RNAi-mediated gene silencing in tumour tissue using replication-competent retroviral vectors

Abstract

RNAi represents a powerful technology to specifically downregulate the expression of target genes. For cancer research and therapy, an efficient in vivo delivery system is supposed to distribute RNAi to all tumour cells upon systemic administration. We present replication-competent murine leukaemia virus (MLV) vectors, which deliver RNAi to tumour tissue upon tail vein injection. In HT1080 cells stably expressing GFP or luciferase, GFP expression was suppressed by more than 80% and luciferase (luc) activity by more than 90%, even when only 0.1% of the cells were initially infected with reporter gene specific vectors. To demonstrate its potential, PLK1-and MMP14-specific small hairpin RNA expression cassettes were applied in the system. Upon infection, PLK1 and MMP14 levels were reduced on mRNA and protein level. MLV-shPLK1-infected cells were arrested in the G2-phase and underwent apoptosis. MLV-shMMP14-infected cells showed reduced MMP2 activity, as well as substantially reduced invasion and tumour growth. In vivo, MLV-shLuc silenced luc expression in HT1080-luc tumour tissue by more than 80% and MLV-shPLK1 reduced tumour growth substantially, demonstrating the therapeutic relevance of this system. This RNAi vector system allows long-term downregulation of target gene expression as well as efficient delivery to and distribution throughout tumour tissue in vivo.

Inhibition of Sox2 Expression in the Adult Neural Stem Cell Niche In Vivo by Monocationic-based siRNA Delivery

“…This is an advantage for transient study, as when using plasmids, even non-integrative plasmids, overexpression or downregulation of corresponding RNA and proteins can last several days. Although stable expression, such as obtained with integrative viruses, may be in many cases an advantage (e.g., for tumor targeting), 24 in other cases, it is not, as it is not possible to study how the population returns to equilibrium. 24 Finally, a transient knockdown better mimics the transcriptional regulations occurring in physiological conditions.…”

“…Although stable expression, such as obtained with integrative viruses, may be in many cases an advantage (e.g., for tumor targeting), 24 in other cases, it is not, as it is not possible to study how the population returns to equilibrium. 24 Finally, a transient knockdown better mimics the transcriptional regulations occurring in physiological conditions. Thus, transient siRNA-applied in vivo has the advantage of resembling physiological responses and regulations, an advantage that can be exploited.…”

Retroviral Replicating Vector Delivery of miR-PDL1 Inhibits Immune Checkpoint PDL1 and Enhances Immune Responses In Vitro

“…We generated human U6 promoter driven-MLV-based RRV expressing conventional shRNA (RRV-shGFP) or microRNA30-based shRNA (RRV-miRGFP) targeted to GFP (Figure 1). We tested two sequences (shGFP1, shGFP2, miRGFP1, and miRGFP2) that have been reported to have at least 75% GFP downregulation activity 18, 22. RRVs were produced by transient transfection in 293T cells, and viral titer values were determined on PC3 cells by qPCR as previously described 23 (Table S1).…”

“…Several groups compared the biological process and function between shRNA versus miRshRNA and reported that miRshRNA are processed more efficiently than shRNA and have less cytotoxicity linked to saturation of endogenous RNAi machinery 10, 13, 14, 15. Since the development of the miRshRNA configuration, it has been widely incorporated into non-viral expression vectors as well viral vectors,13, 16, 17, 18, 19 Schaser et al. have previously shown the feasibility of incorporating a single miRshRNA cassette into a MLV-based RRV and observed anti-tumor function in vivo 18 .…”

“…Since the development of the miRshRNA configuration, it has been widely incorporated into non-viral expression vectors as well viral vectors,13, 16, 17, 18, 19 Schaser et al. have previously shown the feasibility of incorporating a single miRshRNA cassette into a MLV-based RRV and observed anti-tumor function in vivo 18 . However, the vector designed was limited to the commonly used U6 promoter miRshRNA cassette, and vector characterization was also limited in the report.…”