Precise and Sustained Gene Silencing in CD4+ Cells Using Designer Epigenome Modifiers As a Therapeutic Approach to Treat HIV Infection
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Precise and sustained gene silencing in CD4+ cells using designer epigenome modifiers as a therapeutic approach to treat HIV infection Inaugural-Dissertation to obtain the Doctoral Degree Faculty of Biology, Albert-Ludwigs-Universität Freiburg im Breisgau presented by Tafadzwa Mlambo born in Harare, Zimbabwe Freiburg im Breisgau February 2018 Dekanin: Prof. Dr. Bettina Warscheid Promotionsvorsitzender: Prof. Dr. Andreas Hiltbrunner Betreuer der Arbeit: Dr. Claudio Mussolino Referent: Prof. Dr. Toni Cathomen Ko-Referent: Prof. Dr. Peter Stäheli Drittprüfer: Dr. Giorgos Pyrowolakis Datum der mündlichen Prüfung: 27.04.2018 iii Table of Contents TABLE OF CONTENTS TABLE OF CONTENTS III ABSTRACT VIII 1. INTRODUCTION 11 1.1 HIV BURDEN AND EPIDEMIOLOGY 11 1.2 HIV LIFE CYCLE AND TROPISM 12 1.3 HIV TREATMENT 14 1.4 CCR5 AND CXCR4 AS TARGETS OF ANTI-HIV THERAPY 15 1.5 DESIGNER NUCLEASE TECHNOLOGY 17 1.5.1 Zinc finger nucleases 19 1.5.2 Transcription activator-like effector nucleases 20 1.5.3 RNA-guided endonucleases 21 1.6 HIV GENE THERAPY 22 1.7 OFF-TARGET EFFECTS 25 1.8 DELIVERY 27 1.9 EPIGENETIC REGULATION 30 1.9.1 Gene expression 30 1.9.2 Transcriptional regulation of gene expression 31 1.9.3 Targeted transcription activation 32 1.9.4 Targeted transcription repression 33 1.9.5 Epigenome editing 36 1.9.6 DNA methylation 38 iv Table of Contents 1.9.7 Designer epigenome modifiers 40 1.10 AIM AND OBJECTIVES OF PHD THESIS 42 2. MATERIALS AND METHODS 44 2.1 STANDARD MOLECULAR BIOLOGY METHODS 44 2.1.1 Restriction digest 44 2.1.2 Ligation 44 2.1.3 Polymerase Chain Reaction (PCR) 44 2.1.4 Transformation of chemically competent E.coli cells 45 2.1.5 Colony PCR 45 2.1.6 DNA extraction from bacteria (Mini and Midi preparations) 46 2.1.7 Agarose gel electrophoresis 46 2.1.8 PCR purification 47 2.1.9 Gel purification 47 2.1.10 Oligo cloning 47 2.1.11 Gibson Assembly 47 2.2 GENERATION OF THE TALE-BASED DESIGNER TRANSCRIPTION FACTORS 48 2.2.1 Generation of the TALE arrays 48 2.2.2 Generation of the TALE-based designer transcription activators and repressors 48 2.2.3 Generation of DEM expression plasmids 49 2.2.4 Cloning of the Firefly Luciferase reporter 53 2.2.5 Cloning of the EGFP reporter 54 2.3 MRNA PRODUCTION 55 2.3.1 Plasmid linearization 55 2.3.2 In vitro transcription 55 2.4 CELL CULTURE METHODS 56 2.4.1 Culture conditions 56 v Table of Contents 2.4.2 DNA PEI transfection 57 2.4.3 RNA lipofection 58 2.4.4 Dual Luciferase assay 58 2.4.5 Virus production and transduction 59 2.4.6 Generation of reporter cells 60 2.4.7 Reactivation 60 2.4.8 Flow cytometry 61 2.5 PRIMARY CELL CULTURE 62 2.5.1 PBMC extraction from LRS Chamber 62 2.5.2 MACS Isolation of CD4+ cells from PBMCs 63 2.5.3 Thawing and activation of primary human CD4+ T cells 64 2.5.4 Nucleofection of primary CD4+ T cells 65 2.6 EPIGENETIC AND EXPRESSION ANALYSES 66 2.6.1 Bisulfite sequencing 66 2.7 IN SILICO PREDICTION OF DEM #6 OFF-TARGET SITES 68 2.7.1 Bisulfite sequencing via Next Generation Sequencing 69 2.7.2 RNA isolation 71 2.7.3 Reverse transcription 72 2.7.4 Quantitative real-time PCR analysis 72 2.8 CHROMATIN-BASED STUDIES AND WHOLE TRANSCRIPTOME ANALYSIS 73 2.8.1 Chromatin immunoprecipitation 73 2.8.2 ATAC-seq 77 2.8.3 RNA-seq 78 2.9 STATISTICAL ANALYSIS 79 3. RESULTS 80 3.1 FUNCTIONALITY OF THE DESIGNER TRANSCRIPTION ACTIVATORS 81 vi Table of Contents 3.1.1 CCR5 target sites and Firefly Luciferase-based reporter 81 3.1.2 Functionality and synergy of the DTAs 82 3.2 GENERATION OF A GFP-BASED REPORTER AND REPORTER CELL LINE 85 3.3 REPRESSION OF CCR5 EXPRESSION IN A REPORTER CELL LINE 87 3.4 GENERATION OF THE DEM CONSTRUCTS 89 3.5 MRNA PRODUCTION VIA IN VITRO TRANSCRIPTION 90 3.6 LONG-TERM REPRESSION OF EGFP EXPRESSION IN A REPORTER CELL LINE 92 3.6.1 mRNA vs DNA delivery of DEMs resulted in potent silencing of gene expression 93 3.7 DNA METHYLATION ANALYSIS IN A REPORTER CELL LINE 94 3.8 DEM-INDUCED GENE SILENCING IN HEK293T CELLS 98 3.9 DEM-INDUCED GENE SILENCING IN CD4+ CELLS 101 3.9.1 Multiplex gene silencing in CD4+ cells 105 3.9.2 DNA methylation analysis in CD4+ cells 106 3.9.3 DEM-mediated histone modifications 109 3.10 SPECIFICITY PROFILE OF THE DEMS 110 4. DISCUSSION 116 4.1 TRANSCRIPTIONAL REGULATION ACHIEVED WITH THE DESIGNER TRANSCRIPTION FACTORS IS MODEST AND TRANSIENT 116 4.2 DEMS MEDIATE POTENT, STABLE GENE SILENCING ACCOMPANIED BY DNA METHYLATION 120 4.3 DEMS EXHIBIT FUNCTIONALITY AT CLINICALLY RELEVANT, ENDOGENOUS GENES 123 4.4 DEMS EXHIBIT A BENIGN SPECIFICITY PROFILE 129 4.5 THE RELEVANCE OF DEM-BASED APPROACHES FOR HIV GENE THERAPY 132 5. REFERENCES 137 6. APPENDIX 149 vii Table of Contents 6.1 LIST OF ABBREVIATIONS 149 6.2 LIST OF FIGURES 154 6.3 LIST OF TABLES 155 6.4 SUPPLEMENTARY MATERIALS AND METHODS 156 6.5 SUPPLEMENTARY TABLES 159 6.6 CURRICULUM VITAE 198 6.7 ACKNOWLEDGMENTS 200 6.8 DECLARATION 201 viii Abstract ABSTRACT The human immunodeficiency virus (HIV) is a major global health burden which has claimed over 25 million lives in the past 30 years. Although the current treatment strategies have proven to be effective, several concerns remain to be addressed. The CCR5 and CXCR4 co-receptors are necessary for HIV entry into host cells and have therefore gained interest as possible targets for therapeutics against HIV infection. Designer nuclease technology has not only revolutionized the fields of biology and medicine but has also provided tools which enable the specific targeting and inactivation of the CCR5 and CXCR4 genes. However, nuclease-based approaches are associated with significant off-target effects and improving their safety remains an important consideration. Transcriptional repression via epigenetic modification provides a viable alternative to nuclease-mediated gene knock-out and may represent a safer approach as the genomic sequence remains unchanged and the severity of off-target effects may therefore be reduced. To investigate this, designer epigenome modifiers (DEMs) were generated by fusing transcription activator-like-effector (TALE)- based DNA binding domains to effectors capable of inducing gene silencing through DNA methylation and by altering chromatin conformation. DEM constructs targeted to the CCR5 and CXCR4 genes were tested in a reporter cell line harbouring an integrated EGFP expression cassette under control of the CCR5 proximal promoter or in HEK293T cells respectively. EGFP silencing was observed in 80% of the cells and up to 65 days following the delivery of a CCR5-specific DEM. Silencing was associated with up to 80% CpG methylation with minimal spreading from the target site. Furthermore, EGFP expression could be restored via treatment with the non-specific demethylating agent 5-AZA thus demonstrating the reversible nature of DEM-induced silencing. CXCR4-specific DEMs induced a 3.4- and 1.8-fold reduction in CXCR4 mRNA and transcript levels respectively, ix Abstract as measured by quantitative RT-PCR or flow cytometry. Silencing was again accompanied by up to 22% CpG methylation demonstrating that DEM-induced silencing is retained following cell division. To highlight the translational potential of DEMs, their functionality was tested in CD4+ primary T cells. Four days post-delivery, CCR5-specific DEMs resulted in 1.8- or 1.6-fold reduction in CCR5 transcript or protein levels, respectively whereas moderate silencing was observed for CXCR4. Evaluation of the cells at a second time-point 18 days post-transfection revealed a 1.6-fold reduction both in CXCR4 transcript and protein levels whereas CCR5 silencing had been lost, most likely due to the T cell-culturing conditions. Next-generation bisulfite sequencing carried out for CCR5 revealed significant DNA methylation within 5 kb of the target site. Similarly, up to a 12-fold increase in methylation was observed at the CXCR4 gene. Silencing of both genes was associated with the H3K9me3 repressive histone modification as evaluated via chromatin precipitation. To address the safety concerns associated with designer nuclease technology, whole transcriptome analysis via RNA-seq was carried out following delivery of a CCR5-specific DEM into CD4+ cells. In addition to CCR5, 84 genes showed differential expression (more than 1.5-fold) but an evaluation of potential DEM off-target sites revealed that this was not as a result of DEM off-target binding. To evaluate possible changes in chromatin accessibility, ATAC-seq was carried out in cells which received the CCR5-specific DEM. Reduced chromatin accessibility was observed in the region in direct proximity to the target site. In addition, 324 additional sites showed reduced chromatin accessibility but these regions did not correlate with the differentially-expressed genes identified via RNA-seq. Finally, evaluation of DNA methylation at the top 10 computationally-predicted off-target sites revealed no significant differences between DEM- and control-treated cells for all but one site which was later shown to be intergenic and inaccessible in CD4+ cells. Therefore, x Abstract this study establishes DEMs as a powerful epigenome editing tool and provides a possible next step towards developing a safe therapy to treat HIV infection. 11 Introduction 1. INTRODUCTION 1.1 HIV BURDEN AND EPIDEMIOLOGY The human immunodeficiency virus (HIV) is a major global health concern and it is estimated that approximately 36.7 million people were living with HIV and 1 million people had died of acquired immune deficiency syndrome (AIDS)-related illnesses in 2016 (1) (Figure 1.1).