DEVELOPMENT AND ANALYSIS OF RETROVIRAL VECTORS EXPRESSING HUMAN FACTOR-VIII AS A POTENTIAL GENE-THERAPY FOR HEMOPHILIA-A

To develop a potential gene therapy strategy for the treatment of hemo philia A, we constructed several retroviral vectors expressing a B-dom ain-deleted factor WI (FVIII) cDNA, We confirmed previous reports that when the FVIII cDNA is inserted into a retroviral vector, the vector mRNA is decreased resulting in significantly (100- to 1,000-fold) lowe r vector titers. In an attempt to overcome this inhibition we pursued two independent strategies, First, site-directed mutagenesis was emplo yed to change the structure of a putative 1.2-kb FVIII RNA inhibitory sequence (INS), Second, the FVIII gene was transcribed from a retrovir al vector containing a 5' intron, Results demonstrated that the intron increased FVIII expression up to 20-fold and viral titer up to 40-fol d but conservative mutagenesis of the putative FVIII WS region failed to yield a significant increase in FVIII expression or titer, Using th e improved FVIII splicing vector, we transduced a variety of cell type s and were able to demonstrate relatively high FVIII expression (10-60 ng of FVIII/10(6) cells/24 hr), These results underscore the usefulne ss of these transduced cell types for potential in vivo delivery of FV III.