Abstract
Therapeutic retroviral vector integration near the oncogene LMO2 is thought to be a cause of leukemia in X-SCID gene therapy trials. However, no published studies have evaluated the frequency of vector integrations near exon 1 of the LMO2 locus. We identified a high incidence region (HIR) of vector integration using PCR techniques in the upstream region close to the LMO2 transcription start site in the TPA-Mat T cell line. The integration frequency of the HIR was one per 4.46 × 104 cells. This HIR was also found in Jurkat T cells but was absent from HeLa cells. Furthermore, using human cord blood-derived CD34+ cells we identified a HIR in a similar region as the TPA-Mat T cell line. One of the X-linked severe combined immunodeficiency (X-SCID) patients that developed leukemia after gene therapy had a vector integration site in this HIR. Therefore, the descriptions of the location and the integration frequency of the HIR presented here may help us to better understand vector-induced leukemogenesis.
Highlights
Since no integration into the region upstream of -3001 was detected with the indicated primers (Figure 1C and Additional files 2 and 3), we suggest that the high incidence region (HIR) in the TPA-Mat-ecoR cells ranges from -1740 to -3001
We compared the integration pattern in the TPA-Mat-ecoR cells with the integration sites identified in patients (Pt) 4 and 5 who developed leukemia during the gene therapy trials for treatment of X-linked severe combined immunodeficiency (X-SCID) [12,17]
Since CD34+ hematopoietic stem cells have been infected with the MLV vector in the clinical trials, we investigated whether the HIR is found in human CD34+ hematopoietic stem cells
Summary
For 1613 PCR amplifications (1613 μg of sample DNA corresponds to approximately 2.4 × 108 cells) using LTR- and LMO2-specific primers, 65 integration sites were unevenly distributed in an approximately ± 3-kb region surrounding exon 1 of LMO2 in the human genome (Figure 1C and Additional files 2 and 3). Since no integration into the region upstream of -3001 was detected with the indicated primers (Figure 1C and Additional files 2 and 3), we suggest that the HIR in the TPA-Mat-ecoR cells ranges from -1740 (the downstream edge) to -3001 (the upstream edge).
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