Optimization of the Sleeping Beauty Transposon System to Achieve Stable Transgene Expression in Human CD34+ Hematopoietic Progenitor Cells

Sleeping Beauty (SB) transposon-mediated integration has been shown to achieve long-term transgene expression in a wide range of host cells. Transposon-mediated gene integration may have advantages over viral vectors, with a greater transgene carrying capacity and potentially safer integration site profile. Due to these characteristics of SB, there has been great interest in its potential use in hematopoietic stem cell (HSC) gene therapy. In this study, we optimized the SB transposon-mediated gene transfer system to achieve higher stable transgene expression in K562 human erythroleukemia cells, Jurkat human T-lymphoid cells, and primary human CD34+ hematopoietic progenitor cells. The SB transposon system was optimized by two approaches:

1. to increase the transposition efficacy, a hyperactive mutant of SB, HSB16, was used (

   Baus et al.; Mol Ther 12:1148, 2005
   );

2. to optimize the expression of the SB transposase and the transgene cassette carried by the transposon, three different viral and cellular promoters were evaluated, including the modified MPSV long terminal repeat (MNDU3) enhancer-promoter, the human cytomegalovirus (hCMV) immediate-early region enhancer-promoter, and the human elongation factor 1 (hEF1a) promoter.

SB components were delivered in trans into the target cells by nucleoporation. The SB transposon-mediated integration efficacy was assessed by integrated transgene (enhanced green fluorescent protein [eGFP]) expression using fluorescent-activated cell sorting (FACS) analysis over 3–4 weeks. The functional assay showed that HSB16 was a more efficient enzyme compared to the original SB. In purified human cord blood CD34⁺ cells, HSB16 achieved nearly 7-fold higher long-term transgene expression with 90% less plasmid DNA (from 10 mcg of SB reduced to 1 mcg of HSB16) than the original SB transposase. The highest level of stable transgene integration in all three cell types was achieved using the hEF1a promoter to express HSB16 in comparison to either the hCMV or MND promoter. Our data also suggested that optimal GFP reporter gene expression from the integrated transposon was influenced by the type of promoter and the target cell type. Significantly higher levels of eGFP expression (5-fold) were achieved with the hEF1a promoter in Jurkat human T cells, compared to that achieved with the MND promoter; in contrast the MND promoter expressed GFP at the highest level in K562 myeloid cells. In primary human CD34+ cord blood progenitors, optimal transgene integration and expression was achieved using the hEF1a promoter to express the SB transposase combined with the MND promoter to express GFP reporter, when studied under conditions directing myeloid differentiation. Stable transgene expression was achieved at levels up to 27% for over 4 weeks after optimized gene transfer to CD34+ cells (ave=17%, n=4). In vivo studies evaluating engraftment and differentiation of the SB-modified human CD34+ progenitor cells are currently in progress. In conclusion, the optimized SB transposon system in primary human CD34+ hematopoietic progenitors reported here has improved the stable gene transfer efficiency by 29-fold, compared to our prior published data (< 1% -