Extensive in vivo gene transfer studies in animal models and human gene therapy clinical trials with E1-deleted adenovirus vectors have demonstrated transience of transgene expression due to direct cytopathic effects of the vectors and host immune response to virally expressed proteins. In order to overcome these difficulties, we have recently developed packaging cell lines which support the growth of adenovirus vectors containing lethal deletions in both E1 and E4 gene regions. Here we demonstrate that use of E1/E4-deleted adenovirus vectors leads to prolonged in vivo transgene expression due to elimination of cytopathic effects and significant reduction of virus-specific immune response.