Open Research Online The Open University’s repository of research publications and other research outputs Target Site Selection of Retroviral Vectors in the Human Genome: Viral and Genomic Determinants of Non-Random Integration Patterns in Hematopoietic Cells Thesis How to cite: 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. For guidance on citations see FAQs. c 2008 The Author https://creativecommons.org/licenses/by-nc-nd/4.0/ Version: Version of Record Link(s) to article on publisher’s website: http://dx.doi.org/doi:10.21954/ou.ro.0000f263 Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyright owners. For more information on Open Research Online’s data policy on reuse of materials please consult the policies page. oro.open.ac.uk M /v££$lR ICTSS^ CLAUDIA CATTOGLIO TARGET SITE SELECTION OF RETROVIRAL VECTORS IN THE HUMAN GENOME: VIRAL AND GENOMIC DETERMINANTS OF NON-RANDOM INTEGRATION PATTERNS IN HEMATOPOIETIC CELLS PhD thesis in fulfillment of the requirements of the Open University for the degree of Doctor of Philosophy in Molecular and Cellular Biology 2008 Director of studies External Supervisor Prof. Fulvio Mavilio Prof. Andrew M. L. Lever Vita-Salute San Raffaele University Department of Biological and Technological Research San Raffaele Scientific Institute Milan, Italy ProQuest Number: 13837696 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13837696 Published by ProQuest LLC(2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 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 1 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. 2 TABLE OF CONTENTS 1. Introduction ....................................................................................................................... 1 2. A brief review of retroviruses and retroviral vectors ................................................... 4 2.1 Retroviruses ................................................................................................................ 4 2.1.1 Structure and classification ...................................................................................4 2.1.2 Replication cycle ....................................................................................................6 2.1.3 Integration reaction: products and kinetics .........................................................9 2.1.4 Preintegration complex composition ................................................................. 12 2.1.5 Regulation of proviral transcription .................................................................. 14 2.1.5.1 Transcriptional regulation of Mo-MLV ................................................. 16 2.1.5.2 Transcriptional regulation of HIV-1 .......................................................20 2.2 Retroviral vectors and gene therapy .................................................................... 24 3. Retroviral integration features and mechanisms: state of the art ..............................28 3.1 Insertional mutagenesis as a gene therapy adverse event .....................................28 3.2 Non-random integration pattern of retroviral vectors in mammalian genome ...........................................................................................................................................32 3.2.1 Primary DNA sequence and integration site selection .....................................33 3.2.2 Retroviral integration and genes ........................................................................34 3.2.3 Retroviral integration and gene activity ........................................................... 35 3.2.4 Retroviral integration and transcription factor binding sites .......................... 36 3.3 Proposed mechanisms for integration site selection ......................................... 38 3.3.1 Ty retrotransposons: a paradigm for tethered integration ...............................38 3.3.2 Tethering models for retroviral integration .......................................................39 3.3.3 LEDGF/p75: a candidate for lentiviral integration tethering ......................... 40 3.4 In vivo clonal expansion of MLV-transduced human and murine hematopoietic cells ......................................... 43 4. Aim of the study .............................................................................................................48 5. Materials and methods ...................................................................................................53 5.1 Retroviral vectors ....................................................................................................53 5.2 Transduction of target cells .................................................................................... 54 5.3 Sequencing, mapping and annotation of retroviral integration sites ................56 5.4 Gene expression profiling ...................................................................................... 58 5.5 Functional clustering analysis ................................................................................59 5.6 Transcription factor binding site analysis ................................................... 60 5.7 Southern and Western blot analysis ..................................................................... 63 5.8 RNA extraction and RT-PCR analysis .................................................................64 5.9 Chromatin immunoprecipitation assay .................................................................65 6. Results.............................................................................................................................. 67 6.1 Integration preferences of Mo-MLV and HIV-1-based retroviral vectors in human CD34+ HSCs ...................................................................................................... 67 6.1.1 Genome-wide analysis of retroviral integration preferences in human CD34+ HSCs................................................................................................................................ 67 6.1.2 Genes regulating cell growth and proliferation are preferential targets of retroviral integration ...................................................................................................... 75 6.1.3 RV but not LV vectors show a high frequency of integration hot spots ......83 6.1.4 Proto-oncogenes and cancer-associated CISs are hot spots of RV but not LV integration ....................................................................................................................... 88 6.2 Role of LTR and of LTR interactors in the integration site selection of retroviral vectors ............................................................................................................. 90 6.2.1 Collection of integration sites from human hematopoietic cells transduced with LTR-modified retroviral