1. Molecular Long-Term Follow Up in Non-Human Primates That Received Retrovirally Transduced Stem Cells: Despite Proto-Oncogene Insertion No Leukemia or Hematopoietic Abnormalities (2004)

The occurrence of leukemia-like events consequent to vector integration in a successful human gene therapy trial has prompted a reassessment of the insertional mutagenesis risk by retroviral vectors. Recent reports, which used high-throughput sequencing and mapping of vector integration sites, have challenged the notion that retroviruses integrate randomly in the cell genome, and indicated preferential integration into active genes both for HIV- and MLV-based vectors. We used a novel approach based on genetic trapping to score several thousands integration sites at once in transduced cells. To this aim, we constructed HIV- and MLV-based promoter traps and compared the efficiency of promoter trapping using a selectable reporter PuroR-GFP fusion gene. We found that MLV trapped cellular promoters much more efficiently, and targeted more active genes, than HIV. Remarkably, one out of seven MLV integrations trapped an active cellular promoter. We obtained these findings in human and murine cells, including human cord blood CD34+ cells. When we delivered vector traps by CaPi-DNA transfection, we did not find significant differences in trapping efficiency of MLV and HIV traps, indicating that the retroviral integration machinery was responsible for the differences observed in transduction experiments. RNA analysis of transduced cells showed that HIV transcripts had a much wider size range as compared with those of MLV, which did not significantly exceed the length of the vector backbone. These results indicated that MLV integrations resulting in reporter expression occurred in proximity to the transcription start site, or within the first intron, of active genes. Interestingly, analysis of puromycin-selected cells showed strong enrichment in HIV transcripts of size similar to those of MLV, indicating that the few HIV integrations resulting in efficient reporter expression were those occurring proximal to a promoter. The average reporter expression level of these two selected cell populations remained higher for MLV traps, confirming that, on average, MLV vectors targeted genes more active than those targeted by HIV. These results provide functional evidence that MLV vectors preferentially integrate close to highly active promoters, and can efficiently exploit them for proviral expression. Such a strategy may have evolved in the parental viruses to provide a fraction of their rogeny with a supplementary cellular promoter, less susceptible to silencing by genome surveillance mechanisms than the one embedded in the LTR. The proximity and functional relationship established between MLV proviruses and flanking cellular promoters suggests that integration of wild-type MLV may easily up-regulate transcription from cellular promoters and overexpress the cognate genes, including proto-oncogenes. HIV vectors did not show the integration biases observed for MLV. Whether this behavior of HIV, together with the advanced vector engineering that fully inactivates its LTR, provide a safer integrating tool for gene therapy remains to be assessed.