91. Retroviral Vector Integrations Relate to Hematopoietic Stem Cell Gene Expression Patterns

Abstract

Top of pageAbstract The oncogenic risk of retrovirus mediated gene transfer has received major attention and is a considerable safety concern. To analyze the mechanisms involved we have identified integration sites in life-long monitored primary and secondary recipient mice of bone marrow cells transduced with an MLV retroviral vector. In these mice, clones with integrants by definition originate from longterm repopulating stem cells. Vector integrations were identified by LAM-PCR. To determine gene expression levels in the target population for transduction, we performed microarray analyses of Lin-/Sca-1+/c-kit+ cells, which are highly enriched for hematopoietic stem cells. This was done both for unstimulated cells and for cells subjected to 2 days of stimulation with Flt3-ligand, TPO and SCF, similar to the standard transduction protocol. Subsequently, we analyzed the integration sites relative to the gene expression profile of these immature cells. The results of the analysis showed that (i) as much as 81% (80% in growth factor stimulated cells) of the retrovirus integrations occur near genes expressed in mouse hematopoietic stem cells and that, in addition, (ii) the probability of integration is strongly correlated with the expression levels of the surrounding genes. This was observed for genes within 10 kb as well as up to 100 kb upstream or downstream of virus integration sites. Frequently hit genes were Evi1, the RNA-binding protein PCBP1 and the B-cell receptor-associated protein Bcap29. Furthermore, (iii) genes upregulated by growth factor stimulation did not display a higher frequency of retroviral landing than other expressed genes. An initial network-based analysis demonstrated that (iv) virus integrations clustered near genes involved in cancer, cell cycling, signal transduction, connective tissue development and function and, perfectly in line with the targeted stem cells, hematopoietic development and function. Thus, retroviral vector integrations in cells capable of long-term hematopoietic reconstitution occur preferentially near highly expressed genes with specific functions in immature hematopoietic stem cells.