Genetic correction of inherited muscle diseases, such as Duchenne muscular dystrophy, will require long term expression of the recombinant protein following gene transfer. We have shown previously that a new adenoviral vector that lacks all viral genes expressed both full-length dystrophin and ␤-galactosidase in mdx (dystrophin-deficient) mouse muscle. We observed a significant histologic improvement of vector-transduced mdx muscle before the eventual loss of vector-encoded transgene expression. In this study, we investigated whether an immunological response against vector-encoded ␤-galactosidase contributed to the loss of vector expression and affected vector persistence in muscle. Intramuscular vector injection in control normal mice resulted in an early and complete loss of ␤-galactosidase expression accompanied by predominantly CD4 ؉ and CD8؉ lymphocytic infiltration and a significant loss of vector DNA. In contrast, intramuscular vector injection in lacZ transgenic mice resulted in persistent expression of ␤-galactosidase for at least 84 days with no evidence of inf lammation or significant loss of vector DNA. Our studies demonstrate that, in the absence of an immune response induced by ␤-galactosidase expression, an adenoviral vector lacking all viral genes is stably maintained in muscle.

Previous studies of the use of adenoviral vectors in animal models of gene therapy have focused on the immune response against transduced cells as the major limiting factor to long-term transgene expression. In this study we eliminated the variable of immunity induced by expression of the transgene in order to investigate vector DNA stability of both first-generation and high-capacity adenoviral vectors after gene transfer to skeletal muscle. Transgene expression from a high-capacity adenoviral vector remained at a high level for at least 20 weeks and was accompanied by persistence of intact vector genomes. In contrast, transgene expression from a first-generation adenoviral vector markedly diminished by 6 weeks after gene transfer and was accompanied by mild and variable inflammatory cell infiltrates. Surprisingly, despite this loss of transgene expression, the first-generation adenoviral vector genomes persisted like the high-capacity adenoviral vector genomes. Therefore, in the absence of immunity to transgene proteins, loss of expression from the first-generation vector was due to inhibition of transgene expression rather than to the elimination of vector-containing cells. DNA stability and persistent expression of the high-capacity adenoviral vector supports the potential of this vector for clinical applications of muscle gene transfer.