Cattoglio, C, Pellin, D, Rizzi, E, Maruggi, G, Corti, G, Miselli, F et al.. High-definition mapping of retroviral integration sites identifies active regulatory elements in human multipotent hematopoietic progenitors. Blood 116: 5507-5517

Istituto Scientifico H. San Raffaele, Milan, Italy.
Blood (Impact Factor: 10.45). 12/2010; 116(25):5507-17. DOI: 10.1182/blood-2010-05-283523
Source: PubMed


Integration of retroviral vectors in the human genome follows nonrandom patterns that favor insertional deregulation of gene expression and increase the risk of their use in clinical gene therapy. The molecular basis of retroviral target site selection is still poorly understood. We used deep sequencing technology to build genomewide, high-definition maps of > 60 000 integration sites of Moloney murine leukemia virus (MLV)- and HIV-based retroviral vectors in the genome of human CD34(+) multipotent hematopoietic progenitor cells (HPCs) and used gene expression profiling, chromatin immunoprecipitation, and bioinformatics to associate integration to genetic and epigenetic features of the HPC genome. Clusters of recurrent MLV integrations identify regulatory elements (alternative promoters, enhancers, evolutionarily conserved noncoding regions) within or around protein-coding genes and microRNAs with crucial functions in HPC growth and differentiation, bearing epigenetic marks of active or poised transcription (H3K4me1, H3K4me2, H3K4me3, H3K9Ac, Pol II) and specialized chromatin configurations (H2A.Z). Overall, we mapped 3500 high-frequency integration clusters, which represent a new resource for the identification of transcriptionally active regulatory elements. High-definition MLV integration maps provide a rational basis for predicting genotoxic risks in gene therapy and a new tool for genomewide identification of promoters and regulatory elements controlling hematopoietic stem and progenitor cell functions.

Download full-text


Available from: Giorgio Corti,
30 Reads
  • Source
    • "It has become widely established that retroviral vector integration favors nucleosomal over naked DNA, including the epigenetic promoter and enhancer markers H3K4Me3 and H3K4Me/H3K27Ac, respectively [18-24]. Importantly, the studies by De Rijck et al. and Sharma et al. have exposed a fundamental mechanism for integration of MLVs at TSSs mediated by BET proteins [29,30]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background Insertional mutagenesis screens of retrovirus-induced mouse tumors have proven valuable in human cancer research and for understanding adverse effects of retroviral-based gene therapies. In previous studies, the assignment of mouse genes to individual retroviral integration sites has been based on close proximity and expression patterns of annotated genes at target positions in the genome. We here employed next-generation RNA sequencing to map retroviral-mouse chimeric junctions genome-wide, and to identify local patterns of transcription activation in T-lymphomas induced by the murine leukemia gamma-retrovirus SL3-3. Moreover, to determine epigenetic integration preferences underlying long-range gene activation by retroviruses, the colocalization propensity with common epigenetic enhancer markers (H3K4Me1 and H3K27Ac) of 6,117 integrations derived from end-stage tumors of more than 2,000 mice was examined. Results We detected several novel mechanisms of retroviral insertional mutagenesis: bidirectional activation of mouse transcripts on opposite sides of a provirus including transcription of unannotated mouse sequence; sense/antisense-type activation of genes located on opposite DNA strands; tandem-type activation of distal genes that are positioned adjacently on the same DNA strand; activation of genes that are not the direct integration targets; combination-type insertional mutagenesis, in which enhancer activation, alternative chimeric splicing and retroviral promoter insertion are induced by a single retrovirus. We also show that irrespective of the distance to transcription start sites, the far majority of retroviruses in end-stage tumors colocalize with H3K4Me1 and H3K27Ac-enriched regions in murine lymphoid tissues. Conclusions We expose novel retrovirus-induced host transcription activation patterns that reach beyond a single and nearest annotated gene target. Awareness of this previously undescribed layer of complexity may prove important for elucidation of adverse effects in retroviral-based gene therapies. We also show that wild-type gamma-retroviruses are frequently positioned at enhancers, suggesting that integration into regulatory regions is specific and also subject to positive selection for sustaining long-range gene activation in end-stage tumors. Altogether, this study should prove useful for extrapolating adverse outcomes of retroviral vector therapies, and for understanding fundamental cellular regulatory principles and retroviral biology.
    Retrovirology 05/2014; 11(1):36. DOI:10.1186/1742-4690-11-36 · 4.19 Impact Factor
  • Source
    • "Since that time, more chromatin features have been identified, along with tens of thousands of integration sites (13). This has led to the inference that murine leukemia virus (MLV) integrates near regulatory elements like promoters and enhancers (14). Consistent with this, previous studies have detected associations between MLV integration and individual chromatin marks (15,16). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Retroviruses integrate into the host genome in patterns specific to each virus. Understanding the causes of these patterns can provide insight into viral integration mechanisms, pathology and genome evolution, and is critical to the development of safe gene therapy vectors. We generated murine leukemia virus integrations in human HepG2 and K562 cells and subjected them to second-generation sequencing, using a DNA barcoding technique that allowed us to quantify independent integration events. We characterized >3 700 000 unique integration events in two ENCODE-characterized cell lines. We find that integrations were most highly enriched in a subset of strong enhancers and active promoters. In both cell types, approximately half the integrations were found in <2% of the genome, demonstrating genomic influences even narrower than previously believed. The integration pattern of murine leukemia virus appears to be largely driven by regions that have high enrichment for multiple marks of active chromatin; the combination of histone marks present was sufficient to explain why some strong enhancers were more prone to integration than others. The approach we used is applicable to analyzing the integration pattern of any exogenous element and could be a valuable preclinical screen to evaluate the safety of gene therapy vectors.
    Nucleic Acids Research 01/2014; 42(7). DOI:10.1093/nar/gkt1399 · 9.11 Impact Factor
  • Source
    • "This lowers, but does not completely alleviate, the risk of genotoxicity according to studies addressing the oncogenic potential of these vectors either in vitro or in vivo (Modlich et al, 2009; Montini et al, 2006, 2009). The common consensus drawn by these studies is that viral vector integration is an active process catalysed by the tethering of the viral preintegration complex to open chromatin regions in the host cell genome as characterized by DNaseI hypersensitive sites and epigenetics marks (Cattoglio et al, 2010; Deichmann et al, 2011; Felice et al, 2009). For example, the host‐cell encoded LEDGF/p75 binds to the lentiviral integrase to direct integration to active transcription units. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The first gene therapy clinical trials were initiated more than two decades ago. In the early days, gene therapy shared the fate of many experimental medicine approaches and was impeded by the occurrence of severe side effects in a few treated patients. The understanding of the molecular and cellular mechanisms leading to treatment- and/or vector-associated setbacks has resulted in the development of highly sophisticated gene transfer tools with improved safety and therapeutic efficacy. Employing these advanced tools, a series of Phase I/II trials were started in the past few years with excellent clinical results and no side effects reported so far. Moreover, highly efficient gene targeting strategies and site-directed gene editing technologies have been developed and applied clinically. With more than 1900 clinical trials to date, gene therapy has moved from a vision to clinical reality. This review focuses on the application of gene therapy for the correction of inherited diseases, the limitations and drawbacks encountered in some of the early clinical trials and the revival of gene therapy as a powerful treatment option for the correction of monogenic disorders.
    EMBO Molecular Medicine 11/2013; 5(11). DOI:10.1002/emmm.201202287 · 8.67 Impact Factor
Show more