Replication-defective gammaretroviral vectors have successfully been used for gene marking and therapy studies. Recently, we and others have shown that proto-oncogene upregulation due to retroviral vector insertion may trigger malignant transformation of engineered cells. We have now investigated whether insertional mutagenesis may also influence the contribution of individual cell clones to normal long-term hematopoiesis. Therefore, using LM-PCR we retrieved retroviral insertion sites from hematopoietic cells of mice exhibiting progression to oligoclonal or monoclonal hematopoiesis upon serial bone marrow transplantation. From 28 mice analyzed (12 primary, 16 secondary) we obtained a total of 67 insertions that matched the murine genome. In samples harvested from primary recipients seven months after transplantation of gene-marked cells a number of intriguing hits (HoxB4, Evi1, Ly78, Ccnd3, Pip5k2a) was found obviously already reflecting selection for long-term repopulating ability. Strikingly, almost all clones analyzed five months after serial transplantation revealed hits in genes with an established or potential role in the self-renewal process of hematopoietic stem cells (e.g., Vegfa, HoxB4, CyclinD3, Evi1) or other genes involved in the regulation of cell survival (Ly78, Pip5k2a, Irf2bp, Cflar). Two (Evi1, Ccnd3) among the 13 insertions associated with serial repopulation activity were already established common integration sites (CIS) in the context of mouse leukemias elicited by replication-competent retroviruses (RTCGD database). Using RT-PCR transcriptional activation involving fusion transcripts originating from the retroviral LTR into exons could be detected for the two genes (HoxB4, Vegfa) tested. Interestingly, Evi1 was the only gene targeted several times - with distinct insertions in 5 different clones. However, despite the fact that Evi1 was also the only established proto-oncogene found to be marked, four of the clones detected in primary recipients were lost upon serial transplantation. This indicates that insertion into the Evi1 locus, although having the potential to promote engraftment and/or survival of a given clone, was not sufficient for malignant transformation. Thus our data demonstrate a delicate regulatory balance following insertional mutagenesis by replication-deficient retroviral vectors. These findings have major implications for interpreting results from diagnostic gene marking experiments, for gene therapy, and the discovery of genes regulating stem cell turnover.

(The first two authors contributed equally to this work)

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