Adenovirus vectors based on a new helper dependent system are being developed for use in gene therapy. Because cloning capacity of the vectors is greater than 35 kb and all viral genes are deleted, these vectors are well suited for gene therapy, and will be used to produce vectors carrying genes for therapy of cardiovascular disease. Both """"""""first generation"""""""" E1, E3 deleted viruses and helper dependent vectors will be used to identify and study gene regulatory elements that can improve duration of expression while retaining ability to express transgenes at high levels. A disadvantage of adenovirus vectors for some applications is the failure of the viral DNA to integrate a high frequency. To achieve integration of transgenes into mammalian DNA, the possibility of using components of a eukaryotic transposable element will be explored. The eukaryotic transposable element, mariner, has been shown to encode a transposase that can function in the absence of host cell factors and therefore should be capable of inducing transposition of sequences flanked by mariner terminal repeats when both are introduced into mammalian cells. This transpoase will be expressed in Ad vectors under the control of a T7 promoter with T7 RNA polymerase provided by a second vector or by cells transformed with a T7 pol expression cassette. Initially, enzymatic activity of the transposase expressed in vector infected cells will be studied in cell free systems. To detect transposition in transduced cells, appropriate vectors carrying minimariner elements and selectable markers encoding drug resistance will be constructed, used in combination with vectors expressing transposase to transduce cells, and transformed cells selected for drug resistance will be established and analyzed. Eventually experiments will be done in transgenic mice expressing the T7 pol to obtain transposition in vivo.
