title: Erythroid-specific expression of beta-globin by the sleeping beauty transposon for Sickle cell disease.
abstract: 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.
authors.affiliation: Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
authors.forename: Jianhui
authors.initials: J
authors.lastname: Zhu
authors.forename: Betsy T
authors.initials: BT
authors.lastname: Kren
authors.forename: Chang Won
authors.initials: CW
authors.lastname: Park
authors.forename: Rasim
authors.initials: R
authors.lastname: Bilgim
authors.forename: Phillip Y-P
authors.initials: PY
authors.lastname: Wong
authors.forename: Clifford J
authors.initials: CJ
authors.lastname: Steer
mesh_terms: D000755:Anemia, Sickle Cell / Q000097:blood*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004912:Erythrocytes / Q000378:metabolism*; D005914:Globins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019895:Transposases / Q000097:blood* / Q000235:genetics
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D012313:RNA; D005914:Globins; D019895:Transposases; C494471:sleeping beauty transposase, human