Estimated Comparative Integration Hotspots Identify Different Behaviors of Retroviral Gene Transfer Vectors

Integration of retroviral vectors in the human genome follows non random patterns that favor insertional deregulation of gene expression and may cause risks of insertional mutagenesis when used in clinical gene therapy. Understanding how viral vectors integrate into the human genome is a key issue in predicting these risks. We provide a new statistical method to compare retroviral integration patterns. We identified the positions where vectors derived from the Human Immunodeficiency Virus (HIV) and the Moloney Murine Leukemia Virus (MLV) show different integration behaviors in human hematopoietic progenitor cells. Non-parametric density estimation was used to identify candidate comparative hotspots, which were then tested and ranked. We found 100 significative comparative hotspots, distributed throughout the chromosomes. HIV hotspots were wider and contained more genes than MLV ones. A Gene Ontology analysis of HIV targets showed enrichment of genes involved in antigen processing and presentation, reflecting the high HIV integration frequency observed at the MHC locus on chromosome 6. Four histone modifications/variants had a different mean density in comparative hotspots (H2AZ, H3K4me1, H3K4me3, H3K9me1), while gene expression within the comparative hotspots did not differ from background. These findings suggest the existence of epigenetic or nuclear three-dimensional topology contexts guiding retroviral integration to specific chromosome areas.逆转录病毒载体在人类基因组中的整合遵循非随机模式，此类模式倾向于引发基因表达的插入性失调，在临床基因治疗应用中还可能带来插入性诱变风险。阐明病毒载体整合进入人类基因组的具体机制，是预测此类风险的核心问题。本研究提出一种全新的统计学方法，用于对比不同逆转录病毒载体的整合模式。我们在人类造血祖细胞（hematopoietic progenitor cells）中，鉴定出了人类免疫缺陷病毒（Human Immunodeficiency Virus, HIV）与莫洛尼鼠白血病病毒（Moloney Murine Leukemia Virus, MLV）来源的载体呈现差异化整合行为的位点。研究采用非参数密度估计法识别候选比较热点区域，随后对这些区域进行验证与排序。我们共得到100个具有统计学显著性的比较热点区域，这些区域分布于所有染色体中。相较于莫洛尼鼠白血病病毒载体的热点区域，人类免疫缺陷病毒载体的热点区域范围更广，所包含的基因数量更多。针对人类免疫缺陷病毒整合靶点的基因本体（Gene Ontology, GO）分析显示，参与抗原加工与呈递过程的基因显著富集，这与在6号染色体上的主要组织相容性复合体（Major Histocompatibility Complex, MHC）位点观测到的人类免疫缺陷病毒高整合频率相吻合。在比较热点区域中，四种组蛋白修饰/组蛋白变体的平均密度存在显著差异（H2AZ、H3K4me1、H3K4me3、H3K9me1），而热点区域内的基因表达水平与背景基因组并无显著差异。上述研究结果表明，存在表观遗传或细胞核三维拓扑结构层面的调控环境，引导逆转录病毒载体整合至特定的染色体区域。