Development of Herpesvirus saimiri as a gene therapy vector

The success of human gene therapy relies on efficient delivery and appropriate expression of therapeutic genes which will cure or slow the progression of a disease. However, current viral vectors do not possess the full complement of properties that are generally believed necessary in an ideal gene delivery system. Therefore, alongside attempts to improve current gene delivery vectors, the identification and evaluation of new viral vectors is crucial to the long term success of gene therapy. Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus, originally isolated from its natural host, the squirrel monkey. HVS is capable of infecting a range of human cell lines with high efficiencies, in particular human carcinoma cell lines. Moreover, the HVS viral genome does not integrate into the cellular genome and persists as a high copy number, circular, non-integrated episome which segregate to progeny upon cell division. This allows the virus to stably' transduce a dividing cell population and provide sustained transgene expression for an extended period of time in both in vitro and in vivo studies. Herein, I aim to further develop HVS-based vectors. At present, HVS vectors utilise heterologous promoters such as the IE hCMV promoter. However, these maybe unsuitable for long term expression in vivo, as promoter silencing has been observed in herpesvirus-based vector systems. Ideal regulatory regions would be functional when the herpesvirus genome is maintained as a latent episome. Recent analysis has identified an adjacent set of latency-associated genes encoding ORFs 71,:,,73, which are expressed from a common regulatory region upstream ofthe ORF 73 gene. In Chapter 3, I assess the potential of this regulatory region to provide heterologous gene expression in a wide variety of human cancer cell lines. I demonstrate, utilising transient transfection assays, that the ORF 73 regulatory region can provide transgene expression in a variety of human carcinoma cell lines. Furthermore, incorporation of the minimal ORF 73 regulatory region in a recombinant HVS-based vector provides sustained expression of the green fluorescent protein in both in vitro and in vivo environments. Moreover, analysis has demonstrated that this regulatory region is autoregulated by the ORF 73 protein itself. To further investigate this mechanism we have utilized the yeast 2-hybrid system to identify possible cellular proteins which are involved in the regulation of the ORF 73 promoter. In Chapter 4, I demonstrate that ORF 73 interacts with the cellular protein, p32 and triggers accumulation of p32 into the nucleus. Using reporter gene-based transient transfection assays we demonstrate that ORF 73 and p32 act synergistically to transactivate a number of heterologous promoter constructs and also upregulate the ORF 73 regulatory region. This suggests that p32 may have transcriptional regulatory activity. The binding of ORF 73 to p32 is mediated by an amino-terminal arginine-rich domain, which contains two functionally distinct nuclear localisation signals. The p32 binding domains are required for ORF73 transactivating abilities and for ORF73 to induce nuclear accumulation of p32. These results suggest that ORF 73 can function as a regulator of gene expression and p32 is involved in ORF73-dependent transcriptional activation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo