Erythroid-Specific Expression of β-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 -or γ-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 -globin expression cassette into the human genome for sustained expression of -globin. We initially constructed a -globin expression vector composed of the hybrid cytomegalovirus (CMV) enhancer chicken -actin promoter (CAGGS) and full-length -globin cDNA, as well as truncated forms lacking either the 3′ or 3′ and 5′ untranslated regions (UTRs), to optimize expression of -globin. -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--globin plasmids using a minimal -globin gene driven by hybrid promoter IHK (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human RLCR, ankyrin-1 promoter), IH p (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human RLCR, -globin promoter), or HS3 p (HS3 core element from human LCR, -globin promoter) to establish erythroidspecific expression of -globin. Stable genomic insertion of the minimal gene and expression of the -globin transgene for >5 months at a level comparable to that of the endogenous γ-globin gene were achieved using a SB-Tn -globin cis construct. Interestingly, erythroid-specific expression of -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 -globin.

Nanocapsule-delivered Sleeping Beauty mediates therapeutic Factor VIII expression in liver sinusoidal endothelial cells of hemophilia A mice

“…Purified recombinant DsRed protein (0.4 μg, liver; or 0.08 μg, other tissues) (Clontech) was used as a positive control. The lane loading control, β-actin, was detected using the conditions previously described (67).…”

“…However, any vector that inserts DNA into the host genome has the potential for insertional mutagenesis via gene disruption or activation of flanking chromosomal sequences. To date, the genomic SB-mediated insertion profile, although not entirely random for the TA dinucleotide, suggests that SB preferentially inserts into nontranscribed and intronic regions of the genome (33,66,67). This may be related to its preference for TA-rich sequences (68,69) and/or the preferential affinity of the transposase for heterochromatin (70).…”

Gene Therapy with the Sleeping Beauty Transposon System

“…In vitro, human cell lines [140] Tail vain hydrodynamic injection of naked DNA In vivo, mouse liver [141] Transfection…”

Nonviral Gene Delivery with the Sleeping Beauty Transposon System

“…One way to achieve this is to equip the transposon-contained expression cassette with tissue-specific promoters; this was shown to confer erythroid-specific expression of b-globin in a sickle cell disease model (Zhu et al, 2007). Another strategy for targeted expression of therapeutic gene constructs is their targeted delivery into tissues of interest.…”