Lentiviral gene transfer into primary and secondary NOD/SCID repopulating cells

The ability of lentiviral vectors to transfer genes into human hematopoieti c stem cells was studied, using a human immunodeficiency virus 1 (HIV-1)-de rived vector expressing the green fluorescence protein (GFP) downstream of the phosphoglycerate kinase (PGK) promoter and pseudotyped with the G prote in of vesicular stomatitis virus (VSV). High-efficiency transduction of hum an cord blood CD34(+) cells was achieved after overnight incubation with ve ctor particles. Sixteen to 28 percent of individual colony-forming units gr anulocyte-macrophage (CFU-GM) colonies derived from cord blood CD34(+) cell s were positive by polymerase chain reaction (PCR) for the GFP gene. The tr ansduction efficiency of SCID-repopulating cells (SRC) within the cord bloo d CD34(+) population was assessed by serial transplantation into nonobese d iabetic/severe combined immunodeficient (NOD/SCID) mice. When 400 000 cord blood CD34(+) cells were transplanted into primary recipients, all primary and secondary recipients contained and expressed the transgene. Over 50% of CFU-GM colonies derived from the bone marrow of these primary and secondar y recipients contained the Vector on average as deter mined by PCR. Transpl antation of transduced cells in limiting dilution generated GFP(+) lymphoid and myeloid progeny cells that may have arisen from a single SRC, Inverse PCR analysis was used to amplify vector-chromosomal junctional fragments in colonies derived from SRC and confirmed that the vector was integrated. Th ese results show that lentiviral vectors can efficiently transduce very pri mitive human hematopoietic progenitor and stem cells. (C) 2000 by The Ameri can Society of Hematology.