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Prepublished online as a Blood First Edition Paper on May 17, 2002; DOI 10.1182/blood-2002-01-0219. This Article Full Text (PDF) All Versions of this Article: 2002-01-0219v1 100/6/2012    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Emery, D. W Articles by Stamatoyannopoulos, G. Search for Related Content PubMed PubMed Citation Articles by Emery, D. W Articles by Stamatoyannopoulos, G. Social Bookmarking                   What's this? Submitted January 28, 2002 Accepted March 18, 2002 Development of virus vectors for gene therapy of ß chain hemoglobinopathies: flanking with a chromatin insulator reduces -globin gene silencing in vivo David W Emery*, Evangelia Yannaki, Julie Tubb, Tamon Nishino, Qiliang Li, and George Stamatoyannopoulos Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, WA, USA Gene and Cell Therapy Center, Hematology Department and Bone Marrow Transplantation Unit, George Papanikolaou General Hospital, Thessaloniki, Greece * Corresponding author; email: demery{at}u.washington.edu. We have previously described the development of oncoretrovirus vectors for human -globin using a truncated ß-globin promoter, modified -globin cassette, and -globin enhancer. However, one of these vectors is genetically unstable and both vectors exhibit variable expression patterns in cultured cells, common characteristics of oncoretrovirus vectors for globin genes. In order to address these problems, we identified and removed the vector sequences responsible for genetic instability and flanked the resulting vector with the chicken ß-globin HS4 chromatin insulator in order to protect expression from chromosomal position effects. After determining that flanking with the cHS4 element allowed higher, more uniform levels of -globin expression in MEL cell lines, we tested these vectors using a mouse bone marrow transduction and transplantation model. When present, the -globin cassettes from the uninsulated vectors were expressed in only 2-5% of RBC long-term, indicating they are highly sensitive to epigenetic silencing. In contrast, when present the -globin cassette from the insulated vector was expressed in 49±20% of RBC long-term. RNase protection analysis indicated that the insulated -globin cassette was expressed at 23±16% per copy of mouse -globin in transduced RBC. These results demonstrate that flanking a globin vector with the cHS4 insulator increases the likelihood of expression nearly 10-fold, which in turn allows for -globin expression approaching the therapeutic range for sickle cell anemia and ß thalassemia in transplanted mice. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: F. Park Lentiviral vectors: are they the future of animal transgenesis? Physiol Genomics, October 19, 2007; 31(2): 159 - 173. [Abstract] [Full Text] [PDF] J. C. Cheng, K. M. Sakamoto, E. M. Horwitz, S. L. Karsten, L. Shoemaker, H. I. Kornblumc, and P. Malik Report on the Workshop "New Technologies in Stem Cell Research," Society for Pediatric Research, San Francisco, California, April 29, 2006 Stem Cells, April 1, 2007; 25(4): 1070 - 1088. [Abstract] [Full Text] [PDF] D. Rund and E. Rachmilewitz {beta}-Thalassemia N. Engl. J. Med., September 15, 2005; 353(11): 1135 - 1146. [Full Text] [PDF] Q. Li, D. W. Emery, H. Han, J. Sun, M. Yu, and G. Stamatoyannopoulos Differences of globin transgene expression in stably transfected cell lines and transgenic mice Blood, April 15, 2005; 105(8): 3346 - 3352. [Abstract] [Full Text] [PDF] P. Malik and P. I. Arumugam Gene Therapy for {beta}-Thalassemia Hematology, January 1, 2005; 2005(1): 45 - 50. [Abstract] [Full Text] [PDF] G. Puthenveetil, J. Scholes, D. Carbonell, N. Qureshi, P. Xia, L. Zeng, S. Li, Y. Yu, A. L Hiti, J.-K. Yee, et al. Successful correction of the human {beta}-thalassemia major phenotype using a lentiviral vector Blood, December 1, 2004; 104(12): 3445 - 3453. [Abstract] [Full Text] [PDF] C. Baum, J. Dullmann, Z. Li, B. Fehse, J. Meyer, D. A. Williams, and C. von Kalle Side effects of retroviral gene transfer into hematopoietic stem cells Blood, March 15, 2003; 101(6): 2099 - 2113. [Abstract] [Full Text] [PDF] D. A. Persons, P. W. Hargrove, E. R. Allay, H. Hanawa, and A. W. Nienhuis The degree of phenotypic correction of murine beta -thalassemia intermedia following lentiviral-mediated transfer of a human gamma -globin gene is influenced by chromosomal position effects and vector copy number Blood, March 15, 2003; 101(6): 2175 - 2183. [Abstract] [Full Text] [PDF]

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