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Critical Factors Influencing Stable Transduction of Human CD34+ Cells with HIV-1-Derived Lentiviral Vectors

2000; Elsevier BV; Volume: 2; Issue: 1 Linguagem: Inglês

10.1006/mthe.2000.0094

1525-0024

Dennis L. Haas, Scott S. Case, Gay M. Crooks, Donald B. Kohn,

CRISPR and Genetic Engineering

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. 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.