Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure.
Retrovirus-mediated gene transfer of the human beta-globin gene into hematopoietic stem cells is an attractive approach to the therapy of human beta-globin gene disorders. However, expression of the transduced beta-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 beta-locus control region (beta-LCR), organized in four major DNase I hypersensitive sites far upstream of the human beta-like globin gene cluster, provided a potential means to achieve a high level of expression of a linked human beta-globin gene, but initial attempts to incorporate beta-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 beta-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 beta-LCR derivatives in dimethylsulfoxide-induced murine erythroleukemia cells and to achieve ratios of human beta-globin/murine beta maj-globin mRNA, on a per gene basis, as high as 80%.
Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA.
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P
Leboulch
G M
GM
Huang
R K
RK
Humphries
Y H
YH
Oh
C J
CJ
Eaves
D Y
DY
Tuan
I M
IM
London
D000818:Animals; D001483:Base Sequence; D001854:Bone Marrow Cells; D002460:Cell Line; D004279:DNA, Viral; D015672:Erythroid Precursor Cells / Q000378:metabolism; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics*; D006801:Humans; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011533:Proviruses / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D012045:Regulatory Sequences, Nucleic Acid; D012190:Retroviridae / Q000235:genetics*
D004279:DNA, Viral; D012333:RNA, Messenger; D005914:Globins