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Abstract

Pluripotent stem cells are considered a prime source of cells for regenerative therapies and gene therapy applications because of their extensive proliferation, the potential for self-renewal and their capability for multi-lineage differentiation. A great advantage of induced pluripotent stem cells (iPSCs) is their derivation from a patient’s somatic cells, which can be isolated using non-invasive techniques, thus eliminating not only ethical concerns associated with embryonic stem cells but also the risk of immune rejection. Therefore, iPSCs are an attractive tool for personalised medicine, drug screening and to generate disease models. Typically, the modification of pluripotent cells is done by using integrating viral vectors. Although vectors based on modified viruses are unquestionably the most effective gene delivery systems in use today, their efficacy at gene transfer is, however, tempered by their potential integration and genotoxicity. Non-viral DNA vectors are attractive alternatives to viral gene delivery systems because of their low toxicity, relatively easy production and great versatility. However, their efficiency is still regarded as below the requirements for realistic in vivo gene therapy due to deficient delivery exacerbated by the merely transient gene expression of plasmid DNA in vivo.

Thus, the development of safer, more efficient and easily and economically prepared persistently expressing genetic vectors remains one of the main strategic tasks of gene therapy research and is the crucial prerequisite for its successful clinical application. An ideal vector for the genetic modification of cells should deliver sustainable therapeutic levels of gene expression without compromising the viability of the host in any way. Permanently maintained, episomal and autonomously replicating DNA vectors, which comprise entirely human elements, might provide the most suitable method for achieving these goals.

This thesis presents the development of a non-viral, non-integrating and autonomously replicating DNA vector system based on the use of a Scaffold Matrix Associated Region (S/MAR), for the persistent genetic modification of differentiating and dividing cells, including but not limited to murine and human Stem Cells (SCs). Although this DNA Vector is among the best of its class, one of its limitations is that as it is produced in bacteria it comprises a large proportion of bacterial sequences which are unnecessary and undesirable for clinical application. Accordingly, the vector system has been refined, updated and all aspects of its functionality have been improved whilst also reducing its impact on cells following its delivery, resulting in higher levels of more sustained expression than previous versions. Molecular and genetic analysis of S/MAR-labelled cells revealed that the vectors are kept at low copy numbers, are present in their episomal forms and do not modify or genetically damage the cells or their progeny, as the cells fully retain their pluripotent capabilities and are able to generate chimeric mice. This new vector system is also used to generate iPSCs from murine or patient-derived fibroblasts.

For the first time, this work shows that genetic modification with this DNA vector system provides robust transgene expression which is sustained through the reprogramming and differentiation process in vitro and in vivo.