Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice.

Tekmira Pharmaceuticals Corporation, 100-8900 Glenlyon Parkway, Burnaby, British Columbia, Canada.
The Journal of clinical investigation (Impact Factor: 15.39). 03/2009; 119(3):661-73. DOI: 10.1172/JCI37515
Source: PubMed

ABSTRACT siRNAs that specifically silence the expression of cancer-related genes offer a therapeutic approach in oncology. However, it remains critical to determine the true mechanism of their therapeutic effects. Here, we describe the preclinical development of chemically modified siRNA targeting the essential cell-cycle proteins polo-like kinase 1 (PLK1) and kinesin spindle protein (KSP) in mice. siRNA formulated in stable nucleic acid lipid particles (SNALP) displayed potent antitumor efficacy in both hepatic and subcutaneous tumor models. This was correlated with target gene silencing following a single intravenous administration that was sufficient to cause extensive mitotic disruption and tumor cell apoptosis. Our siRNA formulations induced no measurable immune response, minimizing the potential for nonspecific effects. Additionally, RNAi-specific mRNA cleavage products were found in tumor cells, and their presence correlated with the duration of target mRNA silencing. Histological biomarkers confirmed that RNAi-mediated gene silencing effectively inhibited the target's biological activity. This report supports an RNAi-mediated mechanism of action for siRNA antitumor effects, suggesting a new methodology for targeting other key genes in cancer development with siRNA-based therapeutics.

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    ABSTRACT: Introduction RNAi, which is commonly understood as RNA interference, refers to a member of non-coding RNA (ncRNA). The term non-coding RNA (ncRNA) is used for RNA that are not translated into protein; however, this does not mean that non-coding RNA delivers no performance. 1 New evidence suggests that a majority of the mammalian genome is transcribed into ncRNA and exclusively 2% of it is transcribed into mRNA and translated into protein. 2-10 RNA sequencing studies showed that the origin of ncRNAs is in the transcript antisense protein-coding genes, bidirectional promoter transcripts, enhancer and repeated sequences areas transcription, Intronic transcripts. 11 Viruses and other double-stranded RNA microorganisms insert their genome into their host cells or artificially synthesize double-stranded RNA. 12-14 Non-coding RNA is divided into two groups: 1-Small regulatory RNA and 2-Long non-coding RNA (Table 1). RNA interference (RNAi) is part of a small regulatory RNA, including siRNA and miRNA. 15 The discovery of RNA interference molecules indebted to Mr. Fire's and Mello's research into the C.elegans in 1998. 12 The advances in RNAi were made in following years leading eventually to the Physiology and Medicine Nobel Prize for Fire and Mello in 2006. Specific gene expression silencing by RNAi is a mechanism of transcriptional regulation in the eukaryotic cell. This is mediated by small RNA with 21-23 nucleotides length called siRNA and is conserved in terms of evolution among eukaryotes. RNAi seems to protect against not only exogenous genes such as microbial organisms genes including viral, bacterial genes, but also endogenous genes such as transposons. The other roles of these molecules in cells involving gene expression regulation and cell growth control have been demonstrated. 16-19 On this regard, 3 mechanisms have been identified embodying: 1) heterochromatin formation changes 2) Inhibition of translation of target mRNA 3) Degradation of the target mRNA. The second and third mechanisms are more divulged. Cancer is one of the main targets for RNAi-based therapy. Several studies conducted invivo and invitro showed that RNAi-based therapy can be used for treating single-gene disorders and those with overexpression of proteins. 20
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    ABSTRACT: Alterations in cellular metabolism and bioenergetics are vital for cancer cell growth and motility. Here, the role of the mitochondrial protein voltage-dependent anion channel (VDAC1), a master gatekeeper regulating the flux of metabolites and ions between mitochondria and the cytoplasm, in regulating the growth of several cancer cell lines was investigated by silencing VDAC1 expression using small interfering RNA (siRNA). A single siRNA specific to the human VDAC1 sequence at nanomolar concentrations led to some 90% decrease in VDAC1 levels in the lung A549 and H358, prostate PC-3, colon HCT116, glioblastoma U87, liver HepG2, and pancreas Panc-1 cancer cell lines. VDAC1 silencing persisted 144 hours post-transfection and resulted in profound inhibition of cell growth in cancer but not in noncancerous cells, with up to 90% inhibition being observed over 5 days that was prolonged by a second transfection. Cells expressing low VDAC1 levels showed decreased mitochondrial membrane potential and adenoside triphosphate (ATP) levels, suggesting limited metabolite exchange between mitochondria and cytosol. Moreover, cells silenced for VDAC1 expression showed decreased migration, even in the presence of the wound healing accelerator basic fibroblast growth factor (bFGF). VDAC1-siRNA inhibited cancer cell growth in a Matrigel-based assay in host nude mice. Finally, in a xenograft lung cancer mouse model, chemically modified VDAC1-siRNA not only inhibited tumor growth but also resulted in tumor regression. This study thus shows that VDAC1 silencing by means of RNA interference (RNAi) dramatically inhibits cancer cell growth and tumor development by disabling the abnormal metabolic behavior of cancer cells, potentially paving the way for a more effective pipeline of anticancer drugs.
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    ABSTRACT: RNA interference (RNAi) has been proven in recent years to be a newly advanced and powerful tool for development of therapeutic agents toward various unmet medical needs such as cancer, in particular, a great attention has been paid to the development of antineoplastic agents. Recent success in clinical trials related to RNAi-based therapeutics on cancer and ocular disease has validated that small interfering RNAs (siRNAs) constitute a new promising class of therapeutics. Currently, a great wealth of multi-target based siRNA structural modifications is available for promoting siRNA-mediated gene silencing with low side effects. Here, the latest developments in RNAi-based therapeutics and novel structural modifications described for siRNAs-in particular multi-target siRNAs-are reviewed.
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