Abstract
BackgroundDelivery of small interfering RNA (siRNA) to tumours remains a major obstacle for the development of RNA interference (RNAi)-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV) OncoVEXGM-CSF, we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV.MethodsTo evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA) or artificial microRNA (miRNA) against the reporter genes green fluorescent protein (eGFP) and β-galactosidase (lacZ). These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting.ResultsOur results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective.ConclusionsThis preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen in human clinical trials.
Highlights
Delivery of small interfering RNA to tumours remains a major obstacle for the development of RNA interference (RNAi)-based therapeutics
For the first time, that short-hairpin RNA (shRNA) and artificial miRNA can be expressed from an oncolytic herpes simplex virus (HSV) virus and results in effective silencing of reporter genes in vitro in non-tumour cells that are highly susceptible and tumour cells that are moderately susceptible to HSV infection and in vivo in tumours that are not cured by oncolytic virus treatment alone
ShRNA can be efficiently expressed from the CMV polymerase II promoter when embedded into endogenous miRNA sequences and these artificial miRNAs have been shown to induce specific degradation of target mRNAs similar to transfected small interfering RNA (siRNA) [34,35,36]
Summary
Delivery of small interfering RNA (siRNA) to tumours remains a major obstacle for the development of RNA interference (RNAi)-based therapeutics. RNAi can be induced by synthetic siRNAs introduced directly into the cell or by plasmid and viral vectors that express short-hairpin RNA (shRNA) or artificial miRNAs, which have been termed the new generation RNAi triggers [7,8,9]. A variety of strategies, such as chemical modifications, liposomes, nanoparticles, and antibodies or cell-surface receptors, have been employed to increase siRNA stability and delivery to specific cell types [10], in vivo delivery of siRNAs remains a major obstacle for the development of RNAi-based cancer therapeutics. Lentiviral vectors have been employed to silence a number of tumourassociated genes, including Tiam, resulting in suppression of cancer cell growth in vitro and in vivo [12]. Herpes simplex virus (HSV) amplicon vectors have been shown to silence genes in tumour cells both in vitro and in vivo [16,17]
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