Many applications for human gene therapy would be facilitated by high levels and long duration of physiologic gene expression. Adenoviral vectors are frequently used for gene transfer because of their high cellular transduction efficiency in vitro and in vivo. Expression of viral proteins and the low capacity for foreign DNA limits the clinical application of first- and second-generation adenoviral vectors. Adenoviral vectors with all viral coding sequences deleted offer the prospect of decreased host immune responses to viral proteins, decreased cellular toxicity of viral proteins and increased capacity to accommodate large regulatory DNA regions. Currently most vectors used in vivo for preclinical and clinical studies express cDNAs under the control of heterologous eukaryotic or viral promoters. Using an adenoviral vector with all viral coding sequences deleted and containing the complete human alpha1-antitrypsin (PI) locus, we observed tissue-specific transcriptional regulation in cell culture and in vivo; intravenous injection in mice resulted in high levels of very stable expression for more than ten months and decreased acute and chronic toxicity. These results indicate significant advantages of regulated gene expression using genomic DNA for gene transfer and of adenoviral gene transfer vectors devoid of all viral coding sequences.

The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration of transgene expression, which can be related to immune responses and to toxic effects of viral proteins. In addition, readministration is usually ineffective unless the animals are immunocompromised or a different adenovirus serotype is used. Recently, adenoviral vectors devoid of all viral coding sequences (helper-dependent or gutless vectors) have been developed to avoid expression of viral proteins. In mice, liver-directed gene transfer with AdSTK109, a helper-dependent adenoviral (Ad) vector containing the human ␣1-antitrypsin (hAAT) gene, resulted in sustained expression for longer than 10 months with negligible toxicity to the liver. In the present report, we have examined the duration of expression of AdSTK109 in the liver of baboons and compared it to first-generation vectors expressing hAAT. Transgene expression was limited to approximately 3-5 months with the first-generation vectors. In contrast, administration of AdSTK109 resulted in transgene expression for longer than a year in two of three baboons. We have also investigated the feasibility of circumventing the humoral response to the virus by sequential administration of vectors of different serotypes. We found that the ineffectiveness of readministration due to the humoral response to an Ad5 first-generation vector was overcome by use of an Ad2-based vector expressing hAAT. These data suggest that long-term expression of transgenes should be possible by combining the reduced immunogenicity and toxicity of helperdependent vectors with sequential delivery of vectors of different serotypes.