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 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/10(6) cells/24 hr). These results underscore the usefulness of these transduced cell types for potential in vivo delivery of FVIII.
Chuah
M K
MK
Gene Transfer Technology Section, National Institutes of Health, Bethesda, MD 20892, USA.
VandenDriessche
T
T
Morgan
R A
RA
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D005169:Factor VIII / Q000235:genetics*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006467:Hemophilia A / Q000628:therapy*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry; D012190:Retroviridae / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004274:DNA, Recombinant; D012313:RNA; D005169:Factor VIII