Activation of transgene expression by early region 4 is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo

Abstract

The persistence of transgene expression has become a hallmark for adenovirus vector evaluation in vivo.Although not all therapeutic benefit in gene therapy is reliant on long-term transgene expression, it is assumed that the treatment of chronic diseases will require significant persistence of expression. To understand the mechanisms involved in transgene persistence, a number of adenovirus vectors were evaluated in vivo in different strains of mice. Interestingly, the rate of vector genome clearance was not altered by the complete deletion of early region 4 (E4) in our vectors. The GV11 (E1 ؊ E4 ؊ ) vector genome cleared with a similar kinetic profile as the GV10 (E1 ؊ ) vector genome in immunocompetent and immunocompromised mice. These results suggest that the majority of adenovirus vector genomes are eliminated from transduced tissue via a mechanism(s) independent of T-cell, B-cell, and NK cell immune mechanisms. While the levels of persistence of transgene expression in liver or lung transduced with GV10 and GV11 vectors expressing ␤-galactosidase, cystic fibrosis transmembrane conductance regulator, or secretory alkaline phosphatase were similar in immunocompetent mice, a marked difference was observed in immunocompromised animals. Levels of transgene expression initially from both GV10 and GV11 vectors were the same. However, GV11 transgene expression correlated with loss of vector genome, while GV10 transgene expression persisted at a high level. Coadministration and readministration of GV10 vectors showed that E4 provided in trans could activate transgene expression from the GV11 vector genome. While transgene expression activity per genome from the GV10 vector is clearly activated, expression from a cytomegalovirus promoter expression cassette in a GV11 vector appeared to be further inactivated as a function of time. Understanding the molecular mechanisms underlying these expression effects will be important for developing persistent adenovirus vectors for chronic applications.

Biology of Adenovirus and Its Use as a Vector for Gene Therapy

“…It was thus theorized that removal of all or part of the E4 transcription unit would impair viral replication and gene expression such that an immune response would not be triggered. Rodent models have suggested that the deletion of some or all E4 proteins may affect the length and level of transgene expression; however, this regulation appears to be both tissue and promoter specific (Armentano et al, 1997;Brough et al, 1997;Dedieu et al, 1997;Wang et al, 1997;Lusky et al, 1998;Grave et al, 2000).…”

Helper‐Dependent Adenoviral Vectors

“…Additional deletions of viral genes (E2 or E4) were implemented in so-called second generation Ad vectors in an attempt to overcome this problem. The advantages of second generation Ad over FGAd remain controversial as some studies show them to be superior in terms of toxicity and longevity of transgene expression [28][29][30][31][32][33][34][35][36] while others do not [22,[37][38][39][40][41].…”

Generation of an Adenovirus Vector Lacking E1, E2a, E3, and All of E4 except Open Reading Frame 3

“…First, we had a cell line that complemented only E1 and E2a functions (17) and transient-transfection experiments with 293 cells demonstrated that expression of E4 ORF3 alone could support growth of a virus with E4 deleted, dl1011 (5), although not as well as a virus with the entire E4. Second, previous studies suggested that E4 gene products helped maintain vector transgene expression (2,6,10).…”

“…Quantitative analysis of in vitro ␤-galactosidase expression. A remarkable feature that emerged from infection with the adenoviral vectors with E1 and E4 deleted was that expression of a reporter gene under the control of the cytomegalovirus (CMV) or RSV promoter was influenced by E4 protein expression (2,6,10). Thus, a quantitative ␤-galactosidase assay was used to evaluate transgene expression in a noncomplementing A549 cell background, by comparing the ␤-galactosidase expression of the Av3nBg vector (E4 ϩ ) to that of the Av4orf3nBg vector.…”

“…This type of vector grows in packaging cell lines that complement the deleted viral genes. Adenovirus vectors have been constructed with the E1 gene deleted (28,36,37,45) and with E1 plus E2a, E2b, or E4 deleted (1,2,6,10,13,14,16,17,28,40). Studies carried out in vitro to evaluate adenovirus vectors with double deletions consistently feature an absence of detectable replication and late gene expression (1,17).…”