Development and analysis of retroviral vectors expressing human factor VIII as a potential gene therapy for hemophilia A.

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/10(6) cells/24 hr). These results underscore the usefulness of these transduced cell types for potential in vivo delivery of FVIII.

• Authors: M K Chuah, T VandenDriessche, R A Morgan
• Journal: Human gene therapy (Hum Gene Ther) Vol. 6 Issue 11 Pg. 1363-77 (Nov 1995) ISSN: 1043-0342 [Print] United States
• PMID: 8573610 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• DNA, Recombinant
• RNA
• Factor VIII

Topics

• 3T3 Cells
• Animals
• Base Sequence
• Cell Line
• Cloning, Molecular
• DNA, Recombinant
• Factor VIII (genetics)
• Gene Transfer Techniques
• Genetic Therapy
• Genetic Vectors
• Hemophilia A (therapy)
• Humans
• Mice
• Molecular Sequence Data
• Mutagenesis
• Nucleic Acid Conformation
• RNA (chemistry)
• Retroviridae (genetics)
• Tumor Cells, Cultured