The overall goal of this proposal is to develop a new viral hybrid vector for treatment of genetic liver disease. In order to obtain life- long expression of a therapeutic gene, the vector must transduce hepatocytes efficiently without associated toxic or immunologic side effects and integrate into predictable sites into the host genome. In an attempt to combine the advantages of adenoviral vectors (high titer, infectivity, large capacity) and the integration capability of recombinant AAV, AAV ITRs flanking a reporter gene cassette were incorporated into E1/E3 deleted adenoviral vectors. During replication of these hybrid vectors in 293 cells, a 5.5kb genome (deltaAd.AAV1) was efficiently generated and packaged into adenovirus capsids. The deltaAd.AAV1 genome contained only the AAV-vector cassette linked on both sides to the adenoviral packaging signal and ITRs deltaAd.AAV1 was produced at high titer and purity. This hybrid vector devoid of all viral genes transduced cells in vitro and in vivo with frequencies comparable to rAAV integrating into the host genome randomly. One objective of this proposal is to further characterize the in vivo properties of deltaAd.AAV1. To construct a site-specific integrating deltaAd.AAV vector, the functions of AAV Rep68/78 will be utilized to mediate rescue and integration of a transgene cassette flanked by AAV ITRs into a specific site in human chromosome 19. The central questions of this proposal are: 1) whether the Rep 68/78 functions can be incorporated into deltaAd.AAV without affecting the ability to produce high titer vector. To address this problem, the unique structure of deltaAd.AAV will be applied to bring the rep gene in a transcriptionally active position under the control of a hepatocyte specific promoter only at late stages of virus replication in 293 cells. 2) whether this modified hybrid vector will transfer the Rep68/78 function of site- specific integration transiently to human target cells in vitro and in vivo without toxic side effects. Our hypothesis is that Rep-mediated vector integration occurs via the AAV ITRs without the rep gene, which was placed outside the AAV-ITR-transgene cassette, thus eliminating rep expression upon integration. 3) whether site-specific vector integration can be mediated by Rep protein molecules co-packaged into deltaAd.AAV capsids. Initial transduction experiments will be performed in human and murine cell cultures. For initial in vivo studies, immunodeficient mice with hepatic tumors derived from transformed human cells that can be targeted by systemic vector application will be used.