708. Retroviral Vector Integration into the Genome of Rhesus Macaque Long-Term Repopulating Cells

Replication defective retroviruses are very attractive tools for hematopoietic stem cell (HSC) gene therapy largely because of their ability to integrate into genomic DNA. While the risk of insertional mutagenesis has long been recognized, reports of neoplasia consecutive to proto-oncogenes' activation in mice and humans have refocused attention on the safety limitations of these vectors. Mapping studies of retroviral integration sites (RIS) in human cell lines have suggested that retroviral vectors favor integration near transcriptional start sites. Medical applications using these vectors are aimed at HSC, and thus large-scale analysis of RIS in a relevant pre-clinical situation is essential to assess the safety of HSC gene therapy. Here we report the mapping of 423 RIS in a long-term model of autologous retrovirally-transduced HSC transplantation in the non-human primate (Macaca mulatta). Circulating granulocytes and mononuclear cells from 22 rhesus macaques, transplanted with G-CSF + SCF mobilized CD34+ peripheral blood stem cells transduced with an amphotropic Moloney murine leukemia-derived vector containing a neomycin-resistance marker gene, have been analyzed between one and five years after transplantation. Genomic regions adjacent to RIS have been retrieved using linear amplification-mediated PCR followed by shotgun TA cloning, and mapped to the NCBI build 34 of the human genome sequence. Out of the 423 RIS retrieved, 37% landed between the transcriptional start and stop codons of a member of the well-defined RefSeq set of genes, 55% were located in non-coding regions of the genome and 8% were found to be in interspersed repeats and therefore not mappable. Analysis of the targeted genomic regions provides evidence for non-random insertion: 8 independent RIS, recovered from 6 animals, have been mapped within the 2 first introns of the myelodysplasia syndrome 1 gene (MDS1), and seven other genes have 2 independent insertions. Among these 8 genes, it is noteworthy that three are known to be involved in chromosomal rearrangements observed in spontaneous acute human leukemias: the proto-oncogene runt-related transcription factor 1 (RUNX1), the fms-related tyrosine kinase 3 (FLT3) and MDS1. The presence of the fusion sequence between the genomic MDS1 region and the 5'-LTR of the vector has been confirmed using insertion-specific primers for 6 out of the 8 events, and clonal evolution as well as expression of the targeted gene are being analyzed. These observations suggest that retroviral integration in long-term repopulating HSC is highly non-random and is favored at specific loci. Systematic mapping of RIS after HSC gene transfer in the non-human primate will provide critical pre-clinical data necessary for risk assessment of such vectors, and offers also a powerful means for identifying genes involved in the biology of HSC.