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

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

Recent evidence that TADs and chromatin loops are dynamic structures.

Mammalian genomes are folded into spatial domains, which regulate gene expression by modulating enhancer-promoter contacts. Here, we review recent studies on the structure and function of Topologically Associating Domains (TADs) and chromatin loops. We discuss how loop extrusion models can explain TAD formation and evidence that TADs are formed by the ring-shaped protein complex, cohesin, and that TAD boundaries are established by the DNA-binding protein, CTCF. We discuss our recent genomic, biochemical and single-molecule imaging studies on CTCF and cohesin, which suggest that TADs and chromatin loops are dynamic structures. We highlight complementary polymer simulation studies and Hi-C studies employing acute depletion of CTCF and cohesin, which also support such a dynamic model. We discuss the limitations of each approach and conclude that in aggregate the available evidence argues against stable loops and supports a model where TADs are dynamic structures that continually form and break throughout the cell cycle

Guided nuclear exploration increases CTCF target search efficiency.

The enormous size of mammalian genomes means that for a DNA-binding protein the number of nonspecific, off-target sites vastly exceeds the number of specific, cognate sites. How mammalian DNA-binding proteins overcome this challenge to efficiently locate their target sites is not known. Here, through live-cell single-molecule tracking, we show that CCCTC-binding factor, CTCF, is repeatedly trapped in small zones that likely correspond to CTCF clusters, in a manner that is largely dependent on an internal RNA-binding region (RBRi). We develop a new theoretical model called anisotropic diffusion through transient trapping in zones to explain CTCF dynamics. Functionally, transient RBRi-mediated trapping increases the efficiency of CTCF target search by ~2.5-fold. Overall, our results suggest a 'guided' mechanism where CTCF clusters concentrate diffusing CTCF proteins near cognate binding sites, thus increasing the local ON-rate. We suggest that local guiding may allow DNA-binding proteins to more efficiently locate their target sites

A DNA Repair Complex Functions as an Oct4/Sox2 Coactivator in Embryonic Stem Cells

SummaryThe transcriptional activators Oct4, Sox2, and Nanog cooperate with a wide array of cofactors to orchestrate an embryonic stem (ES) cell-specific gene expression program that forms the molecular basis of pluripotency. Here, we report using an unbiased in vitro transcription-biochemical complementation assay to discover a multisubunit stem cell coactivator complex (SCC) that is selectively required for the synergistic activation of the Nanog gene by Oct4 and Sox2. Purification, identification, and reconstitution of SCC revealed this coactivator to be the trimeric XPC-nucleotide excision repair complex. SCC interacts directly with Oct4 and Sox2 and is recruited to the Nanog and Oct4 promoters as well as a majority of genomic regions that are occupied by Oct4 and Sox2. Depletion of SCC/XPC compromised both pluripotency in ES cells and somatic cell reprogramming of fibroblasts to induced pluripotent stem (iPS) cells. This study identifies a transcriptional coactivator with diversified functions in maintaining ES cell pluripotency and safeguarding genome integrity.PaperCli

Determining cellular CTCF and cohesin abundances to constrain 3D genome models.

Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved. Here, we report the quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells. Extending our previous imaging studies (Hansen et al., 2017), we estimate bounds on the density of putatively DNA loop-extruding cohesin complexes and CTCF binding site occupancy. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) suggest the presence of cohesin dimers and/or oligomers. Finally, based on our cell lines with accurately measured protein abundances, we report a method to conveniently determine the number of molecules of any Halo-tagged protein in the cell. We anticipate that our results and the established tool for measuring cellular protein abundances will advance a more quantitative understanding of 3D genome organization, and facilitate protein quantification, key to comprehend diverse biological processes

Genomic Analysis of Sleeping Beauty Transposon Integration in Human Somatic Cells

The Sleeping Beauty (SB) transposon is a non-viral integrating vector system with proven efficacy for gene transfer and functional genomics. However, integration efficiency is negatively affected by the length of the transposon. To optimize the SB transposon machinery, the inverted repeats and the transposase gene underwent several modifications, resulting in the generation of the hyperactive SB100X transposase and of the high-capacity \u2018\u2018sandwich\u2019\u2019 (SA) transposon. In this study, we report a side-by-side comparison of the SA and the widely used T2 arrangement of transposon vectors carrying increasing DNA cargoes, up to 18 kb. Clonal analysis of SA integrants in human epithelial cells and in immortalized keratinocytes demonstrates stability and integrity of the transposon independently from the cargo size and copy number-dependent expression of the cargo cassette. A genome-wide analysis of unambiguously mapped SA integrations in keratinocytes showed an almost random distribution, with an overrepresentation in repetitive elements (satellite, LINE and small RNAs) compared to a library representing insertions of the first-generation transposon vector and to gammaretroviral and lentiviral libraries. The SA transposon/SB100X integrating system therefore shows important features as a system for delivering large gene constructs for gene therapy application

Long-term Engraftment of Single Genetically Modified Human Epidermal Holoclones Enables Safety Pre-assessment of Cutaneous Gene Therapy

Predicting the risks of permanent gene therapy approaches involving the use of integrative gene-targeting vectors has become a critical issue after the unfortunate episode of a clinical trial in children with X-linked severe combined immunodeficiency (X-SCID). Safety pre-assessment of single isolated gene-targeted stem cells or their derivative clones able to regenerate their tissue of origin would be a major asset in addressing untoward gene therapy effects in advance. Human epidermal stem cells, which have extensive proliferative potential in vitro, theoretically offer such a possibility as a method of assessment. By means of optimized organotypic culture and grafting methods, we demonstrate the long-term in vivo regenerative capacity of single gene-targeted human epidermal stem cell clones (holoclones). Both histopathological analysis of holoclone-derived grafts in immunodeficient mice and retroviral insertion site mapping performed in the holoclone in vitro and after grafting provide proof of the feasibility of pre-assessing genotoxicity risks in isolated stem cells before transplantation into patients. Our results provide an experimental basis for previously untested assumptions about the in vivo behavior of epidermal stem cells prospectively isolated in vitro and pave the way for a safer approach to cutaneous gene therapy

Retroviral Integration Process in the Human Genome: Is It Really Non-Random? A New Statistical Approach

Retroviral vectors are widely used in gene therapy to introduce therapeutic genes into patients' cells, since, once delivered to the nucleus, the genes of interest are stably inserted (integrated) into the target cell genome. There is now compelling evidence that integration of retroviral vectors follows non-random patterns in mammalian genome, with a preference for active genes and regulatory regions. In particular, Moloney Leukemia Virus (MLV)–derived vectors show a tendency to integrate in the proximity of the transcription start site (TSS) of genes, occasionally resulting in the deregulation of gene expression and, where proto-oncogenes are targeted, in tumor initiation. This has drawn the attention of the scientific community to the molecular determinants of the retroviral integration process as well as to statistical methods to evaluate the genome-wide distribution of integration sites. In recent approaches, the observed distribution of MLV integration distances (IDs) from the TSS of the nearest gene is assumed to be non-random by empirical comparison with a random distribution generated by computational simulation procedures. To provide a statistical procedure to test the randomness of the retroviral insertion pattern, we propose a probability model (Beta distribution) based on IDs between two consecutive genes. We apply the procedure to a set of 595 unique MLV insertion sites retrieved from human hematopoietic stem/progenitor cells. The statistical goodness of fit test shows the suitability of this distribution to the observed data. Our statistical analysis confirms the preference of MLV-based vectors to integrate in promoter-proximal regions

High-Definition Mapping of Retroviral Integration Sites Defines the Fate of Allogeneic T Cells After Donor Lymphocyte Infusion

The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34+ hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction

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