Cellular carriers of viral vectors for turmour selective targeting of cancer gene therapy

• Main Author: Naik, Jay Dolatrai
• Published: University of Leeds 2009
• Subjects: 616.99406
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505080

Cancer gene therapy holds promise for patients, yet key issues involving the delivery of vector and achieving tumour selective cytotoxicity, has limited progress over recent years. In this thesis a combination of cell-based carriers, viral vectors and tumour selective promoters are assessed to tackle these two important issues directly. Initially two retroviral systems were considered: intracellular carriage of a rapamycin-inducible retrovirus and extracellular carriage ('hitchhiking') of a self-inactivating (SIN) retroviral vector. Both systems controlled transgene expression using the tumour selective human telomerase reverse transcriptase and telomerase RNA promoters (hTERTp & hTRp). Direct transduction of target cells with SIN marker virus, expressing enhanced Green Fluorescent Protein (eGFP) under the internal control of human telomerase reverse transcriptase promoter (hTERTp) demonstrated similar activity to a positive control virus, with some evidence of telomerase selectivity. Hitchhiking of the same SIN marker vectors also mediated eGFP expression in target cells. In contrast the therapeutic SIN vectors containing suicide transgenes did not achieve a reliable direct telomeraseltumour selective cytotoxic effect, with no discernible toxicity seen with hitchhiked vector. The rapamycin-inducible vectors in contrast to the SIN vectors only demonstrated retroviral genome expression following single cell cloning of a positive-control producer cell line. The retrovirus from this producer successfully mediated low-level target cell eGFP expression, however the data did not support development of therapeutic vectors in,corporating either telomerase promoter. Incorporating oncolytic viruses into the system allowed in vitro therapy. An eGFP expressing oncolytic vesicular stomatitis virus (VSV) and wild-type reovirus, showed direct cytotoxicity against a range of target cell lines. T-cell and non-T-cell peripheral blood mononuclear cell (PBMC) fractions were resistant to both viruses indicating their suitability as oncolytic viral carriers. Finally VSV was successfully hitchhiked on donor T-Iymphocytes leading to the release of VSV .and widespread replication within the malignant Molt-4 cell line.