Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607515
Title: A study of mechanisms of genotoxicity in mammalian cells by retrovirus vectors intended for gene therapy
Author: Reja, Safia
Awarding Body: Brunel University
Current Institution: Brunel University
Date of Award: 2013
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Abstract:
Retrovirus gene therapy vectors can deliver therapeutic genes to mammalian cells in a permanent manner by integrating their genome into host chromosomes and therefore provide the potential for long term therapeutic gene expression. Retrovirus integration, however, can be oncogenic. Apart from insertional mutagenesis (IM) genotoxicity may be caused by other factors including DNA damage following infection and integration and epigenetic effects related to incoming viral particles. Thus, using retrovirus and lentivirus infected murine tumour tissue and infected cell lines in vitro this thesis was directed at investigating whether virus infection and integration could cause genotoxicity by alternative route(s) other than IM. Using clonally derived liver tumours that developed in mice, and normal liver and kidney tissues, following EIAV and HIV delivery in utero, comparative genome hybridisation methodology was used to examine for copy number variation. This showed amplification and deletions only in EIAV derived tumours. Real time Q-PCR analysis was then used to measure gene expression changes relating to genes contained within or near to amplifications observed in two tumours of individual mice. The STRING database was then used to find networks linking genes with differential expression profiles and genes in one of these tumours identified with provirus insertions that were also differentially expressed. These data provided preliminary data implicating a role for LV in Hepatocellular carcinoma (HCC). DNA damage is known to cause chromosomal instability that can lead to tumour development. The relationship between double strand breaks (DSB) and virus infection was also investigated in-vitro to find alternative routes to genotoxicity other than IM. Cell viability analysis demonstrated cells with a defective DNA damage response (DDR) have decreased cell viability compared with cells with intact DDR when infected with RV or LV vectors. DSB assays showed RV and LV infection to generate foci over a 6 hour period followed by DDR. Where no viral integrase is present, no DDR appears, however, where the vector is used with or without a genome to infect cells, DDR occurs as shown by the presence of 53BP1 foci indicative of DNA damage. The relationship between DNA damage and methylation was also investigated. Global methylation was found elevated in the genomic DNA of LV and RV infected cells and not in control uninfected cells. In contrast, methylation changes were not found in infected cells lacking the NHEJ repair pathway. These data suggest the DNA damage response is linked to genome methylation. The E2F transcription factor plays a key role in regulating expression of genes known to control oncogenesis and cancer, and E2F is regulated by methylation of its related target gene promoters. Taking into account all genes in the human genome the number of genes that bind E2F is 32.77%. However, using microarray to represent genes differentially expressed after infection, 59% of these were E2F targets. Overall, taking the data obtained in this thesis into account it may be suggested that RV and LV infection causes a number of potentially related changes to cells that include DNA damage and repair and methylation changes that could influence E2F that is an important factor involved in oncogenesis. Combining this with IM, attenuated RV and LV currently in use for gene therapy may cause genotoxicity to infected cells and increase the risk of oncogenesis especially where DNA damage is not correctly repaired. Further work is required to show in greater detail the extent of this genotoxicity, possible by whole genome sequencing of treated host genomes or cell transformation assays linked to the genotoxicity assays presented here. Collectively these data show that alternative factors to IM might exist that could act independently or synergistically to IM.
Supervisor: Themis, M. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.607515  DOI: Not available
Keywords: Genoxtoxicity ; Retrovirus ; Gene therapy ; Double strand breaks ; Vector
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