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

Philippe Leboulch, George M.S. Huang, R. Keith Humphries, Young H. Oh, Connie J. Eaves, Dorothy Y.H. Tuan, Irving M. London

Research output: Contribution to journalArticle

146 Scopus citations

Abstract

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%.

Original languageEnglish (US)
Pages (from-to)3065-3076
Number of pages12
JournalEMBO Journal
Volume13
Issue number13
DOIs
StatePublished - 1994

Keywords

  • Gene therapy
  • Locus control region
  • Rearrangements
  • Retroviruses
  • β-globin

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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