RNAi against Virally-Induced Disease

Norliana Khairuddin (2010). RNAi against Virally-Induced Disease PhD Thesis, Diamantina Institute for Cancer, Immunology and Metabolic Medicine, The University of Queensland.

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Author Norliana Khairuddin
Thesis Title RNAi against Virally-Induced Disease
School, Centre or Institute Diamantina Institute for Cancer, Immunology and Metabolic Medicine
Institution The University of Queensland
Publication date 2010-09
Thesis type PhD Thesis
Supervisor A/P Dr Nigel McMillan
Dr Wenyi Gu
Dr Graham Leggatt
Total pages 184
Total colour pages 27
Total black and white pages 157
Subjects 06 Biological Sciences
Abstract/Summary RNA interference (RNAi) using synthetic small RNA molecules to elicit gene knockdown has been a popular tool of choice in determining gene function. Increased understanding of the molecular mechanisms behind RNAi has allowed the development of these RNA molecules for gene therapy. In particular, small interfering RNAs (siRNAs) have been the most successfully translated into the clinic, with an ongoing Phase II clinical trial using siRNA against vascular endothelial growth factor (VEGF) to treat age-related macular degeneration (AMD). However, the Achilles‟ heel of using RNAi-based therapy is the need to determine a target gene of interest which is specific in causing a particular disease. This has been overcome in certain cases through advancements in linking genes to diseases, for example Bcl2 for melanoma and oncogenes such as the Human Papillomavirus (HPV) E6 and E7 for cervical cancer. Furthermore, in the case of virally-induced cervical cancer, maintenance of the cancer state depends on the continued expression of E6/E7. Suppression of these oncogenes has been shown to cause cells to undergo apoptosis or senescence, therefore targeting E6/E7 could potentially lead to cancer cell clearance and reduction of tumours. Although originally thought to not trigger innate immune responses, siRNAs have been shown to demonstrate immunostimulatory abilities through recognition by Toll-like receptors (TLRs). Such siRNA-induced immunostimulation would cause induction of pro-inflammatory cytokines, hence bridging innate and adaptive immune responses. TLR activation leads to the induction of Type I interferons, for example IFNα, which has been used clinically as immunotherapy against cancer. Thus, siRNAs that function to both silence target gene and activate immune responses, provide a single molecule approach with bifunctional action, that is beneficial in the development of an RNAi-based therapy against virally-induced disease such as cervical cancer. In this study, a panel of Dicer substrate siRNAs (D-siRNAs) against HPV16 E6/E7 was designed to theoretically cause target gene knockdown and siRNA-induced immunostimulation. Screens on the functionality of these D-siRNAs, however, showed that modification to the conventional siRNA structure alone is not significant; rather, specific sequences were found to be better targets for knockdown. Nonetheless, D-siRNAs were shown to be able to knockdown E6/E7 mRNA levels up to 93%, and E7 protein levels up to 96% compared to scrambled treated cells. Transfection of cells with D-siRNAs was observed to induce p53; however this induction was not concurrent with E6/E7 knockdown. On the other hand, reduced E6/E7 knockdown was shown to cause cells to undergo senescence and also caused reduced colony formation specific to HPV16-positive cells. TLR7 sequence-specific sensing of D-siRNAs also occurred to varying levels. SiRNA-induced immunostimulation correlated with the motif “UXUCU” on the antisense strand of the D-siRNAs. From the screens, a D-siRNA was identified as fitting the criteria of 'bifunctional siRNA', i.e. having both gene knockdown and immunostimulatory abilities. This study next investigated the functionality of these D-siRNAs in vivo. A single pre-treatment of cells with D-siRNAs prior to implantation demonstrated slower and smaller tumour growth specific to E6/E7 knockdown. Systemic administration of D-siRNAs also suggested similar results; however the bifunctionality of D-siRNA was not superior to E6/E7 knockdown alone. Furthermore, non-targeting D-siRNA initially used as a control was demonstrated to also have the ability to reduce tumour growth, albeit not persistently. This ability correlated with siRNA-induced immunostimulation and was supported by a finding in this study that shows induction of pro-inflammatory cytokines by immunostimulatory siRNAs to be transient, lasting 4-8 hours. Indeed, modifications made to the D-siRNAs by addition of 2'-O-methyl groups (2'OMe) to the antisense strand‟s backbone was demonstrated to remove immunostimulatory ability and tumour reduction ability of these D-siRNAs. The study revealed a threshold between siRNA-induced immunostimulation with gene knockdown, where the effects on tumour reduction were more dependent on immunostimulation at an early time point, but the effects of gene knockdown were found to be more persistent and long-lasting. Additionally, TLR-independent induction of monocyte chemo-attractant protein-1 (MCP-1, also known as CCL2) was observed when any siRNA was delivered systemically. A microarray analysis was also carried out in this study on cells transfected with D-siRNAs, to determine the localised effects of RNAi-based therapy. Target gene knockdown was demonstrated to occur at 20 hours post-transfection and could still be observed after 36 hours. This concurs with data from microarray analysis, where off-target effects by D-siRNAs were shown to correlate with time post-transfection. D-siRNAs caused differentially expressed genes in the immune response, cell-cycle and cancer maintenance pathways. These effects were further clustered to siRNA-specific or downstream E6/E7 knockdown effects. However, off-target effects were not related to seed region pairing. Nevertheless, this analysis was able to determine the localised effects of siRNA delivery and identify downstream targets of E6/E7 knockdown. In conclusion, this study illustrated the potential of D-siRNAs for RNAi therapy incorporating siRNA-induced immunostimulation on virally-induced cervical cancer. SiRNA sequence-biases were shown to be preferred over structural modifications to optimize knockdown and immunostimulation. This study also revealed for the first time that siRNA-induced immunostimulation is transient, lasting 4-8 hours in vivo. Single transfection of D-siRNAs was shown to have prolonged effects on tumour growth, potentially through promoting cell senescence. Tumour reduction was shown to depend on a threshold between siRNA-induced immunostimulation with efficient gene knockdown. Localised effects were identified through microarray and differential expressions of genes were clustered into early, intermediate and late time points. This research thus lays the foundation for the development of RNAi-based therapy for virally-induced diseases.
Keyword bifunctional siRNA
cervical cancer
HPV16 E6/E7
virally-induced disease
Additional Notes Pages in colour (pdf page number): 18,20,26,28,33,36,43,72,81,82,84,85,86,88,92,98,101,104,112,125,129,130,132, 133, 136, 139, 145 Pages as landscape (pdf page number): 20, 33, 73, 74, 136 Pages 81-82 and 84 should be printed on photo-quality paper.

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Created: Tue, 21 Dec 2010, 13:27:56 EST by Ms Norliana Khairuddin on behalf of Library - Information Access Service