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• W3002247662 abstract "We recently discovered that coxsackievirus B3 (CVB3) is a potent oncolytic virus against KRAS mutant lung adenocarcinoma. Nevertheless, the evident toxicity restricts the use of wild-type (WT)-CVB3 for cancer therapy. The current study aims to engineer the CVB3 to decrease its toxicity and to extend our previous research to determine its safety and efficacy in treating TP53/RB1 mutant small-cell lung cancer (SCLC). A microRNA-modified CVB3 (miR-CVB3) was generated via inserting multiple copies of tumor-suppressive miR-145/miR-143 target sequences into the viral genome. In vitro experiments revealed that miR-CVB3 retained the ability to infect and lyse KRAS mutant lung adenocarcinoma and TP53/RB1-mutant SCLC cells, but with a markedly reduced cytotoxicity toward cardiomyocytes. In vivo study using a TP53/RB1-mutant SCLC xenograft model demonstrated that a single dose of miR-CVB3 via systemic administration resulted in a significant tumor regression. Most strikingly, mice treated with miR-CVB3 exhibited greatly attenuated cardiotoxicities and decreased viral titers compared to WT-CVB3-treated mice. Collectively, we generated a recombinant CVB3 that is powerful in destroying both KRAS mutant lung adenocarcinoma and TP53/RB1-mutant SCLC, with a negligible toxicity toward normal tissues. Future investigation is needed to address the issue of genome instability of miR-CVB3, which was observed in ~40% of mice after a prolonged treatment. We recently discovered that coxsackievirus B3 (CVB3) is a potent oncolytic virus against KRAS mutant lung adenocarcinoma. Nevertheless, the evident toxicity restricts the use of wild-type (WT)-CVB3 for cancer therapy. The current study aims to engineer the CVB3 to decrease its toxicity and to extend our previous research to determine its safety and efficacy in treating TP53/RB1 mutant small-cell lung cancer (SCLC). A microRNA-modified CVB3 (miR-CVB3) was generated via inserting multiple copies of tumor-suppressive miR-145/miR-143 target sequences into the viral genome. In vitro experiments revealed that miR-CVB3 retained the ability to infect and lyse KRAS mutant lung adenocarcinoma and TP53/RB1-mutant SCLC cells, but with a markedly reduced cytotoxicity toward cardiomyocytes. In vivo study using a TP53/RB1-mutant SCLC xenograft model demonstrated that a single dose of miR-CVB3 via systemic administration resulted in a significant tumor regression. Most strikingly, mice treated with miR-CVB3 exhibited greatly attenuated cardiotoxicities and decreased viral titers compared to WT-CVB3-treated mice. Collectively, we generated a recombinant CVB3 that is powerful in destroying both KRAS mutant lung adenocarcinoma and TP53/RB1-mutant SCLC, with a negligible toxicity toward normal tissues. Future investigation is needed to address the issue of genome instability of miR-CVB3, which was observed in ~40% of mice after a prolonged treatment. Lung cancer is the leading cause of cancer-related death among both men and women worldwide.1Siegel R.L. Miller K.D. Jemal A. Cancer statistics, 2018.CA Cancer J. Clin. 2018; 68: 7-30Crossref PubMed Scopus (5158) Google Scholar There are two major forms of lung cancer, non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), with the former constituting ~85% of all lung cancer cases. Among them, adenocarcinoma is the most common type of lung cancer, responsible for almost half of all lung cancers, and is associated with both smokers and non-smokers.2Reck M. Rabe K.F. Precision diagnosis and treatment for advanced non-small-cell lung cancer.N. Engl. J. Med. 2017; 377: 849-861Crossref PubMed Scopus (311) Google Scholar Genetic mutations play a critical role in the development of lung adenocarcinoma. The well-identified oncogenic driver mutations in lung adenocarcinoma include those in epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and Kirsten rat sarcoma viral oncogene homolog (KRAS), which occur in ~15%, ~7%, and ~30% of lung adenocarcinoma, respectively.3Ding L. Getz G. Wheeler D.A. Mardis E.R. McLellan M.D. Cibulskis K. Sougnez C. Greulich H. Muzny D.M. Morgan M.B. et al.Somatic mutations affect key pathways in lung adenocarcinoma.Nature. 2008; 455: 1069-1075Crossref PubMed Scopus (2009) Google Scholar SCLC accounts for ~15% of all lung cancers and is almost exclusive to smokers. Between 60% and 90% of SCLC cases feature mutations in gene encoding tumor protein p53 (TP53mut) and/or retinoblastoma protein (RB1mut).4Koinis F. Kotsakis A. Georgoulias V. Small cell lung cancer (SCLC): no treatment advances in recent years.Transl. Lung Cancer Res. 2016; 5: 39-50PubMed Google Scholar Although lung adenocarcinoma associated with EGFR mutations (EGFRmut) or ALK translocations can be clinically treated using tyrosine kinase inhibitors,2Reck M. Rabe K.F. Precision diagnosis and treatment for advanced non-small-cell lung cancer.N. Engl. J. Med. 2017; 377: 849-861Crossref PubMed Scopus (311) Google Scholar lung adenocarcinoma with KRAS mutations (KRASmut) and SCLC are currently undruggable and associated with a poor prognosis.4Koinis F. Kotsakis A. Georgoulias V. Small cell lung cancer (SCLC): no treatment advances in recent years.Transl. Lung Cancer Res. 2016; 5: 39-50PubMed Google Scholar,5Tomasini P. Walia P. Labbe C. Jao K. Leighl N.B. Targeting the KRAS pathway in non-small cell lung cancer.Oncologist. 2016; 21: 1450-1460Crossref PubMed Scopus (62) Google Scholar Highly effective and innovative treatment modalities for these subsets of advanced lung cancer are therefore urgently needed. Recent advances in oncolytic virotherapy provide a promising new treatment approach.6Bell J. McFadden G. Viruses for tumor therapy.Cell Host Microbe. 2014; 15: 260-265Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar,7Lawler S.E. Speranza M.C. Cho C.F. Chiocca E.A. Oncolytic viruses in cancer treatment: a review.JAMA Oncol. 2017; 3: 841-849Crossref PubMed Scopus (234) Google Scholar Oncolytic viruses are a group of viruses that are genetically engineered or naturally occurring to specifically destroy cancer cells while sparing normal tissues. Their unique tumor-destructive mechanism lies in their ability of lytic replication, resulting in the lysis of cancer cells and the release of viral progeny to infect neighboring cells. Moreover, oncolytic viruses can overcome the immunosuppressive effects of tumors and have the ability to initiate anti-tumor immunity.6Bell J. McFadden G. Viruses for tumor therapy.Cell Host Microbe. 2014; 15: 260-265Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar,7Lawler S.E. Speranza M.C. Cho C.F. Chiocca E.A. Oncolytic viruses in cancer treatment: a review.JAMA Oncol. 2017; 3: 841-849Crossref PubMed Scopus (234) Google Scholar In October 2015, the US Food and Drug Administration (FDA) approved the first genetically modified herpes simplex virus 1 (talimogene laherparepvec [T-VEC]) for the treatment of melanoma.8Poh A. First oncolytic viral therapy for melanoma.Cancer Discov. 2016; 6: 6Crossref PubMed Scopus (23) Google Scholar During the past decades, several oncolytic viruses (both RNA and DNA viruses), including coxsackievirus A21,9Coxsackievirus A21 synergizes with checkpoint inhibitors.Cancer Discov. 2017; 7: OF9Crossref Scopus (4) Google Scholar reovirus,10Villalona-Calero M.A. Lam E. Otterson G.A. Zhao W. Timmons M. Subramaniam D. Hade E.M. Gill G.M. Coffey M. Selvaggi G. et al.Oncolytic reovirus in combination with chemotherapy in metastatic or recurrent non-small cell lung cancer patients with KRAS-activated tumors.Cancer. 2016; 122: 875-883Crossref PubMed Scopus (30) Google Scholar vaccinia virus,11Quoix E. Ramlau R. Westeel V. Papai Z. Madroszyk A. Riviere A. Koralewski P. Breton J.L. Stoelben E. Braun D. et al.Therapeutic vaccination with TG4010 and first-line chemotherapy in advanced non-small-cell lung cancer: a controlled phase 2B trial.Lancet Oncol. 2011; 12: 1125-1133Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar adenovirus,12Lei J. Li Q.H. Yang J.L. Liu F. Wang L. Xu W.M. Zhao W.X. The antitumor effects of oncolytic adenovirus H101 against lung cancer.Int. J. Oncol. 2015; 47: 555-562Crossref PubMed Scopus (12) Google Scholar measles virus,13Fujiyuki T. Yoneda M. Amagai Y. Obayashi K. Ikeda F. Shoji K. Murakami Y. Sato H. Kai C. A measles virus selectively blind to signaling lymphocytic activation molecule shows anti-tumor activity against lung cancer cells.Oncotarget. 2015; 6: 24895-24903Crossref PubMed Scopus (17) Google Scholar Newcastle disease virus,14Meng S. Zhou Z. Chen F. Kong X. Liu H. Jiang K. Liu W. Hu M. Zhang X. Ding C. Wu Y. Newcastle disease virus induces apoptosis in cisplatin-resistant human lung adenocarcinoma A549 cells in vitro and in vivo.Cancer Lett. 2012; 317: 56-64Crossref PubMed Scopus (32) Google Scholar and vesicular stomatitis virus,15Patel M.R. Jacobson B.A. Ji Y. Drees J. Tang S. Xiong K. Wang H. Prigge J.E. Dash A.S. Kratzke A.K. et al.Vesicular stomatitis virus expressing interferon-β is oncolytic and promotes antitumor immune responses in a syngeneic murine model of non-small cell lung cancer.Oncotarget. 2015; 6: 33165-33177Crossref PubMed Scopus (59) Google Scholar have been tested in clinical trials for the treatment of lung cancer. However, overall anti-cancer efficacy and specificity remain low and there is still no FDA-approved virotherapy for lung cancer. Coxsackievirus B3 (CVB3) is an enterovirus in the family of Picornaviridae.16Fung G. Luo H. Qiu Y. Yang D. McManus B. Myocarditis.Circ. Res. 2016; 118: 496-514Crossref PubMed Scopus (191) Google Scholar It is a small, non-enveloped virus that contains a positive RNA genome encoding a single open reading frame flanked by 5′ and 3′ untranslated regions (UTRs). Although CVB3 infection can be severe in children and immunocompromised individuals, causing myocarditis, pancreatitis, and meningitis, infection in adults is generally asymptomatic or causes mild flu-like symptoms.16Fung G. Luo H. Qiu Y. Yang D. McManus B. Myocarditis.Circ. Res. 2016; 118: 496-514Crossref PubMed Scopus (191) Google Scholar Our recent study has demonstrated that CVB3 is an extremely potent oncolytic virus against KRASmut lung adenocarcinoma, while sparing normal lung epithelial cells, and EGFRmut lung adenocarcinoma.17Deng H. Liu H. de Silva T. Xue Y. Mohamud Y. Ng C.S. Qu J. Zhang J. Jia W.W.G. Lockwood W.W. Luo H. Coxsackievirus type B3 is a potent oncolytic virus against KRAS-mutant lung adenocarcinoma.Mol. Ther. Oncolytics. 2019; 14: 266-278Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Despite this promising discovery, we found that wild-type (WT)-CVB3 causes damage to multiple organs, particularly to the heart, in immunodeficient mice.17Deng H. Liu H. de Silva T. Xue Y. Mohamud Y. Ng C.S. Qu J. Zhang J. Jia W.W.G. Lockwood W.W. Luo H. Coxsackievirus type B3 is a potent oncolytic virus against KRAS-mutant lung adenocarcinoma.Mol. Ther. Oncolytics. 2019; 14: 266-278Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar In the current study, we aimed to use microRNA (miRNA) targeting to modify the CVB3 genome to lessen its toxicity to normal tissues while maintaining oncolytic properties specifically in cancer cells. miRNAs are a class of endogenous small non-coding RNAs that are evolutionarily conserved and act as key regulators in a wide range of fundamental cellular functions, including cell proliferation, differentiation, and apoptosis, by binding to the mRNAs with complementary sequences. Subsequently, they promote either mRNA degradation or suppression of translation.18Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (27032) Google Scholar Recent evidence suggests that miRNAs also play a key role in tumorigenesis and progression of cancers.19Gregory R.I. Shiekhattar R. MicroRNA biogenesis and cancer.Cancer Res. 2005; 65: 3509-3512Crossref PubMed Scopus (538) Google Scholar,20Ventura A. Jacks T. MicroRNAs and cancer: short RNAs go a long way.Cell. 2009; 136: 586-591Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar miRNAs are commonly downregulated in different types of cancer tissues in comparison with normal tissues.21Lu J. Getz G. Miska E.A. Alvarez-Saavedra E. Lamb J. Peck D. Sweet-Cordero A. Ebert B.L. Mak R.H. Ferrando A.A. et al.MicroRNA expression profiles classify human cancers.Nature. 2005; 435: 834-838Crossref PubMed Scopus (7741) Google Scholar This unique feature of cancer cells can be exploited to develop miRNA-sensitive, tumor-specific oncolytic viruses. In this study, we showed that inclusion of tumor-suppressive miRNA complementary target sequences into the CVB3 genome markedly reduces its virulence to normal tissues without compromising its anti-tumor potency. Moreover, we demonstrated that, in addition to KRASmut lung adenocarcinoma, CVB3 also acts as a potent oncolytic virus against TP53mut/RB1mut SCLC. As alluded to above, our recent in vitro and in vivo studies discovered that WT-CVB3 effectively destroys KRASmut lung adenocarcinoma.17Deng H. Liu H. de Silva T. Xue Y. Mohamud Y. Ng C.S. Qu J. Zhang J. Jia W.W.G. Lockwood W.W. Luo H. Coxsackievirus type B3 is a potent oncolytic virus against KRAS-mutant lung adenocarcinoma.Mol. Ther. Oncolytics. 2019; 14: 266-278Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Nonetheless, it was observed that the efficient tumor suppression is accompanied by damage to normal tissues, particularly the heart in immunocompromised mice. In this study, we aimed to genetically engineer the CVB3 genome to decrease its toxicity to normal tissues. The miRNAs miR-145 and miR-143 have been reported to be tumor suppressive and significantly downregulated in lung cancer tissues.22Guan P. Yin Z. Li X. Wu W. Zhou B. Meta-analysis of human lung cancer microRNA expression profiling studies comparing cancer tissues with normal tissues.J. Exp. Clin. Cancer Res. 2012; 31: 54Crossref PubMed Scopus (151) Google Scholar,23Volinia S. Calin G.A. Liu C.G. Ambs S. Cimmino A. Petrocca F. Visone R. Iorio M. Roldo C. Ferracin M. et al.A microRNA expression signature of human solid tumors defines cancer gene targets.Proc. Natl. Acad. Sci. USA. 2006; 103: 2257-2261Crossref PubMed Scopus (4696) Google Scholar To confirm their relative abundance in lung cancer versus normal tissues, quantitative PCR (qPCR) was conducted to measure the levels of miR-145 and miR-143 in various lung cancer and normal cells. As shown in Figures 1A and 1B , the expression of both miR-145 and miR-143 was significantly downregulated in lung cancer cells, including KRASmut lung adenocarcinoma cells (H2030, H23, and A549) and TP53mut/RB1mut SCLC cells (H524, H526), than in normal lung epithelial cells (BEAS2B and primary lung epithelial cells) and cardiomyocytes (mouse HL-1 cardiomyocytes and human induced pluripotent stem cell [iPSC]-derived cardiomyocytes [iCMs]). Also note that the levels of miR-145 and miR-143 in HeLa cells, in which WT-CVB3 and miR-CVB3 were grown and titered, were also very low. Our data suggest that miR-145 and miR-143 serve as candidate targets for restricting oncolytic CVB3 replication to tumor cells. We then engineered several miRNA-modified CVB3s, in which multiple copies of miR-145 target sequences alone (either in its forward or reverse orientation) or in combination with miR-143 target sequences (the core sequences of miR-145/miR-143 between mice and humans are 100% identical) were inserted into the 5′ UTR or 3′ UTR of the CVB3 genome to promote tumor-targeted viral replication. Among them, we found that miRNA-regulated CVB3s, in which four copies of miR-145 and two copies of miR-143 target sequences were inserted into either the 5′ UTR or 3′ UTR of the CVB3 genome (Figure 1C), displayed the least cardiotoxicity and the highest anti-tumor potency in vitro (Figure S1). In this study, we used miRNA (5′ UTR)-modified CVB3 (denoted hereinafter by miR-CVB3) to generate virus stocks for subsequent experiments. To test the safety of the newly generated recombinant CVB3, we assessed the cytotoxicity of miR-CVB3 toward mouse HL-1 cardiomyocytes, as this cell line has been extensively used to study CVB3-induced cardiac damage,24Wong J. Zhang J. Yanagawa B. Luo Z. Yang X. Chang J. McManus B. Luo H. Cleavage of serum response factor mediated by enteroviral protease 2A contributes to impaired cardiac function.Cell Res. 2012; 22: 360-371Crossref PubMed Scopus (26) Google Scholar and it highly expresses miR-145 and miR-143 (Figures 1A and 1B). After a 72-h viral infection over a range of multiplicity of infection (MOI) of 0.01 to 100, we demonstrated a significant reduction of cytotoxicity in miR-CVB3-treated cells in comparison with WT-CVB3-treated cells, as assessed through morphological observation (Figure 2A), crystal violet staining (Figure 2B), and a cell viability assay (Figure 2C). The decreased cardiotoxicity of miR-CVB3 was further verified in human cardiomyocytes (iCMs) by the cell viability assay (Figure 2D). We next sought to determine whether inclusion of miRNA targets to the CVB3 genome affects its lytic ability against lung cancer cells. Consistent with our previous observation that KRASmut lung adenocarcinoma cells are acutely permissive to WT-CVB3,17Deng H. Liu H. de Silva T. Xue Y. Mohamud Y. Ng C.S. Qu J. Zhang J. Jia W.W.G. Lockwood W.W. Luo H. Coxsackievirus type B3 is a potent oncolytic virus against KRAS-mutant lung adenocarcinoma.Mol. Ther. Oncolytics. 2019; 14: 266-278Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar we found that WT-CVB3 efficiently killed KRASmut lung adenocarcinoma cells (H2030, H23, A549) in a dose-dependent manner (Figure 3). Moreover, we found that miR-CVB3 retained its ability and potency to lyse KRASmut cells, although at a slightly reduced level compared to WT-CVB3. As discussed earlier, SCLC is the most aggressive subtype of lung cancer and is associated with poor prognosis.4Koinis F. Kotsakis A. Georgoulias V. Small cell lung cancer (SCLC): no treatment advances in recent years.Transl. Lung Cancer Res. 2016; 5: 39-50PubMed Google Scholar Most patients with SCLC are diagnosed late when the cancer is already advanced and there is no targeted therapy for SCLC. In the current study, we extended our previous work on NSCLC to investigate the ability of both WT-CVB3 and miR-CVB3 in killing SCLC. H524 and H526, two SCLC cell lines carrying TP53/RB1 mutations characteristic of this disease, were utilized in this study. Following a 72-h viral infection at an MOI of 0.1, 1, and 10, both WT-CVB3 and miR-CVB3 caused severe cytopathic effects at all viral doses (Figure 4A). Furthermore, cell viability assays revealed that miR-CVB3 dose-dependently destroyed these cells in a comparable manner to WT-CVB3 (Figure 4B). Lastly, BEAS2B, a normal human lung epithelial cell line that expresses elevated levels of miR-145 and miR-143 (Figures 1A and 1B), was used to examine the possible cytotoxicity of miR-CVB3 toward normal lung cells. In agreement with our earlier report,17Deng H. Liu H. de Silva T. Xue Y. Mohamud Y. Ng C.S. Qu J. Zhang J. Jia W.W.G. Lockwood W.W. Luo H. Coxsackievirus type B3 is a potent oncolytic virus against KRAS-mutant lung adenocarcinoma.Mol. Ther. Oncolytics. 2019; 14: 266-278Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar infection with WT-CVB3 caused a marginal cytopathic effect and cell death at the higher MOIs examined (Figures 4C and 4D). However, as anticipated, we observed that miRNA modification of CVB3 significantly reduced the toxicity induced by WT-CVB3 to normal lung epithelial cells (Figure 4D). We further determined whether reduced cytotoxicity in cardiomyocytes and normal lung epithelial cells is a consequence of decreased viral infectivity in these cells. A median tissue culture infection dose (TCID50) assay was performed on supernatant collected from various cell types treated with WT-CVB3 or miR-CVB3 at an MOI of 0.1 for 36 h as indicated to measure infectious viral titers. As shown in Figure 5A, viral titers were strikingly decreased in miR-CVB3-treated HL-1 cardiomyocytes in comparison with WT-CVB3-treated cells. Although a significant decrease in miR-CVB3 titers in H526 TP53mut/RB1mut cells was also observed, the ratio of reduction was much less as compared to that in cardiomyocytes. We also examined the kinetics of viral RNA replication (Figure 5B) and titers (Figure 5C) in different cell lines incubated with WT-CVB3 or miR-CVB3 at an MOI of 10 for indicated times by qPCR and a TCID50 assay, respectively. We showed that starting at 5–7 h post-infection, viral RNA copy numbers and titers were considerably lower in miR-CVB3-treated as compared to WT-CVB3-treated HL-1 cardiomyocytes (Figures 5B and 5C, left panels). It was also found that viral RNA copies and titers were decreased in H2030 KRASmut and H526 TP53mut/RB1mut cells upon miR-CVB3 treatment, but at a much lesser extent (Figures 5B and 5C, middle and right panels). Taken together, these data suggest that the miR-modified version of CVB3 has an increased therapeutic window between cancer and normal cells than does the WT counterpart. After verification of tumor specificity and efficacy of the miR-CVB3 in vitro, we next characterized its safety and effectiveness in vivo using a non-obese diabetic (NOD)-severe combined immunodeficiency (SCID) xenograft mouse model. Since CVB3 infection triggers more severe inflammation in males than in females,25Fairweather D. Cooper Jr., L.T. Blauwet L.A. Sex and gender differences in myocarditis and dilated cardiomyopathy.Curr. Probl. Cardiol. 2013; 38: 7-46Crossref PubMed Scopus (127) Google Scholar only male mice were used in this study to determine the oncolytic efficacy and toxicity of miR-CVB3. We first tested the possible systemic toxicity of miR-CVB3 in NOD-SCID mice without a prior tumor implantation. Mice were intraperitoneally inoculated with either WT-CVB3 (n = 4) or miR-CVB3 (n = 5) at a single dose of 1 × 108 plaque-forming units (PFU) for 14 days, which represents the peak time of CVB3-induced tissue injuries. Various mouse organs were then harvested for the analysis of tissue damage and viral infection. Consistent with our early report,17Deng H. Liu H. de Silva T. Xue Y. Mohamud Y. Ng C.S. Qu J. Zhang J. Jia W.W.G. Lockwood W.W. Luo H. Coxsackievirus type B3 is a potent oncolytic virus against KRAS-mutant lung adenocarcinoma.Mol. Ther. Oncolytics. 2019; 14: 266-278Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar NOD-SCID mice treated with WT-CVB3 showed severe toxicity, and only one mouse survived (25% survival rate) throughout the time course, while all mice treated with miR-CVB3 survived (100% survival rate) to the end of the experiment (Figure 6A). Hematoxylin and eosin (H&E) staining (Figure 6B) and pathological quantitation (Figure 6C) revealed a massive inflammatory infiltration and necrosis in the heart of mice treated with WT-CVB3. Remarkably, we observed no apparent cardiac pathology in mice treated with miR-CVB3. There were also no evident damages to other organs, including the lung, liver, and spleen, in both WT-CVB3- and miR-CVB3-treated mice. Modest pancreatic pathology was observed in both groups (Figures 6B and 6C). Viral quantitation by VP1 immunostaining (Figures 6D and 6E) and plaque assay (Figure 6F) demonstrated a significant reduction in viral protein VP1 expression (almost undetectable) and viral titers (i.e., ~1 million-fold lower) in the heart of miR-CVB3 mice as compared to WT-CVB3 mice, indicating that decreased cardiovirulence in miR-CVB3 mice is mainly due to reduced viral replication. It was also observed that VP1 expression (nearly undetectable) and viral titers were significantly decreased in the pancreas of miR-CVB3 mice compared to WT-CVB3 mice (Figures 6D–6F). Finally, a different cohort of NOD-SCID mice was used to generate the H526-derived TP53mut/RB1mut SCLC xenograft model to determine the anti-tumor potency and the long-term toxicity of miR-CVB3. The mouse model was established through subcutaneous injection of H526 cells (1 × 107 cells) into the left and right flank of the mice. After ~10 days, the implanted tumor reached a palpable size. WT-CVB3 or miR-CVB3 was then given via intraperitoneal injection as described above. Mice treated with PBS were used as controls. Similar to the observations made in cultured cells (Figures 1A and 1B), we showed that the levels of miR-145 and miR-143 were significantly lower in implanted SCLC as compared to normal mouse tissues, including heart, pancreas, lung, liver, spleen, kidney, intestine, and brain (Figures 7A and 7B ). Kaplan-Meier survival analysis revealed that all mice treated with WT-CVB3 succumbed to death or had to be euthanized due to severe morbidity associated with viral infection at or prior to day 15 post-injection, whereas 100% of miR-CVB3-treated mice survived until day 35 post-infection, and the overall survival rate in the miR-CVB3 group at day 56 post-infection was 57.14% (Figure 7C). In the sham group, six out of eight PBS-treated mice were euthanized prior to day 25 because of the exceeded tumor size (>20 mm in diameter) according to animal care guidelines. Tumor size analysis showed that implanted H526-derived tumors in PBS-treated mice continued to grow until the end of experiment, while upon WT-CVB3 or miR-CVB3 treatment the tumor volumes were markedly reduced in a comparable manner between the two groups (Figure 7D). Tumor weight measurement on day 25 also showed a substantial reduction in mice treated with miR-CVB3 compared with PBS treatment (Figure 7E). Tumor volumes remained very small or undetectable in miR-CVB3-treated mice until the experimental endpoint (i.e., day 56 post-infection, data not shown). Viral titers were extremely low in the heart and lung, but moderately high in the pancreas and tumor, of mice injected with miR-CVB3 on day 25 (Figure 7F). There was no evident tissue damage in mice treated with miR-CVB3 or PBS on day 25 post-treatment (Figure 7G, pathological scores were not shown due to undetectable pathology). For long-term toxicity analysis (i.e., after day 35 post-infection), we found that mice that survived the entire experimental period (i.e., day 56) displayed no tissue damage and VP1 positive staining, similar to the data shown in Figure 7C. However, evident cardiotoxicity and positive VP1 staining was observed in the heart and pancreas of mice that died between day 35 and 56 post-treatment (Figure S2A). To determine whether the recurrence of cardiotoxicity is due to genome instability of the miR-CVB3, sequencing was conducted, confirming the loss of inserted miRNA target sequences (Figure S2B). Future investigation is needed to address this issue. Taken together, our data suggest that miR-CVB3 retains the oncolytic effectiveness against TP53mut/RB1mut SCLC, but with substantially decreased tissue toxicity. In this study, we genetically modified the CVB3 genome through a miRNA-targeting approach. Using both in vitro cell culture and in vivo mouse xenograft models, we demonstrated that miR-CVB3 infection is specific to KRASmut lung adenocarcinoma and TP53mut/RB1mut SCLC cells with little to no observable damage to normal lung or heart cells, leading to significant tumor regression and improved overall survival. The oncolytic effect of miR-CVB3 is very powerful, as evidenced by the observation that one dose of viral injection through systemic administration results in more than 90% reduction of tumor volume. The advantage of systemic delivery of oncolytic virus is apparent. In addition to being clinically feasible, it also has potential to kill metastatic tumors. The selection of miR-145/miR-143 for the present study is based on our prior experience using their target sequences to augment tumor selectivity of oncolytic herpes simplex virus-1,26Lee C.Y. Rennie P.S. Jia W.W. MicroRNA regulation of oncolytic herpes simplex virus-1 for selective killing of prostate cancer cells.Clin. Cancer Res. 2009; 15: 5126-5135Crossref PubMed Scopus (80) Google Scholar,27Li J.M. Kao K.C. Li L.F. Yang T.M. Wu C.P. Horng Y.M. Jia W.W. Yang C.T. MicroRNA-145 regulates oncolytic herpes simplex virus-1 for selective killing of human non-small cell lung cancer cells.Virol. J. 2013; 10: 241Crossref PubMed Scopus (28) Google Scholar and on our current evidence that expression levels of miR-145/miR-143 are particularly low in various types of lung cancer cells in comparison to normal lung epithelial cells and cardiomyocytes. This differential expression pattern enables selective viral replication in lung cancer cells. miR-145/miR-143, together with many other miRNAs, have been revealed to function as tumor suppressors by negatively regulating the expression of multiple oncogenes.28Shenouda S.K. Alahari S.K. MicroRNA function in cancer: oncogene or a tumor suppressor?.Cancer Metastasis Rev. 2009; 28: 369-378Crossref PubMed Scopus (543) Google Scholar Oncogenic protein RAS has been shown to transcriptionally suppresses miR-145/miR-143,29Kent O.A. Chivukula R.R. Mullendore M. Wentzel E.A. Feldmann G. Lee K.H. Liu S. Leach S.D. Maitra A. Mendell J.T. Repression of the miR-143/145 cluster by oncogenic Ras initiates a tumor-promoting feed-forward pathway.Genes Dev. 2010; 24: 2754-2759Crossref PubMed Scopus (243) Google Scholar whereas activation of tumor suppressor protein p53 causes upregulation of this miRNA cluster.30Suzuki H.I. Yamagata K. Sugimoto K. Iwamoto T. Kato S. Miyazono K. Modulation of microRNA processing by p53.Nature. 2009; 460: 529-533Crossref PubMed Scopus (907) Google Scholar As a RNA virus, the CVB3 genome is less stable than that of DNA viruses. To reduce the potential loss of miRNA target function due to viral RNA mutation, we used different miRNA targets and" @default.
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• W3002247662 title "MicroRNA Modification of Coxsackievirus B3 Decreases Its Toxicity, while Retaining Oncolytic Potency against Lung Cancer" @default.
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