β-Globin LCR and Intron Elements Cooperate and Direct Spatial Reorganization for Gene Therapy

et al. (2008) b-Globin LCR and Intron Elements Cooperate and Direct Spatial Reorganization for Gene Therapy. PLoS Genet 4(4): e1000051. doi:10.1371/journal.pgen.1000051 b-Globin LCR and Intron Elements Cooperate and Direct Spatial Reorganization for Gene Therapy Alla Buzina 0 Mandy Y. M. Lo 0 Angela Moffett 0 Akitsu Hotta 0 Eden Fussner 0 Rikki R. 0 Bharadwaj 0 Peter Pasceri 0 J. Victor Garcia-Martinez 0 David P. Bazett-Jones 0 James Ellis 0 Wendy A. Bickmore, MRC Human Genetics Unit, United Kingdom 0 1 Developmental and Stem Cell Biology Program, SickKids , Toronto, Ontario , Canada , 2 Department of Molecular Genetics, University of Toronto , Toronto, Ontario , Canada , 3 Program in Genetics and Genome Biology, SickKids , Toronto, Ontario , Canada , 4 Department of Biochemistry, University of Toronto , Toronto, Ontario , Canada , 5 Department of Internal Medicine, Division of Infectious Diseases, University of Texas Southwestern Medical Center , Dallas, Texas , United States of America The Locus Control Region (LCR) requires intronic elements within b-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional bglobin intron 2 elements that rescue LCR activity directed by 59HS3. Evaluation in transgenic mice demonstrates that an Oct1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igm 39MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igm 39MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 59HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 59HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of hemoglobinopathies using single copy vectors. - Funding: AM was supported by a University of Toronto Open Masters Studentship and MYML was supported by an Ontario Student Opportunity Trust Fund (OSOTF) Studentship from the SickKids Restracomp. DPB-J holds a Canada Research Chair in Molecular and Cellular Imaging. This work was funded by grants from CIHR to JE and to DPB-J (MOP 14311), from the NIH Comprehensive Sickle Cell Center Program 5-U54-HL070588 to JVG and JE, the Anemia Institute for Research and Education to JE, and Phi Beta Sigma International Endowment Fund to JE. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The b-globin gene is regulated by a Locus Control Region (LCR) that interacts with gene proximal elements to activate erythroid specific expression. Many studies demonstrate that individual LCR hypersensitive sites (HS) loop out the intervening DNA to interact with the globin genes [14], and that transcriptional activation is accompanied by movement of the gene away from heterochromatin at the nuclear periphery towards transcription factories located more centrally [58]. While the LCR is not required to establish open chromatin at the endogenous locus [9], it is able to open chromatin at ectopic bglobin transgene integration sites to activate high level transcription [1012]. This ability of the LCR, in particular its 59HS3 element [13], to open chromatin at all integration sites has made it a widely used component of gene therapy expression cassettes designed for treatment of b-thalassemia or Sickle Cell Anemia. Several groups have corrected these diseases in mouse models using lentivirus mediated delivery of LCR b-globin vectors regulated, at least in part, by 59HS3 [1421]. One current limitation to LCR b-globin gene therapy cassettes is that high titer virus transmission requires the deletion of AT-rich (ATR) sequences in b-globin intron 2 [22,23]. We have shown that these intronic ATR sequences are required for LCR activity by 59HS3 when assayed in transgenic mice [24]. These data indicate that LCR b-globin sequences used in gene therapy cassettes may compromise chromatin opening and transcriptional enhancement activities. Thus, vectors containing the ATR deletion may require more than 1 integration per cell to ensure expression, as has been observed during gene therapy [25,26]. Although increased vector copy number will direct therapeutic levels of expression, it also increases the likelihood of insertional activation of surrounding oncogenes [27]. In order to reduce potential genotoxicity, it would be advantageous to include insulator elements in the LTRs to Expression of the b-globin gene is regulated by interactions between a distant Locus Control Region (LCR) and regulatory elements in or near the gene. We previously showed that LCR activity requires specific b-globin intron elements to consistently activate transgene expression in mice. These important intronic elements fail to transmit through lentivirus vectors designed for gene therapy of Sickle Cell Anemia. In this study, we identify intron modifications that reveal functional cooperation between the b-globin intronic enhancer and an intronic Oct-1 site. LCR activity in transgenic mice is also potentiated by an intronically located Igm 39MAR element. During induction of erythroid gene expression, the modified intron directs relocalization of the transgene away from the nuclear periphery towards more central neighbourhoods, and this movement mimics relocalization by the endogenous bglobin locus. Lentivirus vectors with the modified intron produce high titer virus stocks that express the transgene to therapeutic levels in erythroid cells. These findings have implications for understanding the mechanism of LCR activity, and for designing safe and effective lentivirus vectors for gene therapy. block activation events [2832] while also reducing the copy number load by (...truncated)