Hemophilia A is an X-linked hereditary bleeding disorder resulting from the absence of Factor VIII, a blood clotting protein. Because the protein for FVIII is controlled by a single gene product and even a slight increase oSf FVIII in a patients blood stream can improve his/her quality of life, Hemophilia A is a prime candidate for gene therapy. One of the many goals for treating Hemophilia using gene therapy is to find a cell line which can effectively express FVIII. One possible cell type is megakaryocytes, human cells which form into platelets. It is hypothesized that if megakaryocytes can be targeted for FVIII expression, platelets will be able to secrete FVIII at a sight of bleeding and help clot the blood. In this study, Human Erythroid Leukemia (HEL) cells, a model for megakaryocyte cells, were transduced with Ad/AAV viral vector containing either the marker gene EGFP/Neo or FVIII. Optimal conditions for infection of HEL cells with the hybrid Ad/AAV virus using centrifugation were tested. HEL cells were infected with virus containing EGFP/Neo using centrifugation and non-centrifugation methods at various multiplicities of infection (MOIs) (100, 1,000, 10,000). With the use of flow cytometry, it was shown that centrifugation methods increase transduction efficiency 2-fold and that infection at higher MOIs yields an increased efficiency. When transduced at an MOI of 10,000, centrifuged cells have a 92% transduction efficiency, as quantified by flow cytometry and GFP fluorescence. These G418-selected cells were also shown to be stabily transduced for up to three weeks, although the mean fluorescence decreased over time, suggesting loss of transgene expression or cell death. In addition, cells were infected and spun at various centrifugal forces (500g, 1000g, 2000g, 3000g), and for various amounts of time (0.5, 1, 2, 3 hours). With the use of flow cytometry, the optimal infection conditions were established to be an MOI of 10,000, at 2000g, and for 2 hours, yielding a 92% transduction efficiency. Once these settings were determined, HEL cells were transfected at these optimal conditions with Ad/AAV virus containing the gene for FVIII. A COATEST was performed on the supernatant of the transfected cells to determine the level of FVIII activity. In this study, it was found that 0.416% of normal level of FVIII could be generated by 107 cells infected at an MOI of 1000 while 1.828% of normal level of FVIII could be generated by cells transfected at an MOI of 5,000. Thus, HEL cells can generate and secrete functional FVIII. Although a large amount of FVIII was not detected, these results are extremely significant since they suggest a proof-of-principle that megakaryocytes may be a reasonable target cell for a definitive gene correction strategy for FVIII. In the future, the next step is to move into a "primary cell" model system such as human stem cells to evaluate whether comparable results can be obtained. This study was funded by a grant from NIH HL53665.