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IN VIVO IMAGING OF ENGRAFTMENT AND ENRICHMENT OF LENTIVIRAL TRANSDUCED HEMATOPOIETIC BONE MARROW CELLS UNDER MGMT-P140K MEDIATED SELECTION by YUAN LIN Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Stanton L. Gerson Program in Molecular Virology Department of Molecular Biology & Microbiology CASE WESTERN RESERVE UNIVERSITY May, 2011 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Yuan Lin ____________________________________________________ Doctor of Philosophy candidate for the ________________________________degree *. Eric Arts (signed)_______________________________________________ (chair of the committee) George Stark _______________________________________________ Zhenghong Lee _______________________________________________ Cheng-Kui Qu _______________________________________________ Stanton Gerson _______________________________________________ _______________________________________________ 10/7/2010 (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. ii Dedication This work is dedicated to my parents, my wife Phuong and my children, Edwin and Karina, for their continuous support, patient and sacrifice. iii Table of Contents List of Tables 2 List of Figures 3 Acknowledgments 5 List of Abbreviations 7 Abstract 11 Chapter 1. Hematopoietic stem cell gene therapy 13 Chapter 2. Lentiviral vectors in cancer therapy 25 Chapter 3. Bioluminescence imaging of hematopoietic stem cell repopulation in murine models 51 Chapter 4. Imaging stem cell derived persistent foci after in vivo selection of lentiviral MGMT-P140K transduced murine bone marrow cells 69 Chapter 5. Cryo-imaging of hematopoietic bone marrow transplantation 108 Chapter 6. Clonal selection and engraftment of hematopoietic stem and progenitor cells after bone marrow transplantation and in vivo drug selection 130 Chapter 7. Discussion and future directions 149 Appendix 157 Bibliography 160 1 List of Tables Table 1: Advantages and disadvantages of gene transfer vectors 20 Table 2: Lentiviral clinical trials 49 Table 3: Rodent cocktail mixture 57 Table 4: Insertion site analysis of LAM-PCR from CFU colonies of hematopoietic stem and progenitor cells before transplantation and after in vivo selection 94 Table 5: Analysis of CIS of cryo samples and peripheral blood samples 142 2 List of Figures Figure 1. Hematopoiesis 16 Figure 2. Hematopoietic stem cell division 17 Figure 3. HIV-1 provirus and lentiviral particle 29 Figure 4. Schematic representation of lentiviral vector system 35 Figure 5. Regulated transgene expression vectors 36 Figure 6. Lentiviral production and concentration 40 Figure 7. Lentiviral immunotherapy for cancer 44 Figure 8. Myelo-protection with MGMT-P140K 48 Figure 9. Bioluminescence reaction 55 Figure 10. Ventral images of murine hematopoietic stem cell transplant 64 Figure 11. Bioluminescence imaging of murine hematopoietic bone marrow cells 68 Figure 12. Lentiviral vector construct and transgene expression in cell lines 74 Figure 13. Bone marrow cell engraftment appeared in a cell-dose-dependent manner and became less active with time 78 Figure 14. BLI signal correlated to cell numbers 80 Figure 15. BG+TMZ treatment enhanced transgene expression in lethally irradiated and non-myeloablated recipients 84 Figure 16. Spleen of lethally irradiated recipient after in vivo selection 86 Figure 17. Transgene expression measured by BLI and flow cytometry at day 32 after transplantation 87 Figure 18. Semi-quantitative analysis of BLI signal showed increased engraftment and transgene expression after BG+TMZ treatment 89 3 Figure 19. Persistent BLI foci resulted from in vivo drug selection 92 Figure 20. Distribution of persistent foci in recipient animals and insertion analysis of CFU colonies of transduced bone marrow cells in vitro and after in vivo drug selection 95 Figure 21. Cryo-imaging of a whole adult mouse 110 Figure 22. Tricistronic lentiviral vector with MGMT-P140K, GFP and luciferase genes 112 Figure 23. Robust transgene expression in cell line over 7 weeks 113 Figure 24. Case Cryo-imaging system 114 Figure 25. GFP positive transplanted bone marrow cells after transplantation 116 Figure 26. Transgene positive cells in the liver of the recipient 118 Figure 27. Cryo-imaging samples are positive for transgene 119 Figure 28. Adjustment of auto-fluorescence in captured images 120 Figure 29. Correlation of BLI and cryo-imaging 121 Figure 30. 3-D reconstruction from cryo-images of BMT recipient 123 Figure 31. Schema of clonal analysis during in vivo drug selection 134 Figure 32. Enhanced engraftment of LV-PAGAL transduced bone marrow cells with BG+BCNU treatments 136 Figure 33. The method of LAM-PCR 137 Figure 34. Clonal diversity in peripheral blood reduced with drug treatment 139 Figure 35. LAM-PCR of GFP positive cells showed different clonality as in peripheral blood sample 141 4 Acknowledgments I would like to thank Dr. Stanton Gerson for giving me the opportunity to work in his lab, learning the cutting-edge technologies in the field of gene therapy, and for giving me the guidance necessary to develop the skill of critical thinking and understanding of scientific methods. I would also like to thank all members on my thesis committee for their time and for giving me crucial suggestions to my projects. I want to thank Dr. Eric Arts, for his advice on my projects and his service as my committee chair; Dr. Zhenghong Lee for his continuous support, and helps in reviewing manuscripts, and his helpful insight with various imaging studies; Dr. George Stark, for his valuable questions and suggestions on the projects; and Dr. Cheng-kui Qu for his encouragement and scientific input. I would also like to thank Perrin Cheung, David Prabhu, and Dr. David Wilson in the Department of Biomedical Engineering for helping me with early stages of bioluminescence imaging and continuous collaboration on cyro-imaging. Without them, these projects would not be able to get started. I would like to thank everyone in the Gerson lab. Jane Reese, Dr. Youngji Park, Dr. Justin Roth, and Dr. Colin Sweeney for their helpful and intelligent discussions and teaching throughout those years of my study, and also Dr. Yulan Qing, Jonathan Kenyon, Karen Lingas, Min Liu, Amar Desai, Jon Donze, Dr. Lily Liu for their scientific inputs and always friendly personalities, which made my life very enjoyable in the lab. I would also like to thank to all of my friends throughout those years for their friendships and generosity. 5 This work was supported by grants from the National Institutes of Health (NIH-NCI RO1-CA073062) and also in part supported by grants NIH-P30-CA43703 and NIH R24- CA110943. 6 List of Abbreviations AGT: alkyguanine DNA-alkyltransferase APC: antigen presenting cell ATCC: American Type Culture Collection ATP: adenosine triphosphate BCNU: 1,3-bis(2-chloroethyl)-1-nitrosourea BG: O6-benzyguanine BLI: bioluminescence imaging CA: capsid CCD: Charge-coupled device CCR5: C-C chemokine receptor type 5 CD: cytidine deaminase CFC: colony forming cells CMV: cytomegalovirus cPPT: central polypurine tract CT: computed tomography CXCR4: CXC chemokine receptor 4 DC: dendritic cell DHFR: dihydrofolate reductase DMEM: Dulbecco’s Modified Eagle Medium EtOH: Ethyl Alcohol FBS: fetal bovine serum FITC: fluorescein isothiocyanate 7 FL: Flt-3 ligand FOV: field of view GAPDH: glyceraldehyde 3-phosphate dehydrogenase G-CSF: granulocyte-colony stimulating factor GFP: green fluorescent protein H&E: hematoxylin and eosin HIV: human immunodeficiency virus HSC: hematopoietic stem cell HSV-TK: herpes simplex virus thymidine kinase IL-3: interleukin 3 IL-6: interleukin 6 IP: intra-peritoneal LAM-PCR: linear amplification mediated PCR Lin-: lineage negative LTCIC: long term culture initiating cells LTR: long terminal repeat LV: lentivirus MA: matrix MDR-1: multidrug-resistance protein 1 MGMT: methylguanine DNA-methyltransferase MLV: murine leukemia virus MND: MPSV promoter with negative control region deleted MOI: multiplicity of infection 8 MPSV: myeloproliferative sarcoma virus MRI: magnetic resonance imaging MRSI: magnetic resonance spectroscopic imaging MSC: mesenchymal stem cell NC: nucleocapsid NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells NLS: nuclear localization signal NMC: nucleated marrow cells NOD/SCID: non-obese diabetic/severe combined immunodeficiency OCT: optimal cutting temperature ORF: open reading frame PBS (1): phosphate buffered saline PBS (2): primer binding site PE: propidium iodide PET: positron emission tomography PIC: pre-integration complex PPT: polypurine tract RCL: replication competent lentivirus RNA: ribonucleic acid ROI: regions of interest RRE: rev responsive element SA: splice acceptor SB: Sleeping Beauty transposon system 9 SD: splice donor SCF: stem cell factor SIN: self-inactivating SIV: simian immunodeficiency virus SPECT: single photon emission computed tomography SRC: SCID repopulating cell SU: surface unit TAA: tumor-associated antigen TM: transmembrane unit TMZ: temozolomide TPO: thrombopoietin TP53: tumor protein p53 U3: unique 3’ region U5: unique 5’ region VSVG: vesicular stomatitis virus G protein WPRE: woodchuck hepatitis virus post-transcriptional regulatory element 10 In Vivo Imaging of Engraftment and Enrichment of Lentiviral Transduced Hematopoietic Bone Marrow Cells Under MGMT-P140K Mediated Selection Abstract by YUAN
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  • Large-Scale Analysis of Genome and Transcriptome Alterations in Multiple Tumors Unveils Novel Cancer-Relevant Splicing Networks

    Large-Scale Analysis of Genome and Transcriptome Alterations in Multiple Tumors Unveils Novel Cancer-Relevant Splicing Networks

    Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks Endre Sebestyén1,*, Babita Singh1,*, Belén Miñana1,2, Amadís Pagès1, Francesca Mateo3, Miguel Angel Pujana3, Juan Valcárcel1,2,4, Eduardo Eyras1,4,5 1Universitat Pompeu Fabra, Dr. Aiguader 88, E08003 Barcelona, Spain 2Centre for Genomic Regulation, Dr. Aiguader 88, E08003 Barcelona, Spain 3Program Against Cancer Therapeutic Resistance (ProCURE), Catalan Institute of Oncology (ICO), Bellvitge Institute for Biomedical Research (IDIBELL), E08908 L’Hospitalet del Llobregat, Spain. 4Catalan Institution for Research and Advanced Studies, Passeig Lluís Companys 23, E08010 Barcelona, Spain *Equal contribution 5Correspondence to: [email protected] Keywords: alternative splicing, RNA binding proteins, splicing networks, cancer 1 Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Abstract Alternative splicing is regulated by multiple RNA-binding proteins and influences the expression of most eukaryotic genes. However, the role of this process in human disease, and particularly in cancer, is only starting to be unveiled. We systematically analyzed mutation, copy number and gene expression patterns of 1348 RNA-binding protein (RBP) genes in 11 solid tumor types, together with alternative splicing changes in these tumors and the enrichment of binding motifs in the alternatively spliced sequences. Our comprehensive study reveals widespread alterations in the expression of RBP genes, as well as novel mutations and copy number variations in association with multiple alternative splicing changes in cancer drivers and oncogenic pathways. Remarkably, the altered splicing patterns in several tumor types recapitulate those of undifferentiated cells.