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 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 IL2gammac(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.
Division of Pediatric Blood and Marrow Transplantation, Berlin, Germany.
Xingkui
X
Xue
Xin
X
Huang
Sonja E
SE
Nodland
Lajos
L
Mátés
Linan
L
Ma
Zsuzsanna
Z
Izsvák
Zoltán
Z
Ivics
Tucker W
TW
LeBien
R Scott
RS
McIvor
John E
JE
Wagner
Xianzheng
X
Zhou
D000818:Animals; D018952:Antigens, CD34; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D036101:Cord Blood Stem Cell Transplantation; D004251:DNA Transposable Elements; D005260:Female; D005312:Fetal Blood; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006085:Graft Survival / Q000235:genetics; D006412:Hematopoietic Stem Cells; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D013997:Time Factors; D014183:Transplantation, Heterologous; D000097573:Red Fluorescent Protein
D018952:Antigens, CD34; D000943:Antigens, Differentiation; D004251:DNA Transposable Elements; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein