Stable gene transfer and expression in cord blood-derived CD34+ hematopoietic stem and progenitor cells by a hyperactive Sleeping Beauty transposon system

Here we report stable gene transfer in cord blood-derived CD34(+) hematopoietic stem cells (HSCs) using a hyperactive non-viral Sleeping Beauty (SB) transposase (SB100X). In colony forming assays, SB100X mediated the highest efficiency (24%) of stable Discosoma sp. red fluorescent protein (DsRed) reporter gene transfer in committed hematopoietic progenitors compared to both the early-generation hyperactive SB11 transposase and the piggyBac transposon system (1.23% and 3.8%, respectively). In vitro differentiation assays further demonstrated that SB100X-transfected CD34(+) cells can develop into DsRed(+) CD4(+)CD8(+) T (3.17-21.84%, median=7.97%), CD19(+) B (3.83-18.66%, median=7.84%), CD56(+)CD3(-) NK (3.53-79.98, median=7.88%) and CD33(+) myeloid (7.59-15.63%, median=9.48%) cells. SB100X-transfected CD34(+) cells achieved ~46% engraftment in NOD-scid IL2gammac(null) (NOG) mice. Twelve weeks after transplantation, 0.57-28.96% (median=2.79%) and 0.49-34.50% (median=5.59%) of total human CD45(+) cells in the bone marrow and spleen expressed DsRed including CD19(+) B, CD14(+) monocytoid and CD33(+) myeloid cell lineages. Integration site analysis revealed SB transposon sequences in the human chromosomes of in vitro differentiated T, B, NK, and myeloid cells, as well as in human CD45(+) cells isolated from bone marrow and spleen of transplanted NOG mice. Our results support the continuing development of SB-based gene transfer into human HSCs as a modality for gene therapy.