siRNA-induced immunostimulation through TLR7 promotes antitumoral activity against HPV-driven tumors in vivo

Oncogene-specific downregulation mediated by RNA interference (RNAi) is a promising avenue for cancer therapy. In addition to specific gene silencing, in vivo RNAi treatment with short interfering RNAs (siRNAs) can initiate immune activation through innate immune receptors including Toll-like receptors, (TLRs) 7 and 8. Two recent studies have shown that activation of innate immunity by addition of tri-phosphate motifs to oncogene-specific siRNAs, or by co-treatment with CpG oligos, can potentiate siRNA antitumor effects. To date, there are no reports on applying such approach against human papillomavirus (HPV)-driven cancers. Here, we characterized the antitumor effects of non-modified siRNAs that can target a specific oncogene and/or recruit the innate immune system against HPV-driven tumors. Following the characterization of silencing efficacy and TLR7 immunostimulatory potential of 15 siRNAs targeting the HPV type 16 E6/E7 oncogenes, we identified a bifunctional siRNA sequence that displayed both potent gene silencing and active immunostimulation effect. In vivo systemic administration of this siRNA resulted in reduced growth of established TC-1 tumors in C57BL/6 mice. Ablation of TLR7 recruitment via 2′O-methyl modification of the oligo backbone reduced these antitumor effects. Further, a highly immunostimulatory, but non-HPV targeting siRNA was also able to exert antitumoral effects although for less prolonged time compared with the bifunctional siRNA. Collectively, our work demonstrates for the first time that siRNA-induced immunostimulation can have antitumoral effects against HPV-driven tumors in vivo, even independent of gene silencing efficacy

HIV and adolescents: focus on young key populations

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138345/1/jia20076.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138345/2/jia20076-sup-0001.pd

RNAi against Virally-Induced Disease

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

RNAi therapy: Involving the Immune System

RNA interference (RNAi) is a powerful tool for gene silencing. Over the past decade our increased understanding of the molecular mechanisms underlying RNAi has provided an avenue towards utilizing it therapeutically. Of the many RNAi precursors, small interfering RNAs (siRNAs) have been the most successfully translated into the clinic. One of the major issues with RNA-based medicines is the cells' ability to see and respond via pattern-recognition receptors such as Toll-like receptors (TLRs) and retinoic acid inducible gene 1 (RIG-1), inducing innate and adaptive immune responses. We propose that in certain diseases the combination of gene silencing and appropriate immune activation by so-called 'bifunctional siRNA' is a rational therapeutic approach and worthy of further investigation. Here we briefly review the progress in developing siRNA-based therapeutics and its interaction with the immune system, including siRNA design, types of immune activation, RNAi use in cancer and viral infections, as well as pre-clinical applications. We believe that development of immunostimulatory siRNAs provides the means for more potent therapies

RNA interference for the treatment of papillomavirus disease

Human Papillomavirus (HPV)-induced diseases are a significant burden on our healthcare system and current therapies are not curative. Vaccination provides significant prophylactic protection but effective therapeutic treatments will still be required. RNA interference (RNAi) has great promise in providing highly specific therapies for all HPV diseases yet this promise has not been realised. Here we review the research into RNAi therapy for HPV in vitro and in vivo and examine the various targets and outcomes. We discuss the idea of using RNAi with current treatments and address delivery of RNAi, the major issue holding back clinical adoption. Finally, we present our view of a potential path to the clinic

In vivo comparison of local versus systemic delivery of immunostimulating siRNA in HPV-driven tumours

Small interfering RNAs (siRNAs) to inhibit oncogene expression and also to activate innate immune responses via Toll-like receptor (TLR) recognition have been shown to be beneficial as anti-cancer therapy in certain cancer models. In this study, we investigated the effects of local versus systemic delivery of such immune-stimulating Dicer-substrate siRNAs (IS-DsiRNAs) on a human papillomavirus (HPV)-driven tumour model. Localized siRNA delivery using intratumour injection of siRNA was able to increase siRNA delivery to the tumour compared with intravenous (IV) delivery and potently activated innate immune responses. However, IV injection remained the more effective delivery route for reducing tumour growth. Although IS-DsiRNAs activated innate immune cells and required interferon-α (IFNα) for full effect on tumour growth, we found that potent silencing siRNA acting independently of IFNα were overall more effective at inhibiting TC-1 tumour growth. Other published work utilising IS-siRNAs have been carried out on tumour models with low levels of major histocompatibility complex (MHC)-class 1, a target of natural killer cells that are potently activated by IS-siRNA. As TC-1 cells used in our study express high levels of MHC-class I, the addition of the immunostimulatory motifs may not be as beneficial in this particular tumour model. Our data suggest that selection of siRNA profile and delivery method based on tumour environment is crucial to developing siRNA-based therapies