Lentiviral vectors, derived from HIV-1, are promising tools for gene therapy. Recent clinical trials have demonstrated the translation of their effectiveness in laboratory studies to clinical trials. However there are still limitations, relating to vector safety and efficiency of production, that could confine their future use. I investigated the ability of lentiviral vectors to perturb cellular gene expression by insertional mutagenesis (IM), using an in vitro model that detects aberrant splicing from lentiviral vectors to the growth hormone receptor gene (Ghr). The lentiviral vector pHV with full long terminal repeats (LTRs) and an internal spleen focus forming virus promoter (SFFV), was previously found to activate Ghr expression by a fusion mRNA transcript initiated in the HIV LTR (46). I extended this discovery to show that the SFFV promoter was enhancing expression from the HIV LTR, leading to IM (269). Application of our in vitro IM assay to potential clinical lentiviral vectors revealed that the novel ‘UCOE’ (ubiquitously acting chromatin opening element) promoter, within a selfinactivating (SIN) lentiviral vector, could drive UCOE-Ghr mRNA transcripts, causing IM. Mutation of splice donor sites in UCOE alone was insufficient in abrogating IM, however internal deletions around these splice donor sites were more successful. In other work, I made a packaging cell line for lentiviral vectors by stably expressing rev and a modified RD114 env (RDpro) in a cell line expressing HIV gag-pol. This led to the isolation of 57R10E, a cell line that made a titer of over 104 infectious units per ml when a SIN lentiviral vector was transiently or stably expressed. Taken together, this work will broaden the application of lentiviral vectors in clinical gene therapy by reducing both the chances of adverse events and the costs associated with vector production.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:625810 |
| Date | January 2012 |
| Creators | Knight, S. B. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1346459/ |
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