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Production of more stable induced pluripotent stem cells using the Doggybone (dbDNA) vector.

Thornton, Christopher David (2020) Production of more stable induced pluripotent stem cells using the Doggybone (dbDNA) vector. Doctoral thesis (PhD), Manchester Metropolitan University.

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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 4 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.

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