Modification of the E1-pIX region of the adenovirus 5 genome for use in cancer gene therapy
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Currently the use of adenoviruses in cancer gene therapy is limited by efficient delivery of the virus into the tumour cells, detargeting of the virus from the liver, and the efficient spread of the virus within the tumour. Rapid and easy modification of adenoviruses enables expression of different genes from the genome of an oncolytic virus. I developed a system where the E1-pIX region of the adenovirus 5 genome could be mutated via recombination of a recipient virus with the deleted E1-pIX region flanked by a loxP and an attB-site and an “addback” plasmid with the mutated E1-pIX region flanked by a loxP and an attP-site. The recipient virus was found not to be producible even on a pIX-complementing cell line. The pIX was further modified by fusing GFP, FCU1 and MMP7 to the C-terminus with a 2A sequence that enables the ribosome to skip one specific peptide bond enabling the expression of genes flanking this sequence. Two different 2A sequences were used: FMDV 2A (F2A) and PTV-1 2A (P2A). The pIX-P2A-GFP expressing virus was found to have similar heat stability, CPE, burst size and plaque size characteristics as the parental virus, whereas the pIX-F2A-GFP expressing virus was found to have reduced heat stability, CPE, burst size and smaller plaque size. The viruses expressing FCU1 and MMP7 were found only to be producible on a pIX-complementing cell line due to the low expression of pIX from these constructs. I concluded that 2A sequences can be used in the context of adenoviruses but optimisation of the sequence may be needed depending on the fusion partners.
Thesis, MPhil Master of Philosophy
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