Journal
BLOOD
Volume 114, Issue 7, Pages 1319-1330Publisher
AMER SOC HEMATOLOGY
DOI: 10.1182/blood-2009-03-210005
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Funding
- Children's Cancer Research Fund in Minneapolis
- Alliance for Cancer Gene Therapy
- Gabrielle's Angel ( formerly G&P) Foundation for Cancer Research
- Sidney Kimmel Foundation for Cancer Research Kimmel Scholar Program
- University of Minnesota Translational Research Grant
- University Minnesota Medical School Dean's Commitment
- University of Minnesota
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Here we report stable gene transfer in cord blood-derived CD34(+) hematopoietic stem cells using a hyperactive nonviral 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 with 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 approximately 46% engraftment in NOD-scid IL2 gamma c(null) (NOG) mice. Twelve weeks after transplantation, 0.57% to 28.96% (median, 2.79%) and 0.49% to 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 hematopoietic stem cells as a modality for gene therapy. (Blood. 2009;114:1319-1330)
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