High level of retrovirus-mediated gene transfer into dendritic cells derived from cord blood and mobilized peripheral blood CD34+ cells

Hum Gene Ther. 1999 Jan 20;10(2):175-87. doi: 10.1089/10430349950018977.

Abstract

Dendritic cells (DCs), the most potent antigen-presenting cells, can be generated from CD34+ hematopoietic stem cells and used for generating therapeutic immune responses. To develop immunotherapy protocols based on genetically modified DCs, we have investigated the conditions for high-level transduction of a large amount of CD34+-derived DCs. Thus, we have used an efficient and clinically applicable protocol for the retroviral transduction of cord blood (CB) or mobilized peripheral blood (MPB) CD34+ cells based on infection with gibbon ape leukemia virus (GALV)-pseudotyped retroviral vectors carrying the nls-LacZ reporter gene. Infected cells have been subsequently cultured under conditions allowing their dendritic differentiation. The results show that using a growth factor combination including granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor alpha plus interleukin 4 plus stem cell factor plus Flt3 ligand, more than 70% of DCs derived from CB or MPB CD34+ cells can be transduced. Semiquantitative PCR indicates that at least two proviral copies per cell were detected. Transduced DCs retain normal immunophenotype and potent T cell stimulatory capacity. Finally, by using a semisolid methylcellulose assay for dendritic progenitors (CFU-DCs), we show that more than 90% of CFU-DCs can be transduced. Such a highly efficient retrovirus-mediated gene transfer into CD34+-derived DCs makes it possible to envision the use of this methodology in clinical trials.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD34 / immunology*
  • Base Sequence
  • Cell Division
  • Cell Line
  • DNA Primers
  • Dendritic Cells / immunology
  • Dendritic Cells / metabolism*
  • Fetal Blood / cytology*
  • Gene Transfer Techniques*
  • Hematopoietic Stem Cell Mobilization
  • Humans
  • Immunophenotyping
  • Retroviridae / genetics*
  • T-Lymphocytes / cytology
  • Transduction, Genetic

Substances

  • Antigens, CD34
  • DNA Primers