Distinct genomic integration of MLV and SIV vectors in primate hematopoietic stem and progenitor cells

Abstract

Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34(+)cells. Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors.

Figure 1. Comparison of MLV and SIV Integration Events

Shown are integrations that landed within RefSeq gene introns (arrows) in comparison to in silico-generated integration sites (bars). Black indicates MLV and gray indicates SIV. *p < 0.0001 by a Chi² test.

Figure 2. Distribution of MLV and SIV Integration Sites within a 60-kb Window Centered on Transcription Start Sites

The vertical arrow points to 0 kb. Each gray bar corresponds to the percentage of SIV integration sites within a 5-kb interval, and black bars correspond to the percentages of MLV integration sites in a 5-kb interval. The distribution of a set of 65,000 in silico-generated random integration sites is represented by the dashed line.

Figure 3. Distribution and Location of Integration Sites Relative to Chromosomal Gene Density

(A) Distribution of MLV and SIV integration sites relative to gene density within a 1-Mbp window compared to in silico-generated random integration sites. Each bar corresponds to the percentage of integration sites within the corresponding gene density region. (B) Location of MLV and SIV integration sites and gene density on human Chromosome 6. MLV and SIV integrations were aligned to Chromosome 6 (obtained from the UCSC custom annotation track feature) and shown in relation to RefSeq gene density (blue). 73% of the SIV integration events are within the 20-Mbp unique ridge of Chromosome 6, compared to 29% for MLV. Distance between thick black bars is 20 Mbp; centromere is represented by the black circle.