High-efficiency retroviral transduction of fetal liver CD38-CD34++ cells: Implications for in utero and ex utero gene therapy

Objectives: Defining methods for the efficient transduction of fetal stem c ells could lead to novel fetal therapies for blood cell disorders and other birth defects. In this study, we analyzed the effects of various parameter s on the retroviral transduction of primitive hematopoietic progenitors/ste m cells isolated from fetal liver. Methods: Candidate stem cells were isola ted by fluorescence-activated cell sorting from midtrimester human livers b ased on the phenotype CD38-CD34++Iineage- (lineage = glycophorin A, CD3, CD 14, CD19, CD20 and CD56). A murine retroviral vector with a truncated human low-affinity nerve growth factor receptor (Delta NGFR) gene was used to tr ansduce the candidate stem cells. Marker gene expression was monitored by f low cytometry using an anti-NGFR mAb. Candidate stem cells were transduced immediately after isolation or after up to 4 days of culture in serum-depri ved medium containing the growth factors kit ligand and granulocyte-macroph age colony-stimulating factor. The effects on transduction efficiency of th e addition of 4 mug/ml protamine sulfate and/or centrifugation to concentra te the candidate stem cells and virus were tested. After transduction, the cells were expanded for 10-21 days before determining the frequency of NGFR + cells among the different hematopoietic progeny. Results: Efficient trans duction of candidate stem cells, at an average rate of 46%, was achieved af ter 3 days of culture with a single exposure to virus. Longer than 3 days o f culture or repeated exposure to viral supernatant did not significantly i mprove the rate of transduction. The use of centrifugation at 1,200 g for 1 h and the addition of protamine sulfate during the transduction procedure were critical to achieving a high rate of transduction. Marker gene express ion was observed on the progeny of the transduced cells in conjunction with CD34 (progenitors), glycophorin A (erythrocytes), CD14 (monocytes), CD15 ( granulocytes) and CD41 (megakaryocytes). Conclusions: This study demonstrat es that the efficient transduction of fetal candidate stem cells can be ach ieved under defined culture conditions using a retroviral vector. These res ults encourage further examination of in utero and ex utero gene therapy as a means of treating birth defects. Copyright (C) 2001 S. Karger AG, Basel.