Mutagenesis of retroviral vectors transducing human β-globin gene and β-globin locus control region derivatives results in stable transmission of an active transcriptional structure

Retrovirus-mediated gene transfer of the human β-globin gene into hematopoietic stem cells is an attractive approach to the therapy of human β-globin gene disorders. However, expression of the transduced β-globin gene linked to its proximal cis-acting sequences (-0.8 to +0.3 kb from the cap site) is considerably below the level required for a significant therapeutic effect. The discovery of the β-locus control region β-LCR), organized in four major DNase I hypersensitive sites far upstream of the human β-like globin gene cluster, provided a potential means to achieve a high level of expression of a linked human β-globin gene, but initial attempts to incorporate β-LCR derivatives in retroviral vectors resulted in the production of low-titer viruses with multiple rearrangements of the transmitted proviral structures. We now describe how extensive mutagenesis of the transduced β-globin gene, eliminating a 372 bp intronic segment and multiple reverse polyadenylation and splicing signals, increases viral titer significantly and restores stability of proviral transmission upon infection of cell lines and bone marrow-repopulating cells. These optimized vectors have enabled us to analyze the expression properties of various retrovirally transduced β-LCR derivatives in dimethylsulfoxide-induced murine erythroleukemia cells and to achieve ratios of human β-globin/murine β(maj)-globin mRNA, on a per gene basis, as high as 80%.