Efficient Serum-Free Retroviral Gene Transfer into Primitive Human Hematopoietic Progenitor Cells by a Defined, High-Titer, Nonconcentrated Vector-Containing Medium

Abstract

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 et al. achieved low serum conditions by a complex concentration procedure of a retroviral supernatant initially containing 10% fetal bovine serum. The high cost, small volume, possible coenrichment of serum-derived pathogens, limited recovery of vector particles, and low titer of the final diluted medium restrict the clinical application of this procedure. Transduction of primitive hematopoietic progenitor cells was not demonstrated. In the present study, a defined serum-free medium containing high titers of the pseudotyped retroviral vector PG13/LN was generated from PG13/LN producer cells without requiring a physical enrichment procedure. The transduction of committed hematopoietic progenitor 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-initiating cell-derived colonies (LTC-IC-derived colonies) generated from CD34⁺ and CD34⁺/HLA-DR^lo peripheral blood progenitor “stem” cells (PBSCs) increased during 7 days of treatment in this vector-containing medium in the presence of IL-3, SCF, and flt-3 ligand. The described procedure allowed efficient transduction of LTC-IC-derived colonies generated from CD34⁺, CD34⁺/HLA-DR^lo, and CD34⁺/CD38^lo PBSCs. This is the first report to demonstrate an increase in primitive peripheral blood LTC-IC-derived colonies in vitro as well as their efficient transduction in a high-titer, serum-free vector-containing medium that can be produced exclusively from defined pharmaceutical-grade components, making it ideally suited for applications in clinical gene therapy. Defined serum-free conditions are preferable for the clinical application of retroviral gene transfer technology because they improve biological safety and standardization. We have developed a strategy to generate a completely serum-free medium of standardized pharmaceutical-grade composition with high titers of the gibbon ape leukemia virus (GALV) pseudotyped retrovirus vector PG13/LN that does not require a physical enrichment procedure. To our knowledge this is the first report of ex vivo expansion and efficient transduction of primitive human hematopoietic progenitor cells (long-term culture-initiating cell-derived colonies) from the peripheral blood. The described serum-free procedure should have important implications for clinical retroviral gene transfer.