Dataset Information

Erythroid-specific expression of ?-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells.

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-?-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased ?-globin to ?-globin ratio from 0.66±0.08 to 1.05±0.12 (p=0.05), indicating expression of ?-globin from the integrated SB transgene. IHK-?-globin mRNA was found in non-erythroid cell types, similar to native ?-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-?-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-?-globin transgene for gene therapy of sickle cell disease.

SUBMITTER: Sjeklocha LM

PROVIDER: S-EPMC3247234 | biostudies-other | 2011

REPOSITORIES: biostudies-other

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Erythroid-specific expression of β-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells.

Sjeklocha Lucas M LM Park Chang-Won CW Wong Phillip Y-P PY Roney Mark J MJ Belcher John D JD Kaufman Dan S DS Vercellotti Gregory M GM Hebbel Robert P RP Steer Clifford J CJ

PloS one 20111228 12

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-β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature e ...[more]

PMID: 22216176

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Project description:Sickle cell disease (SCD) results predominately from a single monogenic mutation that affects thousands of individuals worldwide. Gene therapy approaches have focused on using viral vectors to transfer wild-type beta- or gamma-globin transgenes into hematopoietic stem cells for long-term expression of the recombinant globins. In this study, we investigated the use of a novel nonviral vector system, the Sleeping Beauty (SB) transposon (Tn) to insert a wild-type beta-globin expression cassette into the human genome for sustained expression of beta-globin. We initially constructed a beta-globin expression vector composed of the hybrid cytomegalovirus (CMV) enhancer chicken beta-actin promoter (CAGGS) and full-length beta-globin cDNA, as well as truncated forms lacking either the 3' or 3' and 5' untranslated regions (UTRs), to optimize expression of beta-globin. Beta-globin with its 5' UTR was efficiently expressed from its cDNA in K-562 cells induced with hemin. However, expression was constitutive and not erythroid-specific. We then constructed cis SB-Tn-beta-globin plasmids using a minimal beta-globin gene driven by hybrid promoter IHK (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, ankyrin-1 promoter), IHbetap (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, beta-globin promoter), or HS3betap (HS3 core element from human betaLCR, beta-globin promoter) to establish erythroid-specific expression of beta-globin. Stable genomic insertion of the minimal gene and expression of the beta-globin transgene for >5 months at a level comparable to that of the endogenous gamma-globin gene were achieved using a SB-Tn beta-globin cis construct. Interestingly, erythroid-specific expression of beta-globin driven by IHK was regulated primarily at the translational level, in contrast to post-transcriptional regulation in non-erythroid cells. The SB-Tn system is a promising nonviral vector for efficient genomic insertion conferring stable, persistent erythroid-specific expression of beta-globin.

Dataset Information

Erythroid-specific expression of ?-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells.

Publications

Erythroid-specific expression of β-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells.

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