Erythroid-Specific Expression of b-globin from Sleeping Beauty- Transduced Human Hematopoietic Progenitor Cells Lucas M. Sjeklocha1, Chang-Won Park1, Phillip Y-P Wong 1 , Mark J. Roney3,4, John D. Belcher3,4, Dan S. Kaufman4,5, Gregory M. Vercellotti3,4, Robert P. Hebbel3,4, Clifford J. Steer1,2* 1 Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America, 2 Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America, 3 Vascular Biology Center, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America, 4 Division of Hematology, Oncology and Transplantation, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America, 5 Stem Cell Institute, University of Minnesota Medical School, University of Minnesota, Minneapolis, Minnesota, United States of America Abstract Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34+ cells with DsRed and a hybrid IHK–b-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased b-globin to c-globin ratio from 0.6660.08 to 1.0560.12 (p = 0.05), indicating expression of b-globin from the integrated SB transgene. IHK–b-globin mRNA was found in non-erythroid cell types, similar to native b- globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK–b-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK–b-globin transgene for gene therapy of sickle cell disease. Citation: Sjeklocha LM, Park C-W, Wong PY-P, Roney MJ, Belcher JD, et al. (2011) Erythroid-Specific Expression of b-globin from Sleeping Beauty-Transduced Human Hematopoietic Progenitor Cells. PLoS ONE 6(12): e29110. doi:10.1371/journal.pone.0029110 Editor: Dimas Tadeu Covas, University of Sao Paulo – USP, Brazil Received September 23, 2011; Accepted November 21, 2011; Published December 28, 2011 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: These studies were supported by National Institutes of Health (NIH) grants R21 HL096469-01 to RPH and CJS and P01 HL55552 to RPH (NIH.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: steer001@umn.edu Introduction Hematopoietic stem cells (HSCs) are an attractive target for genetic modification to treat diseases such as sickle cell anemia, b- thalassemia, and severe combined immunodeficiency (SCID), among others [1,2]. The ability of HSCs to be harvested with relative ease, withstand ex vivo manipulation and their long established clinical use in transplants allows for numerous potential gene therapy strategies. This is supported by a recent report of successful treatment of a b-thalassemia patient using a recombinant hemoglobin-expressing lentivirus to transduce autologous CD34+ cells [3]. However, as in other viral vector clinical trials, significant and potentially problematic clonal expansion was observed. HSCs give rise to a diverse number of cell types and maintain their ability to self-renew [4,5]. The current methods for gene transfer in clinical trials rely principally on the use of modified retroviruses such as Moloney murine leukemia virus (MMLV) and lentivirus, which have been known to insert into actively transcribed genes and potentially promote oncogenesis [6–8]. The development of leukemia in 4 out of 20 patients enrolled in a trial of MLV-based therapy for X-SCID continues to be a cautionary example to the field of gene therapy [9,10]. Non-viral vectors offer a more easily implemented and potentially safer method to genetically modify cells [11–13]. Non-viral vectors, however, are challenged by relatively low gene transfer efficiency and the difficulty of maintaining long-term stable expression [13–15]. Transposons can be utilized to provide an integration mechanism for stable copy number and long-term expression important for non-viral gene therapies [16–19]. The Sleeping Beauty (SB) transposon system has become increasingly useful in fields ranging from cancer biology and stem cell research to gene therapy over the last decade since its resurrection from the salmonid genome [17,20,21]. The recent development of the SB100X transposase and its utility in modifying primary CD34+ cells are evidence of the system’s continued improvements and potential clinical significance [22,23]. In this study, we used the SB transposon to develop an optimal expression system for erythroid-specific gene expression in specific lineages [24]. Typical viral vectors drive erythroid-specific b- globin expression using 2.7 kilobases (kb) from the b-globin locus control region and promoter [25]. This large size makes the approach impractical for the SB system, which loses efficiency in a linear fashion from 2-kb cargos up to a practical limit of 6–10 kb [16,22]. The 1-kb erythroid promoter IHK can provide high-level PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29110