The overall goal of this proposal is to achieve long-term expression of human factor VIII (FVIII) and human alpha1-anti-trypsin (hAAT) at therapeutic levels in mice. Towards this goal, we will utilize hybrid deltaAd.AAV vectors, which are devoid of all adenoviral genes and accommodate transgene cassettes with a maximal size of AAV vectors. Transient expression of AAV rep78 from non-integrating adenoviral vectors stimulates site-specific integration of deltaAd.AAV vectors. We will use two principal gene transfer approaches: (A) transduction of hepatocytes in vivo after systemic vector application and (B) in vitro transduction of bone marrow derived stem cells with subsequent transplantation and liver repopulation. In the in vivo transduction studies (A), we will test i) whether the use of deltaAd.AAV vectors retargeted to hepatocytes will minimize vector related toxic and immunological side effects; ii) whether modified deltaAd.AAV vectors in combination with transient rep78 expression will allow for site-specific integration in hepatocytes; and ii) whether integrated transgenes will provide sustained FVIII and hAAT expression at therapeutic levels. In the in vitro transduction studies with bone marrow derived stem cells (B), we will test i) whether bone marrow cells with liver repopulation capacity can be stably transduced with retargeted deltaAd.AAV vectors; ii) whether transduced cells will engraft in normal or diseased mouse livers, differentiate into hepatocytes, and provide lifelong transgene expression; and iii) whether this approach will circumvent the production of neutralizing antibodies to FVIII or hAAT. For both strategies, we will assess whether transduced cells can be expanded in vivo by dimerizer- drug dependent induction of cell proliferation. In vitro transduction and integration studies will be performed with murine and human hepatocytes. Vector toxicity tests and preliminary in vivo transduction studies will be done in normal mice to select the optimal vector/s and doses for subsequent studies in murine disease models including hemophilic, FVIII knockout mice and hAAT transgenic mice.
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