MicroRNA Regulation of Oncolytic Herpes Simplex Virus-1 for Selective Killing of Prostate Cancer Cells

Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
Clinical Cancer Research (Impact Factor: 8.72). 09/2009; 15(16):5126-35. DOI: 10.1158/1078-0432.CCR-09-0051
Source: PubMed


Advanced castration-resistant prostate cancer, for which there are few treatment options, remains one of the leading causes of cancer death. MicroRNAs (miRNA) have provided a new opportunity for more stringent regulation of tumor-specific viral replication. The purpose of this study was to provide a proof-of-principle that miRNA-regulated oncolytic herpes simplex virus-1 (HSV-1) virus can selectively target cancer cells with reduced toxicity to normal tissues.
We incorporated multiple copies of miRNA complementary target sequences (for miR-143 or miR-145) into the 3'-untranslated region (3'-UTR) of an HSV-1 essential viral gene, ICP4, to create CMV-ICP4-143T and CMV-ICP4-145T amplicon viruses and tested their targeting specificity and efficacy both in vitro and in vivo.
Although miR-143 and miR-145 are highly expressed in normal tissues, they are significantly down-regulated in prostate cancer cells. We further showed that miR-143 and miR-145 inhibited the expression of the ICP4 gene at the translational level by targeting the corresponding 3'-UTR in a dose-dependent manner. This enabled selective viral replication in prostate cancer cells. When mice bearing LNCaP human prostate tumors were treated with these miRNA-regulated oncolytic viruses, a >80% reduction in tumor volume was observed, with significantly attenuated virulence to normal tissues in comparison with control amplicon viruses not carrying these 3'-UTR sequences.
Our study is the first to show that inclusion of specific miRNA target sequences into the 3'-UTR of an essential HSV-1 gene is a viable strategy for restricting viral replication and oncolysis to cancer cells while sparing normal tissues.

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Available from: William WeiGuo Jia, Mar 29, 2014
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    • "These properties of miRNAs have been used in development of virus-based vectors. miRNA target sequences have been inserted into the genomes of vesicular stomatitis virus [37], coxsackievirus [38], poliovirus [39], chimeric tick-borne encephalitis/dengue virus [40], herpes simplex virus 1 [41] and several others. This genetic engineering generates viral tropism toward targeted tissues, limits or enhances viral immunogenicity, decreases cytotoxic side effects of virus infection and increases the safety of the constructed vectors. "
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    ABSTRACT: Due to their broad cell- and tissue-tropism, alphavirus-based replication-competent vectors are of particular interest for anti-cancer therapy. These properties may, however, be potentially hazardous unless the virus infection is controlled. While the RNA genome of alphaviruses precludes the standard control techniques, host miRNAs can be used to down-regulate viral replication. In this study, target sites from ubiquitous miRNAs and those of miRNAs under-represented in cervical cancer cells were inserted into replication-competent DNA/RNA layered vectors of Semliki Forest virus. It was found that in order to achieve the most efficient suppression of recombinant virus rescue, the introduced target sequences must be fully complementary to those of the corresponding miRNAs. Target sites of ubiquitous miRNAs, introduced into the 3' untranslated region of the viral vector, profoundly reduced the rescue of recombinant viruses. Insertion of the same miRNA targets into coding region of the viral vector was approximately 300-fold less effective. Viruses carrying these miRNAs were genetically unstable and rapidly lost the target sequences. This process was delayed, but not completely prevented, by miRNA inhibitors. Target sites of miRNA under-represented in cervical cancer cells had much smaller but still significant effects on recombinant virus rescue in cervical cancer-derived HeLa cells. Over-expression of miR-214, one of these miRNAs, reduced replication of the targeted virus. Though the majority of rescued viruses maintained the introduced miRNA target sequences, genomes with deletions of these sequences were also detected. Thus, the low-level repression of rescue and replication of targeted virus in HeLa cells was still sufficient to cause genetic instability.
    Full-text · Article · Sep 2013 · PLoS ONE
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    • "Four copies of miRNA-145 complementary sequences (miRNA-145T) were constructed as described previously [22]. The ICP27 gene (~1.8 kb) and the miRNA-145T fragments, excised by XhoI and XbaI digestion, were then cloned into the pcDNA3.0-neo "
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    ABSTRACT: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide, and novel treatment modalities to improve the prognosis of patients with advanced disease are highly desirable. Oncolytic virotherapy is a promising approach for the treatment of advanced NSCLC. MicroRNAs (miRNAs) may be a factor in the regulation of tumor-specific viral replication. The purpose of this study was to investigate whether miRNA-145 regulated oncolytic herpes simplex virus-1 (HSV-1) can selectively kill NSCLC cells with reduced collateral damage to normal cells. We incorporated 4 copies of miRNA-145 target sequences into the 3' -untranslated region of an HSV-1 essential viral gene, ICP27, to create AP27i145 amplicon viruses and tested their target specificity and toxicity on normal cells and lung cancer cells in vitro. miRNA-145 expression in normal cells was higher than that in NSCLC cells. AP27i145 replication was inversely correlated with the expression of miRNA-145 in infected cells. This oncolytic HSV-1 selectively reduced cell proliferation and prevented the colony formation of NSCLC cells. The combination of radiotherapy and AP27i145 infection was significantly more potent in killing cancer cells than each therapy alone. miRNA-145-regulated oncolytic HSV-1 is a promising agent for the treatment of NSCLC.
    Full-text · Article · Jul 2013 · Virology Journal
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    • "Herpes Simplex Virus-1 (HSV-1) was engineered to incorporate the binding sites of miR-143 and miR-145 into the 3'-UTR of ICP4 gene. This significantly reduced virulence to normal tissues but maintained the killing of prostate cancer cells [12]. Edges and colleagues incorporated the complementary sequence of let-7 microRNA within Vesicular Stomatitis Virus (VSV), a rhabdovirus, to eliminate undesirable replication and toxicity in normal cells but still grow in cancer cells [13]. "
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    ABSTRACT: Background The microRNA-based gene-silencing machinery has been recognized as a promising approach to control viral replication and used for improving safety for the live attenuated virus vaccines. The effective host microRNA response elements (MREs) have been incorporated into a virus sequence mainly based on the experimental trials for identifying both microRNA binding sites and effective mutations. The design of MREs for viral genomes or with multiple host microRNAs of interest, then, will be time and cost consuming. Results In this paper, we introduced a computational flow that could be used to design MREs of human microRNAs within Influenza A H1N1 virus gene segments. The main steps of the flow includes locating possible binding sites; MREs, of human microRNAs within the viral sequences using a miRNA target prediction tool (miranda), performing various mutations among mismatched binding positions, calculating the binding energy, score, identity, and the effects of changed physical properties of amino acids according to the changed bases in RNA level, and prioritizing the mutated binding sites. The top ranked MREs of human microRNA hsa-miR-93 is consistent with previous literature while other results waited to be experimentally verified. To make the computational flow easily accessible by virologists, we also developed MicroLive, a web server version of the MRE design flow together with the database of miranda-predicted MREs within gene sequences of seven RNA viruses including Influenza A, dengue, hepatitis C, measles, mumps, poliovirus, and rabies. Users may design MREs of specific human microRNAs for their input viral sequences using MRE design tool or optimize the miranda-predicted MREs of seven viruses available on the system. Also, users could design varied number of MREs for multiple human microRNAs to modulate the degree of live vaccine attenuation and reduce the likelihood of escape mutants. Conclusions The computational design of MREs helps reduce time and cost for experimental trials. While the flow was demonstrated using human microRNAs and Influenza A H1N1 virus, it could be flexibly applied to other hosts (e.g., animals) and viruses of interest for constructing host-specific live attenuated vaccines. Also, it could be deployed for engineering tissue-specific oncolytic viruses in cancer virotherapeutics. The MicroLive web server is freely accessible at
    Full-text · Article · Dec 2012 · BMC Genomics
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