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Thesis Submitted in Partial Fulfilment of the Requirements of Manchester Metropolitan University for the Degree of Doctor of Philosophy Thornton, Christopher David (2020) Production of more stable induced pluripotent stem cells using the Doggybone (dbDNA) vector. Doctoral the- sis (PhD), Manchester Metropolitan University. Downloaded from: https://e-space.mmu.ac.uk/626312/ Usage rights: Creative Commons: Attribution-Noncommercial-No Deriva- tive Works 4.0 Please cite the published version https://e-space.mmu.ac.uk Production of more stable induced pluripotent stem cells using the Doggybone (dbDNA) vector. C D THORNTON PhD 2020 1 Production of more stable induced pluripotent stem cells using the Doggybone (dbDNA) vector. Christopher David Thornton A thesis submitted in partial fulfilment of the requirements of Manchester Metropolitan University for the degree of Doctor of Philosophy Department of Life Sciences, Manchester Metropolitan University 2020 2 Abstract: The application of induced pluripotent stem cell (iPSC)-derived cells in clinical trials is in its infancy but the potential is vast. A key asset of iPSCs is the ability to derive autologous cell therapies, but to date most current or approved clinical trials are using fully characterized allogeneic or non-allogeneic cell banks alongside immunosuppressive drugs. Until now, all current or approved clinical trials utilize iPSC generated using EBNA1 expressing plasmids containing the OriP sequence to maintain a self-replicating episome. These vectors are amplified in bacterial hosts and contain bacterial DNA motifs recognized by the transfected cells innate and intrinsic interferon host defense responses. Moreover, the continued forced expression of the Epstein-Barr virus EBNA1 protein is known to cause widespread alterations in gene expression as well as elevated oxidative stress and DNA damage occurrence. Additionally, this method of iPSC derivation incorporates a short-hairpin RNA (shRNA) for the p53 protein; often referred to as the guardian of the genome. The shRNA functions to transiently silence the expression of p53 protein and has been demonstrated to result in an increased persistence of DNA damage in iPSC produced this way. All of these factors have significant implications for the safe clinical use of iPSC generated using oriP/EBNA1 plasmid episomes. The aim of my project was to investigate the function of a novel system in reprogramming and iPSC development. Doggybone DNA (dbDNA) vectors are free of oriP/EBNA1 sequences, bacterial motifs and are produced in a chemically defined, low endotoxin, cGMP compliant manufacture. My results describe efficient iPSC reprogramming by applying equivalent gene sequences transiently expressed from dbDNA vectors in protocols employing both animal-derived and animal-free constituents when using weight equivalents of both systems and not molecular equivalents. In direct comparator experiments with the current state-of-the-art gold standard oriP/EBNA1 episomes, dbDNA vectors produced iPSC colonies with the same efficiency but dbDNA-iPSC displayed evidence of greater stability in terms of maintenance of pluripotency. Differential transcriptomic evaluations by microarray showed that the persistence of oriP/EBNA1 episomes resulted in an elevated interaction with immune system processes and IFN signalling in iPSC when compared to dbDNA generated iPSC. Moreover, an increased susceptibility for DNA damage incitement alongside unwanted spontaneous differentiation in iPSCs incorporating the 3 oriP-EBNA1 were all demonstrated and showed to be intrinsically linked to one-and- other. We propose a potential that utilizing dbDNA vectors presents a safer and more stable approach to iPSC production and development and that this could, with further work, help to bridge the gap between iPSCs and their greater clinical translation. 4 Table of contents: 1.0 Introduction: ................................................................................................................ 13 1.1. Classification of stemness: .................................................................................... 13 1.1.1. Stemness in early embryogenesis: ....................................................................... 14 1.1.2. ESCs – derivation, roadblocks & recovery: ........................................................... 15 1.2. Induced pluripotent stem cells: .............................................................................. 18 1.2.1. History of iPSC development: ............................................................................... 18 1.2.2. Reprogramming: ................................................................................................... 24 1.2.2.1. Phases of reprogramming .................................................................................... 24 1.2.2.2. Transgene modifications: ..................................................................................... 26 1.2.2.3. Vectorology: ......................................................................................................... 28 1.2.2.3.1. Sendai virus (SeV) .............................................................................................. 30 1.2.3.3.2. mRNA: ................................................................................................................ 31 1.2.2.3.2. miRNAs: ………………………………………………………………………………..33 1.2.2.3.3. Recombinant proteins: ........................................................................................ 34 1.2.2.3.4. Transposon Vectors (PiggyBac & Sleeping beauty): ........................................... 35 1.2.2.3.5. Plasmid DNA: ...................................................................................................... 37 1.2.2.3.6. Minicircle:….. ....................................................................................................... 39 1.2.2.3.7. Episomal plasmid – oriP-EBNA1 system: ............................................................ 40 1.3. Doggybone (dbDNA) clinical grade vector:.......................................................... 50 1.3.1. Manufacture: ....................................................................................................... 50 1.3.2. dbDNA reprogramming systems: ........................................................................ 52 2.0. Results: ....................................................................................................................... 54 2.1. dbDNA vector functionality, reprogramming potential & Microarray analysis: ........ 54 2.1.1. Comparison of vector degradation kinetics by quantifying transgene expression: . 56 2.1.2. Functionality and structure of dbDNA reprogramming vectors: .............................. 65 2.1.3. Concurrent IPSC reprogramming experiments & vector efficiency: ....................... 68 2.1.4. Pluripotency characterization of dbDNA/oriP-EBNA1 produced iPSCs: ................. 78 2.1.5. Reprogramming experiment using Xenofree, cGMP compliant protocol: ............... 82 2.1.6. Reprogramming blood and urine-derived cells: ..................................................... 84 2.1.7. Global probed transcriptomic analysis of dbDNA/oriP-EBNA1 produced iPSCs. ... 92 2.1.8. OriP-EBNA1 over-represented gene analysis: .................................................... 103 2.1.8.1. STAT1 signalling and IFN-γ signalling: ............................................................... 107 2.1.8.2. Spontaneous differentiation of iPSCs: ................................................................ 110 2.1.8.3. DNA damage analysis: ....................................................................................... 113 2.1.9. Over-represented genes within dbDNA-iPSCs: ................................................... 124 5 2.1.9.1. Cell cycle analysis: ............................................................................................. 129 2.1.10. Summary: ........................................................................................................... 133 2.1.11. Further work: ....................................................................................................... 137 3.0. Discussion ................................................................................................................ 140 3.1. Somatic cell reprogramming, pluripotency induction and functional capacity: ...... 144 3.2. Retention of the dbDNA system and interactions with host cell innate immunity: . 149 3.3. EBNA1 protein and increased susceptibility for DNA damage: ............................ 157 3.4. dbDNA over-expressed genes and cell cycle analysis: ........................................ 160 3.6. Reprogramming of different primary cell types: ................................................... 161 3.7. Future work: ........................................................................................................ 166 3.8. Summary: ........................................................................................................... 167 4.0. Materials and Methods ............................................................................................. 166 4.1. Cell Culture: ......................................................................................................... 166 4.2. Cell culture methodologies: .................................................................................. 167 4.2.1. The culturing and inactivation of Mouse embryonic fibroblasts (MEFs):................ 167 4.2.2. Culturing and passaging of Pluripotent stem cells (PSCs): ................................... 168 4.2.3. Human Dermal Fibroblast (HDF)
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