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The development of a novel S/MAR DNA vector platform for the stable, persistent and safe genetic engineering of dividing cells

Bozza, Matthias

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Abstract

The development of episomally maintained DNA vectors that are capable of providing safe, persistent and stable modification of cells whilst avoiding the risk of integration-mediated genotoxicity would provide a valuable tool for genetic research. DNA vectors harboring a Scaffold/Matrix Attachment Region (S/MAR) can provide persistent and robust transgene expression in human cancer cell lines which can be used in in vitro, in vivo and ex vivo studies. A prototype S/MAR DNA vector with which we initiated this study replicates episomally, remains unsilenced and unmethylated following the genetic modification of cells. Although, it showed great promise it does have significant limitations which restricts its application. The establishment rate of the original DNA vector is an inefficient passive process and the selection procedure is lengthy and often produces drug-resistant but non-expressing colonies generating a mosaic of cells with differential transgene expression. Thus, these vectors represent a reasonable tool for simple studies such as cell labelling with reporter genes but are not suitable for more sophisticated work such as gene-rescue experiments or for the genetic engineering of primary human cells. In this project, we have refined and enhanced the S/MAR DNA vector system. The range of next-generation DNA vectors that we have produced provide several advances over the original vectors. We have demonstrated that this new S/MAR DNA vector platform is more efficient and stable with improved efficiency in establishing stable cell-lines. We have also demonstrated that the persistence of transgene expression and the molecular integrity of the vector has been improved in a range of cancer cell lines as well as in primary human cells. We have used this next-generation of DNA vector to generate labelled cells suitable for in vitro and in vivo drug screening. We have also generated isogenic tumor models which provide insights into the mechanism of pancreatic cancer development by restoring crucial tumor suppressor genes to the cells without altering the molecular or biochemical integrity of the cells. Additionally, we have utilized the vector system to persistently modify a range of dividing cell types including primary mouse embryonic stem cells and embryonic fibroblasts and primary human fibroblasts. As an ultimate demonstration of the efficacy of this DNA vector we have used it to genetically modify human T-cells for immunotherapy and have demonstrated it to be capable of expressing transgenes in these cells for over 1 month with minimal toxicity. We have demonstrated that this novel class of DNA vector can be used to persistently modify every cell-type tested providing sustained and high levels of transgene expression whilst avoiding the risk of insertional mutagenesis induced by the random integration of genetic material with minimal impact to the cell.

Item Type: Dissertation
Supervisor: Angel, Prof. Dr. Peter
Date of thesis defense: 6 December 2017
Date Deposited: 19 Dec 2017 11:24
Date: 2018
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 420 English
600 Technology (Applied sciences)
610 Medical sciences Medicine
Controlled Keywords: Genetic Engineering, Safe, DNA Vector
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