Development and Analysis of Retroviral Vectors Expressing Human Factor VIII as a Potential Gene Therapy for Hemophilia A

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Abstract
To develop a potential gene therapy strategy for the treatment of hemophilia A, we constructed several retroviral vectors expressing a B-domain-deleted factor VIII (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) lower vector titers. In an attempt to overcome this inhibition we pursued two independent strategies. First, site-directed mutagenesis was employed to change the structure of a putative 1.2-kb FVIII RNA inhibitory sequence (INS). Second, the FVIII gene was transcribed from a retroviral vector containing a 5′ intron. Results demonstrated that the intron increased FVIII expression up to 20-fold and viral titer up to 40-fold but conservative mutagenesis of the putative FVIII INS region failed to yield a significant increase in FVIII expression or titer. Using the improved FVIII splicing vector, we transduced a variety of cell types and were able to demonstrate relatively high FVIII expression (10–60 ng of FVIII/106 cells/24 hr). These results underscore the usefulness of these transduced cell types for potential in vivo delivery of FVIII. Insertion of a human factor VIII cDNA into retroviral vectors results in a 100- to 1,000-fold decrease in vector titer and poor FVIII gene expression. Chuah et al. address this problem using two approaches: (i) conservative mutagenesis of a putative FVIII INS region and (ii) insertion of an intron 5′ to the FVIII cDNA. Results indicated that conservative mutagenesis of the FVIII INS region failed to increase FVIII expression and restore retroviral titer, but the inclusion of an intron in the retroviral backbone led to improved vector titer and FVIII gene expression. Using this improved FVIII retroviral vector, a variety of cell types were transduced, and relatively high FVIII expression levels were observed in cells of epithelial, endothelial, or fibroblastic origin.