Analysis of S/MAR vectors For Gene Therapy In Muscle

Muscular Dystrophy (MD) is a progressive muscle wasting disease which currently has no cure, and is caused by the mutation of the dystrophin gene. A multitude of approaches for the improvement of the muscular pathology caused by this condition are being investigated, one of which is gene therapy. This approach is used to deliver vectors containing therapeutic transgenes such as dystrophin to target muscle cells. One method of gene delivery utilises viral vectors, and although this has resulted in systemic delivery and efficient transgene expression, there are many safety implications which have led to the development of non-viral approaches, such as the direct delivery of naked plasmid DNA. However, the shortcomings of these vectors include an inability to replicate within host cells, resulting in the loss of vector as cells replicate, and the silencing of transgene expression. In an effort to overcome such limitations, a novel system called the ‘pEPI vector’ has been developed. Here, the inclusion of the β-IFN scaffold/matrix-attachment region (S/MAR) element into the open reading frame of an actively transcribed transgene has been found to lead to sustained, long term transgene expression, and to allow the episomal propagation and maintenance of the vector in dividing cells over many generations. The aim of this thesis was to investigate the potential of this vector for use as a gene therapy vector in muscle cells in order to treat MD.
In this study, the long-term expression of the eGFP reporter transgene inserted into the pEPI vector was evaluated, and the pEPI vector’s episomal/integrant status was investigated, in C2C12 murine myoblasts, HeLa, and HepG2 cell lines. 60 days after transfection the vector was found not to have integrated into the host genomes of any of the cell lines. Transgene expression had declined to nearly undetectable levels in fast-replicating C2C12 and HeLa cells, but was at high levels in the relatively slow-dividing HepG2 cells. An attempt to improve long term transgene expression in C2C12 cells by changing the promoter from CMV to CAGG still led to low transgene expression after 60 days.
To address this issue, this study focused on the development of a novel approach to ameliorate long-term transgene expression, based upon the origin of replication and nuclear matrix attachment properties of the S/MAR element, as well as the results obtained from testing the vector in the HeLa and HepG2 cells. It involved the arrest of C2C12 cells in the G0/G1 phase of the cell cycle post transfection with the pEPI vector in order to allow these fast-dividing cells an extended period of time to epigenetically mark pEPI prior to selection. The findings indicated that this novel method of pEPI vector establishment was superior to that which utilises selection alone. However, in spite of the improvement in long-term episomal transgene expression observed using this novel method of establishment of the pEPI vector, transgene expression levels were still relatively low after 35 days of cell proliferation, which led to the conclusion that further development of this vector is essential in order for it to be able to elicit a significant restoration of muscle function in MD patients.
Additionally, two other S/MAR vectors were tested in C2C12 cells. One vector contained the ‘mini-S/MAR’, a shorter version of the β-IFN S/MAR, and the other contained a novel S/MAR derived from the c-myc proto-oncogene. Transgene expression by either vector was nearly undetectable after several weeks of proliferation, and both were found to integrate into the C2C12 host genome, leading to the conclusion that not all S/MAR elements inserted within a plasmid vector can lead to long-term transgene expression, nor confer protection from vector integration.