Rapid retroviral infection of human haemopoietic cells of different lineages: efficient transfer in fresh T cells.
In order to develop a clinically feasible gene marking approach, we have used the recently described PINCO retroviral expression system, composed of the enhanced green fluorescence protein (EGFP) cDNA driven by Moloney MLV LTR and packaged in the Phoenix amphotropic cell line. Two T, five B, one erythromyeloid and three myeloid cell lines were successfully infected with % GFP+ cells ranging from 4% to 79%, showing a lineage-dependent difference in infection susceptibility, with the myeloid cells being the least efficiently infected. We also infected normal mononuclear peripheral cells cultured in PHA and rhIL-2 for 2 d, and obtained an average of 30% GFP+ cells, all present within the CD3+ population, with CD4+ and CD8+ cells being equally infected. Finally, the tonsillar purified B population showed lower levels of infectivity (6%) whereas high susceptibility was shown by normal human umbilical vein endothelial cells (57%). Highly purified CD34+ cells were also susceptible, varying from 6% to 10% GFP+ cells. Immature myeloid/erythroid progenitors have been infected which stably expressed the GFP protein during further differentiation in culture. The GFP+ T cells were FACS-sorted rapidly upon infection, subsequently cultured and the fluorescence intensity monitored. In all cases the difference in percentage of GFP+ cells did not correlate with the percentage of S/G2/M cycling cells as determined at the moment of infection or with the expression levels of Ram-1 amphotrophic receptor. The improved safety of this retroviral system, the rapidity of the technique, the high efficiency of infection with respect to normal T lymphocytes (in this last case higher than previously reported) and the lack of need for in vitro selection make this system favourable for clinical development.
Introna
M
M
Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy.
Barbui
A M
AM
Golay
J
J
Bambacioni
F
F
Schirò
R
R
Bernasconi
S
S
Breviario
F
F
Erba
E
E
Borleri
G
G
Barbui
T
T
Biondi
A
A
Rambaldi
A
A
D018952:Antigens, CD34 / Q000032:analysis; D001402:B-Lymphocytes / Q000821:virology; D015152:Blotting, Northern; D004727:Endothelium / Q000821:virology; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000821:virology*; D006801:Humans; D016130:Immunophenotyping; D008164:Luminescent Proteins / Q000235:genetics; D008979:Moloney murine leukemia virus / Q000235:genetics; D012192:Retroviridae Infections / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016176:T-Lymphocyte Subsets / Q000821:virology*; D014407:Tumor Cells, Cultured
D018952:Antigens, CD34; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins