BALB/c nude mice were given intranasal instillations of 3 × 10⁹ i.u. of either Ad2/βgal-4 or ΔCMVβgal-1. One cohort of animals received ΔCMVβgal-1 in combination with Ad2/CFTR-16. Mice were sacrificed on days 3 and 14 postinstillation, and the β-galactosidase activity in the lung tissue was measured. Each bar represents the average from three animals. Error bars represent the standard deviation. RLU, relative light units.