Doi, K; (2017) A Novel Lentiviral Vector Model to Investigate the Mechanism of Insertional Mutagenesis by Aberrant Splicing. Doctoral thesis , UCL (University College London).

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Abstract

Retrovirus vector-mediated insertional mutagenesis (IM) can lead to serious cancerous risks in gene therapy. Although multiple cases of IM-induced malignancies have been reported in clinical trials with gammaretroviral vectors, no evidence for malignancies has been reported with HIV-derived lentiviral vectors (LVs). However, the 2010 clinical trial for ß-thalassaemia had reported the clonal dominance of erythroblasts in one patient. This was caused by the LV integration into the HMGA2 gene locus and the up-regulation of HMGA2 protein by the formation of host-vector chimeric transcripts (Cavazzana-Calvo et al., 2010). The observed pattern of splicing in the HMGA2 locus, splice-in, is splicing from a host splice donor to a vector splice acceptor. Considering that this literature is the first and only evidence that implies mutagenicity of LVs, We thought that formation of splice-in fusion transcripts could be a key factor to contribute potential LV-mediated IM. Therefore, we set our aim of investigating the mechanism of splice-in caused by vector integration by designing a novel LV model that can induce splice-in fusion transcripts, and by applying to an in vitro IM assay previously established by our group (Bokhoven et al., 2009). The IM assay utilise IL-3 dependent Bcl15 cell transformation into IL-3 independent, which can be analysed for the transformation mechanism via LV integration. Phenotypic analysis of the isolated mutant clone (the IL-3I clone) suggested secretion of autocrine factors and the cause of which was interrogated by molecular analysis. Additionally, the IL-3I clone and two bulk populations from different steps of an IM assay were subjected to RNA sequencing (RNA-Seq) to compare the isolated host genes and investigate chimeric mRNAs. Importantly, RNA-Seq identified the Angpt1 fusion transcript but in the splice-out form using a cryptic vector SD as a possible cause for cell transformation of the IL-3I clone. This gene locus has Retroviral Tagged Cancer Gene Database (RTCGD) hits and was also detected by LM-PCR. A variety of splice sites were used in the detected possible fusion mRNAs. To confirm, Angpt1 loci is the major cause of IL-3 independence and the pattern of splice sites use depend integration site; further analysis is required and discussed in the final chapter.

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