Highly efficient gene transfer into preterm CD34(+) hematopoietic progenitor cells

OBJECTIVE: Retrovirus-mediated gene transfer has been shown to transduce CD 34(+) cells from term gestation umbilical cord blood with relatively high e fficiency. The purpose of this study was to compare the efficiencies of ret rovirus-mediated gene transfer into early (23-28 weeks' gestation) and term (37-41 weeks' gestation) umbilical cord blood CD34+ hematopoietic progenit or cells. STUDY DESIGN: CD34(+) cells were purified from cyropreserved early (23-28 w eeks' gestation) and term (37-40 weeks' gestation) umbilical cord blood spe cimens with fluorescence-activated cell sorting. The CD34(+) cells were the n transduced in virus-containing medium (gibbon ape leukemia virus pseudoty pe vector LAPSN [PG13]) in wells coated with the recombinant human fibronec tin fragment CH-296 and in the presence of multiple hematopoietic growth fa ctors (interleukin 6, stem cell factor, granulocyte colony-stimulating fact or, granulocyte-macrophage colony-stimulating factor, and megakaryocyte gro wth and development factor) and protamine sulfate. The LAPSN (PG13) virus-c ontaining medium was changed every 12 hours for 48 hours, after which time colony-forming cells were assayed in soft agar. The gibbon ape leukemia vir us pseudotype vector LAPSN (PG13) contains the human placental alkaline pho sphatase and neomycin phosphotransferase (neo) genes. The efficiency of gen e transfer was assessed by histochemical staining of colony-forming cells i n agar for expression of heat-stable alkaline phosphatase. RESULTS: Gene transfers, as assessed by alkaline phosphatase staining of co lony-forming cells (granulocyte-macrophage colony-forming units and erythro id burst-forming units), were similar for CD34+ hematopoietic progenitor ce lls from early (58.4% +/- 11.8%) and term (63.2% +/- 12.5%) gestation fetal umbilical cord blood. CONCLUSION: CD34+ hematopoietic progenitor cells from midgestation fetal bl ood can be transduced with high efficiency using techniques optimized for p ostnatal samples with a gibbon ape leukemia virus pseudotype vector. The ea rly fetus may be a preferable target for gene therapy because of the higher number of circulating CD34+ and CD38(-) cells relative to term cord blood, their greater proliferative capacity, and the rapid expansion of the fetal hematopoietic system that occurs from the second trimester to delivery. Be cause in vitro studies of gene transfer into hematopoietic progenitor cells and long-term culture-initiation cells have not been predictive of the eff iciency of gene transfer into marrow-repopulating cells in vivo, studies th at examine clinically applicable approaches to in utero gene therapy in app ropriate animal models are still needed.