EFFICIENT SERUM-FREE RETROVIRAL GENE-TRANSFER INTO PRIMITIVE HUMAN HEMATOPOIETIC PROGENITOR CELLS BY A DEFINED, HIGH-TITER, NONCONCENTRATEDVECTOR-CONTAINING MEDIUM

Defined serum-free conditions have great conceptual advantages for the biological safety and standardization of clinical gene transfer into hematopoietic stem cells. In the only study reported to date, Sekhar e t al, achieved low serum conditions by a complex concentration procedu re of a retroviral supernatant initially containing 10% fetal bovine s erum. The high cost, small volume, possible coenrichment of serum-deri ved pathogens, limited recovery of vector particles, and low titer of the final diluted medium restrict the clinical application of this pro cedure, Transduction of primitive hematopoietic progenitor cells was n ot demonstrated. In the present study, a defined serum-free medium con taining high titers of the pseudotyped retroviral vector PG13/LN was g enerated from PG13/LN producer cells without requiring a physical enri chment procedure. The transduction of committed hematopoietic progenit or cells in the serum-free vector-containing medium was efficient, and similar to that occurring under serum-containing control conditions. The number of primitive human hematopoietic long-term culture-initiati ng cell-derived colonies (LTC-IC-derived colonies) generated from CD34 (+) and CD34(+)/HLA-DRlo peripheral blood progenitor ''stem'' cells (P BSCs) increased during 7 days of treatment in this vector-containing m edium in the presence of IL-3, SCF, and flt-3 ligand, The described pr ocedure allowed efficient transduction of LTC-IC-derived colonies gene rated from CD34(+), CD34(+)/HLA-DRlo, and CD34(+)/CD38(lo) PBSCs, This is the first report to demonstrate an increase in primitive periphera l blood LTC-IC-derived colonies in vitro as well as their efficient tr ansduction in a high-titer, serum-free vector-containing medium that c an be produced exclusively from defined pharmaceutical-grade component s, making it ideally suited for applications in clinical gene therapy.