Critical factors influencing stable transduction of human CD34(+) cells with HIV-1-derived lentiviral vectors

Mol Ther. 2000 Jul;2(1):71-80. doi: 10.1006/mthe.2000.0094.


Lentiviral vectors have been proposed as a more efficient alternative to Moloney murine leukemia virus-based retroviral vectors for transduction of human hematopoietic progenitors and stem cells. These studies were designed to evaluate the conditions that influence transduction frequency of CD34(+) progenitors, with the goal of optimizing efficiency of stable gene transfer with lentiviral vectors. CD34(+) human cord blood cells and 293 cells were transduced with a human immunodeficiency virus (HIV)-1 derived lentiviral vector pseudotyped with vesicular stomatitis virus glycoprotein and carrying an internal human cytomegalovirus promoter driving enhanced green fluorescent protein (eGFP) expression. Using fluorescence-activated cell sorting analysis of eGFP, we observed pseudotransduction beginning at the time of vector addition and lasting up to 24 h in CD34(+) cells and up to 72 h in 293 cells. Integrase-defective lentiviral vector caused transient eGFP expression for up to 10 days in CD34(+) cells and for up to 14 days in 293 cells. Protamine sulfate conferred no increase in transduction efficiency of CD34(+) cells on fibronectin-coated plates. Transduction frequency was related directly to vector concentration and not to multiplicity of infection across the ranges tested. First- and second-generation lentiviral vectors transduced CD34(+) cells equally, demonstrating a lack of dependence on HIV-1 accessory proteins. These findings will be useful for the optimal utilization of this new class of vectors for transduction of human hematopoietic stem cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antigens, CD34 / genetics*
  • Cell Line
  • Cell Separation
  • Cytomegalovirus / genetics
  • Fetal Blood / cytology
  • Fibronectins / metabolism
  • Flow Cytometry
  • Gene Transfer Techniques
  • Genetic Vectors*
  • Green Fluorescent Proteins
  • HIV-1 / genetics
  • Hematopoietic Stem Cells / metabolism*
  • Heparin Antagonists / pharmacology
  • Humans
  • Lentivirus / genetics*
  • Luminescent Proteins / metabolism
  • Membrane Glycoproteins*
  • Plasmids / metabolism
  • Promoter Regions, Genetic
  • Protamines / pharmacology
  • Time Factors
  • Transduction, Genetic*
  • Transfection
  • Viral Envelope Proteins / genetics


  • Antigens, CD34
  • Fibronectins
  • G protein, vesicular stomatitis virus
  • Heparin Antagonists
  • Luminescent Proteins
  • Membrane Glycoproteins
  • Protamines
  • Viral Envelope Proteins
  • Green Fluorescent Proteins