Characterization of Factors Involved in Modulating Persistence of Transgene Expression from Recombinant Adenovirus in the Mouse Lung

Abstract

One potential limitation of adenovirus (Ad)-based vectors for the gene therapy of cystic fibrosis (CF) and other genetic diseases is the transience of expression observed in most in vivo systems. In this study, the influence of various factors on persistence of transgene expression in the lung was investigated. In the absence of immune pressure, such as in the nude mouse, the genomic structure of the vector was found to be predominant in determining the persistence of expression; Ad vector constructs with an E1¯E3⁺E4ORF6⁺ backbone encoding β-galactosidase (β-Gal) or the cystic fibrosis transmembrane conductance regulator (CFTR) produced declining levels of expression while an Ad/CMVβGal vector with an E1¯E3⁺E4⁺ backbone gave rise to sustained, long-term reporter gene expression. The ability of the latter vector to persist was in turn limited in part by the presence of cytotoxic T lymphocytes (CTLs). Adoptive transfer experiments indicated that CTLs directed against either viral proteins or the β-Gal reporter gene product were able to reduce expression in nude C57BL/6 mice stably expressing β-Gal from the E4⁺ vector. Finally, the specificity and strength of the CTL response elicited by Ad vector was found to vary considerably depending on mouse strain haplotype. These results indicate that persistence of transgene expression in a given system is determined by the interplay between several factors including genomic structure of the vector, host background, and immune response. Transience of expression has been observed in most in vivo systems using recombinant adenovirus for gene transfer. In this study, we characterized factors involved in modulating longevity of transgene expression in the mouse lung. In athymic nude mice, the genomic structure of the vector was a predominant factor in determining stability of expression; E1¯E3⁺E4ORF6⁺ vectors encoding β-galactosidase (β-Gal) or cystic fibrosis transmembrane conductance regulator (CFTR) produced declining levels of expression while an E1¯E3⁺E4⁺ CMV βGal-encoding vector gave rise to sustained expression of β-Gal. In immunocompetent animals, persistence of expression was also limited by cytotoxic T lymphocytes (CTL) responses against the vector as determined by results obtained in immunodeficient mice and adoptive transfer experiments. The strength and specificity of the CTL response against vector was, in turn, found to vary considerably depending on mouse haplotype. These results indicate that longevity of expression in a given system is influenced by multiple factors, including genomic structure of the vector, host background, and immune response.