824. Oncoretroviral Vector Integration Biases in Mouse Hematopoietic Progenitors

Although gene transfer vectors based on recombinant retroviruses have been known to have the potential to lead to insertional mutagenesis, that integrations can lead to oncogenic transformation of transduced target cells in a gene therapy setting has led to intensive research on integration site choice. Several groups have reported that oncoretroviral vectors have a slight preference for landing within genes, a strong preference for integration within the region immediately around transcriptional start sites, and a preference for actively transcribed genes. Using primary mouse bone marrow progenitors as target cells for an oncoretroviral vector, we addressed three questions: 1) Are genes preferentially the targets; 2) Are regions surrounding transcriptional starts favored; and 3) Does selection for the vector bias the repertoire of integration sites? We obtained bone marrow from 5-FU treated mice, prestimulated the cells for two days, transduced by co-cultivation on irradiated ecotropic producer lines at a low multiplicity of infection. The retrovirus has a bacterial drug resistance cassette for Zeocin in the long terminal repeat, an MSCV LTR transcribing a GFP reporter, and a separate internal Pgk-Neo cassette (used to select for expression). Progenitor cell colonies were recovered in methylcellulose +/|[minus]| G418 seven days after transduction. Integration junctions were cloned directly from genomic DNA with the Zeocin cassette present in the LTRs, rather than by a PCR based strategy, enabling us to obtain larger genomic segments. Junction sequences were mapped by BLAST to build 33 of the mouse genome in the ENSEMBL database. We identified 130 integration sites from unselected pools and 130 integration sites from selected pools of progenitor cells. We also mapped an equivalent number of simulated integrations; this dataset was treated as the experimental dataset was, with unmappable sites (such as those that landed entirely in gaps or repetitive elements) removed. We have found that MSCV integrations do not occur more frequently into genes (defined as within known transcripts introns and exons, 42%) than random (41%), differing from what was previously reported for MLV integration into HeLa cells. Comparing the integrations that are within 5 kb upstream of a transcription start site, there is a highly significant difference between our experimental dataset (18%) and the simulated dataset (4%), P = < .0001. With the progenitor colonies having undergone selection, the frequency of landing within introns has increased from 35% to 43%. When the overall distribution of integrations is compared to gene density mapped onto the mouse chromosomes, it appears that oncoretroviruses favor gene-rich regions of the mouse genome. Though MSCV retroviruses do not have a preference for integrating within genes, they favor the 5 kb region upstream of transcriptional starts, 18% in this setting, versus 8.5% observed previously in HeLa cells. These results suggest that there may be less of a bias for oncoretrovrus integration within genes in mouse bone marrow progenitor cells compared to other cells types tested to date, and that the use of selection can bias the repertoire of integration sites that are observed following oncoretrovirus transduction.