Retroviral marking of acute myelogenous leukemia progenitors that initiatelong-term culture and growth in immunodeficient mice

Rare primitive progenitors among the malignant cells from most patients wit h AML include AML long-term culture-initiating cells (AML LTC-IC) and NOD/S CID mouse leukemia-initiating cells (NOD/SL-IC). To evaluate the feasibilit y of genetic modification of these progenitors for gene marking and/or gene therapy strategies, cells from patients with newly-diagnosed AML were cocu ltured with retroviral producer cells and then placed in colony (AML-CFC) a ssays, LTC, and injected intravenously into NOD/SCID mice. Southern blottin g demonstrated transfer of the neo(r) gene to 30% to 80% of leukemic blasts when cells were cultured for 48 hours in the presence of IL-3 and steel fa ctor (SF) prior to 48-hour coculture with viral producers. Three of six ret rovirally-infected AML samples showed both engraftment in NOD/SCID mice and the presence of the neo' transgene in mouse tissues 8-15 weeks after injec tion of transduced cells. Thirteen weeks after injection of one of these sa mples, >80% of cells from mouse bone marrow were the progeny of two retrovi rally-transduced AML progenitors. Four of the remaining five samples showed markedly reduced ability to engraft in mice after retroviral infection. Su bsequent experiments demonstrated that the loss of engraftment potential to ok place within 24 hours of culture initiation in the absence of retroviral producers and regardless of the cytokines present. Interestingly, the majo rity of AML-CFC or AML LTC-IC survived the 24-hour culture period. A retrov iral vector containing the murine cell surface marker heat stable antigen ( HSA), which allows purification of transduced cells on immunomagnetic colum ns, was used to obtain an enriched population of gene-modified AML cells fo llowing an infection protocol that eliminated the 48 hours of prestimulatio n in IL-3 and SF and reduced coculture with viral producers to 10-36 hours. These modifications failed to improve engraftment of the infected cells. I n addition, in these experiments more than 10 hours of cocultivation with v iral producer cells was necessary to achieve gene transfer and expression i n AML LTC-IC. These data demonstrate that although retroviral-mediated gene transfer can be achieved to AML progenitors, including NOD/SL-IC, improved culture conditions will be required before substantial numbers of such tra nsduced primitive progenitors can be obtained. In addition, the difference in the ability of AML LTC-IC and NOD/SL-IC to survive ex vivo suggests that these assays may detect different populations of cells or that changes are induced in vitro in primitive cells which can only be detected in the mous e assay. (C) 1999 International Society for Experimental Hematology. Publis hed by Elsevier Science Inc.