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Genetic Mobility and Instability of Retroviral Vector in Vector Producer Cells for Gene Therapy Won-Bin Young Iowa State University

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Genetic Mobility and Instability of Retroviral Vector in Vector Producer Cells for Gene Therapy Won-Bin Young Iowa State University Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1999 Genetic mobility and instability of retroviral vector in vector producer cells for gene therapy Won-Bin Young Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Genetics Commons, Microbiology Commons, and the Molecular Biology Commons Recommended Citation Young, Won-Bin, "Genetic mobility and instability of retroviral vector in vector producer cells for gene therapy" (1999). Retrospective Theses and Dissertations. 12196. https://lib.dr.iastate.edu/rtd/12196 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. 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Bell & Howell Information and Leaming 300 North Zeeb Road, Ann ArtDor, Ml 48106-1346 USA 800-521-0600 Genetic mobility and instability of retroviral vector in vector producer cells for gene therapy by Won-BIn Young A dissertation submitted to the graduate faculty In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Co-majors: Molecular, Cellular and Developmental Biology: Nutritional Physiology Major Professors: Gary L. Lindberg and Charles J. Link, Jr. Iowa State University Ames, Iowa 1999 Copyright © Won-Bin Young, 1999. All rights reserved. DMI Number: 9941786 Copyright 1999 by- Young, Won-Bin All rights reserved. UMI Microform 9941786 Copyright 1999, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. tnvn 300 North Zeeb Road Ann Arbor, MI 48103 ii Graduate College Iowa State University This is to certify that the Doctoral dissertation of Won-Bin Young has met the dissertation requirements of Iowa State University Signature was redacted for privacy. CD Major Professor Signature was redacted for privacy. o Majc^ Professor Signature was redacted for privacy. Signature was redacted for privacy. Signature was redacted for privacy. For the Graduate College iii TABLE OF CONTENTS LIST OF FIGURES v LIST OF TABLES vil ABSTRACT vlii CHAPTER 1. GENERAL INTRODUCTION 1 Introduction 1 Dissertation Organization 11 References 12 CHAPTER 2. RESTRICTION MAPPING OF RETROVIRAL VECTOR MUTATION BY ANALYZING EPISOMAL DNA 19 Introduction 20 Materials and Methods 21 Results 22 Discussion 24 References 24 CHAPTER 3. DNA METHYLATION INCREASES GENETIC INSTABILITY OF RETROVIRAL VECTOR PRODUCER CELLS 33 Abstract 34 Introduction 34 Materials and Methods 37 Results 40 Discussion 48 Acknowledgements 53 References 54 iv CHAPTER 4. CHIMERIC RETROVIRAL HELPER VIRUS AND PICORNAVIRUS IRES SEQUENCE TO ELIMINATE DNA METHYLATION FOR IMPROVED PACKAGING CELLS 80 Abstract 81 Introduction 82 Materials and Methods 84 Results 87 Discussion 92 Acknowledgements 94 References 94 CHAPTER 5. HIGH FREQUENCY RETROTRANSPOSITION OF RETROVIRAL VECTOR IN CULTURED VECTOR PRODUCER CELLS 114 Abstract 115 Introduction 116 Materials and Methods 118 Results 121 Discussion 126 Acknowledgements 128 References 129 CHAPTER 6. GENERAL CONCLUSIONS 142 References 148 APPENDIX A. HUMAN RETROVIRAL PACKAGING CELLS BASED ON A CHIMERIC HELPER VIRUS FOR HIGH VECTOR PRODUCTION 151 ACKNOWLEDGEMENTS 154 V LIST OF FIGURES CHAPTER 1 Figure 1. Retrovlrus-mediated gene transfer 2 Figure 2. Reverse transcription and recombination 4 Figure 3. Endogenous reverse transcriptase activity and retroelements 6 Figure 4. Env-receptor interference and superinfection of vectors on vector producer cells 8 Figure 5. Intracellular retrotransposition of an Env-defective Moloney murine leukemia virus 9 CHAPTER 2 Figure 1. Detection of second vector from LTKOSN.2 vector production 28 Figure 2. Location of episomal DNA in vector producer cells 30 Figure 3. Characterization of HSVtk deleted LTKOSN (ALTKOSN) by episomal DNA restriction mapping 32 CHAPTER 3 Figure 1. Increased copy number of LTKOSN and ALTKOSN is observed in LTKOSN.2 subclones 64 Figure 2. Genetic instability of integrated LTKOSN in VPC genome 66 Figure 3. Differential gene expressions of vectors and helper virus in LTKOSN.2 VPC subclones 68 Figure 4. Superinfection susceptibility of LTKOSN.2 VPC subclones 70 Figure 5. DNA methylation statuses of helper virus and vectors 72 Figure 6. Treatment with 5-aza-C partly restores the vector production ability in subclones 73 Figure 7. Treatment with 5-aza-C reverses the DNA methylation of helper virus 75 Figure 8. Increased DNA methylation in helper virus of subclone #4 over time 77 vi Figure 9. Transcription activity of fielper virus and retroviral vectors in LTK0SN.2 VPC subclone #4 79 CHAPTER4 Figure 1. Construction and cap-independent translation mechanism of chimeric pAM3-iRES-Zeo helper virus 103 Figure 2. Gene expression of pAM3-IRES-Zeo and vectors in LTK0SN.2 VPC subclones 105 Figure 3. DNA methylation of helper virus 5' LTR over time with and without Zeocin™ selection 107 Figure 4. The effectiveness of Zeocin selection on helper virus gene expression in AM IZ cells over time 109 Figure 5. Gene expression of pAM3-IRES-Zeo helper virus and LEIN vector in AMIZ cells transfected with LEIN vector 111 Figure 6. Drug selection eliminates DNA methylation of helper virus and vector from VPC population 113 CHAPTER 5 Figure 1. Retrotransposition assay with pELNIH vector 137 Figure 2. Retrotransposed ELNIH vector integrated into different chromosomal locations 139 Figure 3. Scheme of serial deletion constructs of MoMLV 141 CHAPTER 6 Figure 1. Scheme of cascade reactions initiated by increased DNA methylation of helper virus 5' LTR 144 Figure 2. Alternative pathway of retroviral replication 147 APPENDIX A Figure 1. Variable sensitivity to human serum of vectors produced from different packaging cells 153 VII LIST OF TABLES CHAPTER 3 Table 1. Titer of LTKOSN.2 sub-clones treated with 5-aza-C 62 Table 2. Decreased titer of LTKOSN.2 VPC subclone #4 in continuous cell culture 62 CHAPTER 4. Table 1. Titer of LTKOSN.2 VPC subclones before and after transfection of pAM3-IRES-Zeo helper virus 101 Table 2. Increased resistance of superinfection by Zeocin™ selection 101 Table 3. Titer of AMIZ cells transfected with LEIN vector with continuous drug selection 101 CHAPTER 5. Table 1. Retrotransposition frequencies of pELNIH vector in cultured VPC 135 Table 2. Retrotransposition frequencies of human L1 retroelement (pJMIOI) in VPC 135 viii ABSTRACT A primary biosafety issue of retroviral vector-mediated gene therapy is the genetic instability of retroviral vectors. Reverse transcription of vector RNA genome is initiated by viral reverse transcriptase (RT) in a virion particle after infection of a target celi. During reverse transcription, abnormal template switches between vector and occasionally co- packaged helper virus in a virion particle can therefore enable helper virus to regain replication elements from the vector and revert to replication-competent retrovirus (RCR). This research was undertaken to study the origins of RT enzyme activities and test the hypothesis that RT enzyme activities are contributed by both exogenous RT, which is imported by the re-infection of vector virions, and endogenous RT, which is provided by Intracellular vector virion particles. Superinfection of vector in vector producer cells (VPC) can increase the number of integrated vectors in VPC, and is mainly caused by decreased Env-receptor interference, a consequence of helper virus gene inactivation by host DNA methylation at the 5' LTR promoter region. Suppression of helper virus gene expression by DNA methylation also reduces vector production. A chimeric retroviral helper virus combined with picomavirus IRES (internal ribosome entry site) sequence and a selection marker was therefore constructed to eliminate DNA methylated helper virus from cell populations to maintain active gene expression and enhance Env-receptor interference. Packaging cells established by this chimeric helper virus exhibit high titer production
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