Figure 5. IHK-transduced CD34+ progenitors increase b-globin expression in erythroid progeny. Hypotonic lysates from fetal RBCs from the donor cords, erythrocytes from non-transduced CD34+ cells, and erythrocytes from IHK-transduced CD34+ cells were analyzed by RP- HPLC to determine relative quantities of individual globin chains. Data from three trials are presented as mean 6 SEM, * indicates p,0.05, paired t-test. doi:10.1371/journal.pone.0029110.g005 significant (p,0.001) loss of expression in the non-insulated vectors relative to the insulated vectors (Figure 8A). The change represents a mean loss of 13.1681.6% of b-globin expression in the insulated group and of 66.1650.3% of b-globin expression in the non-insulated group. In summary, the results were similar to those generated with the insulated and non-insulated SB vectors for DsRed. To investigate the effect of the insulators on IHK–b- globin regulation we tested the response of clones in each group to induction with hemin, which stimulates hemoglobin production and upregulation of erythroid-associated genes [37]. Induced clones from the insulated and non-insulated groups showed no significant differences (1.8861.29 fold and 1.7761.75 fold increases, respectively) in b-globin expression upon hemin induction (Figure 8B). Discussion In this study we have shown that primary human CD34+ cells can be transduced with the SB construct, pKT2/meIF-SB100X- IHK-b-globin, can express hemoglobin in mature erythroid progeny, while maintaining tight, erythroid-specific expression. Previous studies have shown that IHK is potentially a more active promoter than the b-globin LCR, albeit in a viral vector. The small size of IHK relative to b-globin LCR/promoter-based vectors makes it well suited for the SB system as a mechanism to drive hemoglobin expression [22,27]. It is important to reduce the overall size of the SB transgene cargo to promote efficient transposition and to lower the number of insertion sites necessary to provide therapeutic expression [22]. An interesting feature of IHK is its use of erythroid enhancer and promoter elements, Erythroid-Specific Expression from Sleeping Beauty Figure 6. Increased b-globin transcript is detectable in non- erythroid progeny of IHK-transduced CD34+ cells. Twenty ng of total RNA was used for RT-PCR specific for spliced b-globin transcript and spliced b-actin. RNA from adult RBCs and erythrocyte-depleted adult WBCs was used as control. doi:10.1371/journal.pone.0029110.g006 which are not directly involved in normal b-globin production. While the IHK acts as a strong promoter in erythroid cells, we have previously shown that the native introns and untranslated regions of the b-globin gene are crucial for high-level expression of protein [26]. Analysis of the transgene content and expression of pKT2/ meIF-SB100X-IHK-b-globin transduced cells showed that each of the nucleofected trials retained the IHK transgene in each lineage after 18 to 28 days of differentiation. In this study, as previously shown, we did find a low-level of transcription in non-erythroid cells, which was translationally suppressed and failed to produce detectable b-globin [26]. In that the inserted transposon still contains IR/DRs necessary for transposition, the potential remobilization of the transposon is a significant concern. We found that SB transposase-coding DNA can be detected at low levels within a transduced population but does not express detectable mRNA as determined by RT-PCR, consistent with a recent study of SB transduced cells [38]. In order to achieve maximum efficiency of transposon insertion into the host genome, we employed an enhanced transposase, SB100X, which was shown to be ,100 times as active as the original version of the Sleeping Beauty transposase [39]. Stable gene transfer efficiencies of almost 50% were achieved with SB100X in the human cells differentiated from transfected hematopoietic CD34+ cells in remarkable contrast to stable gene transfer efficiencies of the original SB transposase (0.2 to 1%) or of second-generation hyperactive SB11 [16]. These previous results support the potential use of SB100X as a gene transfer agent for human CD34+ hematopoietic cells, and further development of the SB system in general. We have also shown that cHS4 insulator elements can mitigate the tendency for SB-inserted transgenes to be inactivated over the long-term [30]. Long-term stable expression of a therapeutic transgene is critical in the successful gene delivery for genetic diseases such as b-thalassemia or sickle cell anemia. There are numerous reports of transgene silencing associated with delivery by either retroviral vectors or non-viral SB vectors [30,40]. Most of this long-term inactivation of transgenes can be attributed to epigenetic changes such as DNA methylation or chromatin modifications in the inserted transgenes [29,30,41]. The chicken HS4 insulator element employed here has previously been shown to block enhancer activity, eliminating or diminishing the influence of enhancer elements on genes in close proximity whether the enhancer is included within the transgene or placed close to the insertion site in the host genome [31,32]. In addition, the cHS4 insulator is known to protect transgene from silencing by PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e29110