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: 8.19). 09/2009; 15(16):5126-35. DOI: 10.1158/1078-0432.CCR-09-0051
Source: PubMed

ABSTRACT 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.


Available from: William WeiGuo Jia, Mar 29, 2014
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Glioblastoma Multiforme (GBM) is an aggressive brain cancer for which there is no effective treatment. Oncolytic HSV vectors (oHSV) are attenuated lytic viruses that have shown promise in the treatment of human GBM models in animals, but their efficacy in early phase patient trials has been limited. Instead of attenuating the virus with mutations in virulence genes, we engineered 4 copies of the recognition sequence for miR-124 into the 3'UTR of the essential ICP4 gene to protect healthy tissue against lytic virus replication; miR-124 is expressed in neurons but not in glioblastoma cells. Following intracranial inoculation into nude mice, the miR-124-sensitive vector failed to replicate or show overt signs of pathogenesis. To address the concern that this safety feature may reduce oncolytic activity, we inserted the miR-124 response elements into an unattenuated, human receptor (EGFR/EGFRvIII)-specific HSV vector. We found that miR-124 sensitivity did not cause a loss of treatment efficiency in an orthotopic model of primary human GBM in nude mice. These results demonstrate that engineered miR-124 responsiveness can eliminate off-target replication by unattenuated oHSV without compromising oncolytic activity, thereby providing increased safety.Molecular Therapy (2014); doi:10.1038/mt.2014.177.
    Molecular Therapy 09/2014; 23(1). DOI:10.1038/mt.2014.177 · 6.43 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The ubiquitin-like interferon (IFN)-stimulated gene 15 (ISG15), and its specific E1, E2 and E3 enzymes are transcriptionally induced by type I Interferons (IFNs). ISG15 conjugates newly synthesized proteins. ISG15 linkage to proteins appears to be an important downstream IFN-signaling event that discrimininates cellular and pathogenic proteins synthesized during IFN stimulation, from existing proteins. This eliminates potentially pathogenic proteins, as the cell attempts to return to normal homeostasis after IFN stressed conditions. However, the molecular events that occur in this process are not well known. Here, we show that the C-terminal LRLRGG of ISG15 interacts with the ubiquitin zinc finger (BUZ) domain of Histone Deacetylase 6 (HDAC6). Since HDAC6 is involved in the autophagic clearance of ubiquitinated (Ub) aggregates, during which SQSTM1/p62 plays a major role as a cargo adapter, we also were able to confirm that p62 binds to ISG15 protein and its conjugated proteins, upon forced expression. Both HDAC6 and p62 colocalize with ISG15 in an insoluble fraction of the cytosol and this colocalization was magnified by the proteasome inhibitor, MG132. In addition, ISG15 was degraded via the lysosome. Overexpression of ISG15, which leads to an increased conjugation level of the cellular proteome, enhanced autophagic degradation, independent of IFN signaling transduction. These results thus indicate that ISG15 conjugation marks proteins for interaction with HDAC6 and p62 upon forced stressful conditions, likely as a step towards autophagic clearance. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 11/2014; DOI:10.1074/jbc.M114.593871 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A novel selectively targeting gene delivery approach has been developed for advanced hepatocellular carcinoma (HCC), a leading cause of cancer mortality whose prognosis remains poor. We combine the strong liver tropism of serotype-8 capsid pseudotyped adeno-associated viral vectors (AAV8) with a liver specific promoter (HLP) and microRNA-122a (miR-122a) mediated post-transcriptional regulation. Systemic administration of our AAV8 construct resulted in preferential transduction of the liver and encouragingly of HCC at heterotopic sites, a finding that could be exploited to target disseminated disease. Tumour selectivity was enhanced by inclusion of miR-122a binding sequences (ssAAV8-HLP-TK-122aT4) in the expression cassette resulting in abrogation of transgene expression in normal murine liver but not in HCC. Systemic administration of our tumour selective vector encoding herpes simplex virus-thymidine kinase (TK) suicide gene resulted in a 7-fold reduction in HCC growth in a syngeneic murine model without toxicity. In summary, we have developed a systemically deliverable gene transfer approach that enables high level expression of therapeutic genes in HCC but not normal tissues, thus improving the prospects of safe and effective treatment for advanced HCC.
    Human Gene Therapy 01/2015; 26(2). DOI:10.1089/hum.2014.052 · 3.62 Impact Factor