RETROVIRAL GENE-TRANSFER TO PRIMITIVE NORMAL AND LEUKEMIC HEMATOPOIETIC-CELLS USING CLINICALLY APPLICABLE PROCEDURES

被引:75
作者
HUGHES, PFD
THACKER, JD
HOGGE, D
SUTHERLAND, HJ
THOMAS, TE
LANSDORP, PM
EAVES, CJ
HUMPHRIES, RK
机构
[1] BRITISH COLUMBIA CANC RES CTR, TERRY FOX LAB, 601 W 10TH AVE, VANCOUVER V5Z 1L3, BC, CANADA
[2] UNIV BRITISH COLUMBIA, DEPT MED, VANCOUVER V6T 1W5, BC, CANADA
[3] UNIV BRITISH COLUMBIA, DEPT MED GENET, VANCOUVER V6T 1W5, BC, CANADA
[4] UNIV BRITISH COLUMBIA, DEPT PATHOL, VANCOUVER V6T 1W5, BC, CANADA
关键词
CHRONIC MYELOGENOUS LEUKEMIA; GROWTH FACTORS; LONG-TERM CULTURE;
D O I
10.1172/JCI115786
中图分类号
R-3 [医学研究方法]; R3 [基础医学];
学科分类号
1001 ;
摘要
Clinical uses of gene transfer to bone marrow transplants require the establishment of a reproducible method for infecting large numbers of very primitive hematopoietic cells at high efficiency using cell-free retrovirus-containing media. In this study we report the results of experiments with preparations of a high-titer (2-5 x 10(7)/ml) helper-free recombinant neo(r) retrovirus that indicate this goal can now be achieved based on measurements of gene transfer efficiencies to cells referred to as long-term culture initiating cells (LTC-IC) because they give rise to clonogenic cells after greater-than-or-equal-to 5 wk in long-term culture (LTC). Intermittent, repeated exposure of normal human marrow mononuclear cells to virus-containing supernatant over a 3-d period of cell maintenance on an IL-3/granulocyte colony-stimulating factor (G-CSF) producing stromal layer resulted in gene transfer efficiencies to LTC-IC of 41%; a level previously obtainable only using co-cultivation infection techniques. Marrow cells enriched greater-than-or-equal-to 500-fold for LTC-IC (1-2% pure) by flow cytometry showed gene transfer efficiencies of 27% when infected in a similar fashion over a shorter period (24 h), but in the presence of added soluble IL-3 and G-CSF without stromal feeders, and this increased to 61% when Steel factor was also present during the infection period. By using a less highly enriched population of LTC-IC obtained by a bulk immunoselection technique applicable to large-scale clinical marrow harvests, gene transfer efficiencies to LTC-IC of 40% were achieved and this was increased to 60% by short-term preselection in G418. Southern analysis of DNA from the nonadherent cells produced by these LTC over a 6-wk period provided evidence of clonal evolution of LTC-IC in vitro. Leukemic chronic myelogenous leukemia LTC-IC were also infected at high efficiency using the same supernatant infection strategy with growth factor supplementation. These data demonstrate the feasibility of using cell-free virus preparations for infecting clinical marrow samples suitable for transplantation, as well as for further analysis of human marrow stem cell dynamics in vitro.
引用
收藏
页码:1817 / 1824
页数:8
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