This thesis is a composition of two investigations. One study involved melanoma gene therapy using a well established HIV-1 based vector and dendritic cells (DCs), while the second study focused on the construction of a novel lentiviral vector for gene delivery to provide alternative vector systems to those based on HIV-1.
Chapter 2 is centred on the transduction of DCs with the replication defective HIV-1 based vector. The transgene of interest is murine tyrosinaserelated protein-2, mTRP-2, which has some significance in a clinically relevant animal model. This protein is a tissue specific self-antigen naturally expressed by normal melanocytes and recently was found to be a tumour-rejection antigen for B16 melanoma. This study showed that HIV-1 based lentiviral vectors were efficient vehicles for mediating transfer of foreign genes to DCs. Proper processing of the transgene, in terms of RNA transcription activity and protein expression, was verified with RT-PCR and specific antibody respectively. A PCR step to detect integration of the vector and replication competent retrovirus (RCR) is also discussed.
Chapter 3 focuses on the immunotherapeutic aspects of mTRP-2 modified DCs. Prophylactic treatment with the mTRP-2 gene modified DCs (DC-HR'CmT2) in C57BL/6 mice resulted in complete protection from tumour challenge. In the therapy model, the results showed DC-HR'CmT2 treated mice had a significantly better survival duration, which was notably improved when the tumour dose was lowered to the minimal tumourigenic portion (1x104 cells/mouse). 4 of 7 mice remained tumour-free for 80 days after tumour inoculation. Also, the presence of both CD4+ and CDS8+ cells was essential for optimal tumour protection during both the priming and challenge phase. Given the potential of DC-HR'CmT2 shown in this study, this mTRP-2 gene transfer to DC approach is potentially beneficial as prophylactic or therapeutic strategies for the therapy of melanoma, especially in the early stage of the disease.
Recent reports from studies with Human Immunodeficiency Virus-based vectors have shown efficient gene delivery to a variety of cell types and sustained gene expression in vivo in animals. However, it must be recognised that the HIV vectors are based on a human pathogen that causes Acquired Immune Deficiency Syndrome (AIDS). While the vector has been disabled of any pathogenic properties, it may be more prudent, practical and safer to use vectors derived from non-human retroviruses. In chapter 4 the development of delivery vectors based on a new, non-primate lentivirus, Jembrana Disease Virus (JDV), is described. Splicing PCR was used to amplify fragments from the JDV genome for the construction of a JDV transfer vector and a packaging construct. Disabled JDV viral particles pseudotyped with the vesicular stomatitis virus glycoprotein G (VSV-G) were produced by co-transfecting 293T cells with a VSV-G expressing plasmid. Viral titre was obtained by transducing the cells with the supernatants harvested from transfectants and determining the number of cells expressing the transgene. PCR was used to detect the presence of potential replication-competent viruses as well as transfer vector integration. The JDV-based vectors were shown to transduce and integrate into the chromosome of a range of primary and transformed cells. This nonprimate lentiviral vector system may be more readily accepted for human gene therapy, especially for genetic conditions.
In gene therapy, "multiplicity of infection (MOI)" is an expression used to describe the average number of viral vectors needed to transduce a single cell. It is probably an ill-suited term to use in this field of science. For lentiviral vectors, particularly the HIV-1 based vectors, the viral stocks are generally produced from a 293T cell line and the titre calculated by determining the number of foci (effect of the marker transgene) produced in a transformable cell line. The titre obtained this way obviously does not represent a good estimate of active vector concentration as it very much depends on vector transduction efficiency of the particular cell line used. Chapter 5 discusses the use of the terminology MOI and looks at different titres and MOIs that could be generated from a single vector preparation when transducing conditions, such as inoculum volume and target cell number were varied.
As a conclusion, lentiviral vectors can be used to efficiently transduce dendritic cells for antigen presentation and a lentiviral vector based on Jembrana Disease Virus may have significant advantages over vectors based on HIV-1 in terms of safety.