Target Site Selection of Retroviral Vectors in the Human Genome: Viral and Genomic Determinants of Non-Random Integration Patterns in Hematopoietic Cells

Cattoglio, Claudia (2008). Target Site Selection of Retroviral Vectors in the Human Genome: Viral and Genomic Determinants of Non-Random Integration Patterns in Hematopoietic Cells. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f263

Abstract

Integration of gamma-retroviruses (RV) and lentiviruses (LV) follows different, non-random patterns in mammalian genomes. To obtain information about the viral and genomic determinants of integration preferences, I mapped > 2,500 integration sites of RV and LV vectors carrying wild type or modified LTRs in human CD34+ hematopoietic cells. Recurrent insertion sites (hot spots) account for > 20% of the RV integration events, while they are significantly less frequent for LV vectors. Genes controlling growth, differentiation and development of the hematopoietic and immune system are targeted at high frequency by RV vectors and further enriched in RV hot spots, suggesting that the cell gene expression program is instrumental in directing RV integration. To investigate the role of transcriptional regulatory networks in directing RV and LV integration, I evaluated the local abundance and arrangement of putative transcription factor binding sites (TFBSs) in the genomic regions flanking integrated proviruses. RV, but not LV integrations are flanked by specific subsets of TFBSs, independently of their location with respect to genes (within genes, outside, or around their transcription start sites). Hierarchical clustering and a Principal Components Analysis of TFBSs flanking integration sites of RV vectors carrying different LTRs, and LV vectors packaged with wild type or RV-LV hybrid integrase, showed that both the protein and the DNA component of the pre-integration complex (PIC) have a causal role in directing proviral integration in TFBS-rich regions of the genome. Chromatin immunoprecipitation analysis indicated that TFs are bound to unintegrated LTR enhancers into the nucleus, and might synergize with the viral integrase in tethering retroviral PICs to specific domains of transcriptionally active chromatin. The results of this project suggest substantial differences in the molecular mechanisms tethering RV and LV PICs to human chromatin, and predict a different insertional oncogenesis risk of RV vs. LV vectors for human gene therapy.

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