Design of retrovirus vectors for transfer and expression of the human beta-globin gene.

Regulated expression of the human beta-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective beta-globin genes. We found regions that interfered with virus production within intron 2 of the beta-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for beta-globin expression. Inclusion of beta-globin introns necessitates an antisense orientation of the gene within the retrovirus vector. However, we found no effect of the antisense beta-globin transcription on virus production. A region downstream of the beta-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10(6) CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human beta-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human beta-globin gene in hematopoietic cells (CFU-S cells) in mice.