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  • RNA polymerase II activity of type 3 Pol III promoters
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-05-08
    Zongliang Gao, Elena Herrera-Carrillo, Ben Berkhout

    In eukaryotes, three RNA polymerases (Pol I, II and III) are responsible for the transcription of distinct subsets of genes. Gene-external type 3 Pol III promoters use defined transcription start and termination sites and are therefore widely used for small RNA expression, including short hairpin RNAs in RNAi applications and guide RNAs in CRISPR-Cas systems. We report that all three commonly used human Pol III promoters (7SK, U6 and H1) mediate luciferase reporter gene expression, which indicates Pol II activity, but to a different extent (H1>>U6>7SK). We demonstrate that these promoters can recruit Pol II for transcribing extended messenger transcripts. Intriguingly, selective inhibition of Pol II stimulates the Pol III activity and vice versa, suggesting that two polymerase complexes compete for promoter usage. Pol II initiates transcription at the regular Pol III start site on the 7SK and U6 promoters, but Pol II transcription on the most active H1 promoter starts 8 nucleotides upstream of the Pol III start site. This study provides functional evidence for the close relationship of Pol II and Pol III transcription. These mechanistic insights are important for optimal use of Pol III promoters and offer additional flexibility for biotechnology applications of these genetic elements.

    更新日期:2018-05-08
  • Circulating miRNAs as predictive biomarkers of type 2 diabetes mellitus development in coronary heart disease patients from the CORDIOPREV study
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-05-08
    Rosa Jimenez-Lucena, Oriol Alberto Rangel-Zúñiga, Juan Francisco Alcalá-Díaz, Javier Lopez-Moreno, Irene Roncero-Ramos, Helena Molina-Abril, Elena Yubero-Serrano, Javier Caballero-Villarraso, Javier Delgado-Lista, Justo Pastor Castaño, Jose Maria Ordovás, Pablo Pérez-Martinez, Antonio Camargo, Jose Lopez-Miranda

    Circulating miRNAs have been proposed as type 2 diabetes biomarkers, and may be a more sensitive way to predict development of the disease than the currently used tools. Our aim was to identify whether circulating miRNAs, added to clinical and biochemical markers, could have better potential predicting type 2 diabetes. The study included 462 non-diabetic patients at baseline in the CORDIOPREV study. After a median follow-up of 60 months, 107 of them developed type 2 diabetes. Plasma levels of 24 miRNAs were measured at baseline by qRT-PCR and other strong biomarkers to predict diabetes were determined. The ROC-analysis identified 9 miRNAs, which added to HbA1c, have a greater predictive value in early diagnosis of type 2 diabetes (AUC = 0.8342) than HbA1c alone (AUC = 0.6950). The miRNAs and HbA1c based model did not improve when the FINDRISC was included (AUC=0.8293). Cox-regression analyses showed that patients with low miR-103, miR-28-3p, miR-29a, miR-9 and high miR-30a-5p and miR-150 circulating levels have higher risk of disease (HR= 11.27; 95% CI: 2.61 – 48.65). Our results suggest that circulating miRNAs could potentially be used as a new tool for predicting the development of type 2 diabetes in clinical practice.

    更新日期:2018-05-08
  • Self-amplifying Replicon RNA delivery to Dendritic Cells by Cationic Lipids
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-05-04
    Pavlos C. Englezou, Cedric Sapet, Thomas Démoulins, Panagiota Milona, Thomas Ebensen, Kai Schulze, Carlos-Alberto Guzman, Florent Poulhes, Olivier Zelphati, Nicolas Ruggli, Kenneth C. McCullough

    Advances in RNA technology during the past two decades have led to the construction of replication-competent RNA termed replicons, RepRNA or self-amplifying mRNA with high potential for vaccine applications. Cytosolic delivery is essential for their translation and self-replication - without infectious progeny generation - providing high levels of antigen expression for inducing humoral and cellular immunity. Synthetic nanoparticle-based delivery vehicles can both protect the RNA molecules and facilitate targeting of dendritic cells – critical for immune defence development. Several cationic lipids were assessed with RepRNA generated from classical swine fever virus encoding nucleoprotein genes of influenza A virus. The non-cytopathogenic nature of the RNA allowed targeting to dendritic cells without destroying the cells – important for prolonged antigen production and presentation. Certain lipids were more effective at delivery and at promoting translation of RepRNA than others. Selection of particular lipids provided delivery to dendritic cells that did result in translation, demonstrating that delivery efficiency could not guarantee translation. The observed translation in vitro was reproduced in vivo by inducing immune responses against the encoded influenza virus antigens. Cationic lipid-mediated delivery shows potential for promoting RepRNA vaccine delivery to dendritic cells, particularly if combined with additional delivery elements.

    更新日期:2018-05-04
  • Parallel genome-wide profiling of coding and non-coding RNAs to identify novel regulatory components in embryonic heart development and maturated heart
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-05-04
    Davood Sabour, Rui S.R. Machado, Jose P. Pinto, Susan Rohani, Raja Ghazanfar Ali Sahito, Jürgen Hescheler, Matthias E. Futschik, Agapios Sachinidis

    Heart development is a complex process, tightly regulated by numerous molecular mechanisms. Key components of the regulatory network underlying heart development are transcription factors (TFs) and microRNAs (miRNAs), yet limited investigation of the role of miRNAs in heart development has taken place. Here, we report the first parallel genome-wide profiling of polyadenylated RNAs and miRNAs in a developing murine heart. These data enable us to identify dynamic activation or repression of numerous biological processes and signaling pathways. More than 200 miRNAs and 25 long non-coding RNAs were differentially expressed during embryonic heart development compared to the mature heart; most of these had not been previously associated with cardiogenesis. Integrative analysis of expression data and potential regulatory interactions suggested 28 miRNAs as novel regulators of embryonic heart development representing a considerable expansion of the current repertoire of known cardiac miRNAs. To facilitate follow-up investigations, we constructed HeartMiR (http://heartmir.sysbiolab.eu), an open access database and interactive visualization tool for the study of gene regulation by miRNAs during heart development.

    更新日期:2018-05-04
  • MicroRNA-125b promotes hepatic stellate cell activation and liver fibrosis by activating RhoA signaling
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-05-03
    Kai You, Song-Yang Li, Jiao Gong, Jian-Hong Fang, Chong Zhang, Min Zhang, Yunfei Yuan, Jine Yang, Shi-Mei Zhuang

    MiR-125b is frequently dysregulated in different diseases. Activation of hepatic stellate cells (HSCs) is a critical event during liver fibrogenesis. However, the function and its underlying mechanism of miR-125b in HSC activation and liver fibrosis are still unknown. Here, we showed that miR-125b was upregulated in HSCs but not in hepatocytes during hepatic fibrogenesis in vivo and upon culture-activation in vitro. Inhibition of miR-125b suppressed the expression of profibrogenic genes in culture-activated primary HSCs and reduced the basal and transforming growth factor beta (TGF-β)-induced α-SMA expression and cell contraction of immortalized HSC cell line. In contrast, ectopic expression of miR-125b promoted α-SMA expression and HSC contraction. Moreover, antagonizing miR-125b in vivo significantly alleviated liver fibrosis in CCl4-treated mice. Mechanistically, overexpression of miR-125b in HSC enhanced RhoA activity by directly targeting StAR-related lipid transfer (START) domain containing 13 (Stard13), a RhoA specific GTPase-activating protein, whereas knockdown of miR-125b abrogated RhoA activation. Furthermore, inhibition of RhoA or its downstream molecules, Mrtf-A and Srf, attenuated the miR-125b-induced α-SMA expression and HSC contraction. Therefore, our findings identify a miR-125b-Stard13-RhoA-α-SMA signaling cascade in HSC and highlight its importance in hepatic fibrosis.

    更新日期:2018-05-03
  • Translating SOD1 gene silencing towards the clinic: A highly efficacious, off-target free and biomarker-supported strategy for familial ALS
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-05-03
    T. Iannitti, J.M. Scarrott, S. Likhite, I.R.P. Coldicott, K.E. Lewis, P.R. Heath, A. Higginbottom, M.A. Myszczynska, M. Milo, G.M. Hautbergue, K. Meyer, B.K. Kaspar, L. Ferraiuolo, P.J. Shaw, M. Azzouz

    Twenty per cent of familial amyotrophic lateral sclerosis (fALS) cases are caused by mutations in the gene encoding human cytosolic Cu/Zn superoxide dismutase (hSOD1). Efficient translation of the therapeutic potential of interfering RNA (RNAi) for the treatment of SOD1-ALS patients requires the development of vectors that are free of significant off-target effects and with reliable biomarkers to discern sufficient target engagement and correct dosing. Using adeno-associated virus serotype 9 to deliver RNAi against hSOD1 in the SOD1G93A mouse model, we found that intrathecal injection of the therapeutic vector via the cisterna magna delayed onset of disease, decreased motor neuron death at end stage by up to 88% and prolonged the median survival of SOD1G93A mice by up to 42%. To our knowledge this is the first report to demonstrate no significant off-target effects linked to hSOD1 silencing, providing further confidence in the specificity of this approach. We also report the measurement of cerebrospinal fluid (CSF) hSOD1 protein levels as a biomarker of effective dosing and efficacy of hSOD1 knockdown. Together, these data provide further confidence in the safety of the clinical therapeutic vector. The CSF biomarker will be a useful measure of biological activity for translation into human clinical trials.

    更新日期:2018-05-03
  • Targeting the IGF1R pathway in breast cancer using antisense lncRNA-mediated promoter cis competition
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-05-03
    Lingling Pian, Xue Wen, Lihua Kang, Zhaozhi Li, Yuanyuan Nie, Zhonghua Du, Dehai Yu, Lei Zhou, Lin Jia, Naifei Chen, Dan li, Songling Zhang, Wei Li, Andrew R. Hoffman, Jingnan Sun, Jiuwei Cui, Ji-Fan Hu

    Aberrant insulin-like growth factor I receptor (IGF1R) signaling pathway serves as a well-established target for cancer drug therapy. The intragenic antisense long noncoding RNA (lncRNA) IRAIN, a putative tumor suppressor, is downregulated in breast cancer cells, while IGF1R is overexpressed, leading to an abnormal IGF1R/IRAIN ratio that promotes tumor growth. To precisely target this pathway, we developed an “antisense lncRNA-mediated intragenic cis competition” (ALIC) approach to therapeutically correct the elevated IGF1R/IRAIN bias in breast cancer cells. We used CRISPR-Cas9 gene editing to target the weak promoter of IRAIN antisense lncRNA and showed that in targeted clones, intragenic activation of the antisense lncRNA potently competed in cis with the promoter of the IGF1R sense mRNA. Notably, the normalization of IGF1R/IRAIN transcription inhibited the IGF1R signaling pathway in breast cancer cells, decreasing cell proliferation, tumor sphere formation, migration, and invasion. Using “nuclear RNA reverse transcription-associated trap” sequencing, we uncovered an IRAIN lncRNA-specific interactome containing gene targets involved in cell metastasis, signaling pathways, and cell immortalization. These data suggest that aberrantly upregulated IGF1R in breast cancer cells can be precisely targeted by cis transcription competition, thus providing a useful strategy to target disease genes in the development of novel precision medicine therapies.

    更新日期:2018-05-03
  • Regulated zinc finger nucleases (ZFNs) by HIV-1 Tat to excise HIV-1 from host genome in infected and latently infected cells
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-05-03
    Haiyan Ji, Panpan Lu, Baochi Liu, Xiying Qu, Yanan Wang, Zhengtao Jiang, Xinyi Yang, Yangcheng Zhong, He Yang, Hanyu Pan, Lin Zhao, Jianqing Xu, Hongzhou Lu, Huanzhang Zhu

    Highly active anti-retroviral therapy (HAART) cannot clear the infected cells harboring HIV-1 proviral DNA from the HIV-1 infected patients. We previously demonstrated that Zinc-finger nucleases (ZFNs) could specifically and efficiently excise HIV-1 proviral DNA from the latently infected human T cells by targeting the long terminal repeats (LTRs), a novel and alternative antiretroviral strategy for eradicating HIV-1 infection. To prevent the unwanted off-target effects from constantly expressed ZFNs, in this study, we engineered the expression of ZFNs under the control of HIV-1 LTR, by which ZFNs expression can be activated by the HIV-1 Tat protein. Our results show that functional expression of ZFNs induced by Tat excised the integrated proviral DNA of HIV-NL4-3-eGFP in approximate 30% population of HIV-1 infected cells. Meanwhile, the results from HIV-1 infected human primary T cells and latently infected T cells treated with the regulated ZFNs further validated proviral DNA could be excised. Taken together, positively regulated expression of ZFNs in the presence of HIV-1 Tat may provide a safer and novel implementation of the genome-editing technology for eradicating the HIV-1 proviral DNA from the infected host cells.

    更新日期:2018-05-03
  • Thermoresponsive bacteriophage nanocarrier as a gene delivery vector targeted to the gastrointestinal tract
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-05-01
    Katawut Namdee, Mattaka Khongkow, Suwimon Boonrungsiman, Naiyaphat Nittayasut, Paladd Asavarut, Sasithon Temisak, Nattika Saengkrit, Satit Puttipipatkhachorn, Amin Hajitou, Kiat Ruxrungtham, Teerapong Yata

    The use of the gastrointestinal tract as a site for the local delivery of DNA is an exciting prospect. In order to obtain an effective vector capable of delivering a gene of interest to target cells to achieve sufficient and sustained transgene expression, with minimal toxicity, we developed a new generation of filamentous bacteriophage. This particular bacteriophage was genetically engineered to display an arginine–glycine–aspartic acid (RGD) motif (an integrin-binding peptide) on the major coat protein pVIII and carry a mammalian DNA cassette. One unanticipated observation is the thermoresponsive behavior of engineered bacteriophage. This finding has led us to simplify the isolation method to purify bacteriophage particles from cell culture supernatant by low-temperature precipitation. Our results showed that in contrast to non-surface modified, the RGD-modified bacteriophage was successfully used to deliver a transgene to mammalian cells. Our in vitro model of the human intestinal follicle-associated epithelium also demonstrated that bacteriophage particles were stable in simulated gastrointestinal fluids and able to cross the human intestinal barrier. In addition, we confirmed an adjuvant property of the engineered bacteriophage to induce nitric oxide production by macrophages. In conclusion, our study demonstrated the possibility of using bacteriophage for gene transfer in the gastrointestinal tract.​

    更新日期:2018-05-02
  • Stable transcriptional repression and parasitism of HIV-1
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-05-01
    Surya Shrivastava, Paige Charlins, Amanda Ackley, Heather Embree, Boro Dropulic, Ramesh Akkina, Marc S. Weinberg, Kevin V. Morris

    Gene-based therapies represent a promising treatment for HIV-1 infection, as they offer the potential for sustained viral inhibition and reduced treatment interventions. One approach developed here involves using conditionally replicating vectors (CR-vectors). CR-vectors utilize HIV-expressed proteins to replicate and disseminate along with HIV into the budding viral particles, thereby co-infecting target cellular reservoirs. We generated and characterized several CR-vectors carrying various therapeutic payloads of non-coding RNAs targeted to HIV-1; both transcriptionally and post-transcriptionally. Both virus and vector expression was followed in cell culture systems and T-cells in the presence and absence of mycophenolic acid (MPA) selection. We find here that CR-vectors functionally suppress HIV expression in a long-term stable manner and that transcriptional targeting of and epigenetic silencing of HIV can be passaged to newly infected cells by the action of the CR-vector, ultimately establishing a sustained parasitism of HIV. Our findings suggest that CR-vectors with modulatory non-coding RNAs may be a viable approach to achieving long-term sustained suppression of HIV-1 leading ultimately to a functional cure.

    更新日期:2018-05-02
  • Increased frataxin expression induced in Friedreich ataxia cells by platinum TALE-VP64s or platinum TALE-SunTag
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-04-27
    Khadija Cherif, Catherine Gérard, Joël Rousseau, Dominique L. Ouellet, Pierre Chapdelaine, Jacques P. Tremblay

    Frataxin gene (FXN) expression is reduced in Friedreich's ataxia patients due to an increase in the number of GAA trinucleotides in intron 1. The frataxin protein, encoded by that gene, plays an important role in mitochondria’s iron metabolism. Platinum TALE (plTALE) proteins targeting the regulatory region of the FXN gene, fused with a transcriptional activator (TA), such as VP64 or P300, were used to increase the expression of that gene. Many effectors plTALEVP64, plTALEp300 and plTALESunTag targeting 14 sequences of the FXN gene promoter or intron 1 were produced. This permitted to select 3 plTALEVP64s and 2 plTALESunTag that increased FXN gene expression by up to 19 folds in different FRDA primary fibroblasts. Adeno-Associated Viruses were used to deliver the best effectors to the YG8R mouse model to validate their efficiencies in vivo. Our results showed that these selected plTALEVP64s or plTALESunTag induced transcriptional activity of the endogenous FXN gene as well as expression of the frataxin protein in YG8R mouse heart by 10 folds and in skeletal muscles by up to 35 folds. The aconitase activity is positively modulated by the frataxin level in mitochondria and was thus increased in vitro and in vivo by the increased frataxin expression.

    更新日期:2018-04-27
  • Sp1 Suppresses MiR-3178 to Promote Metastasis Invasion Cascade via Upregulation of TRIOBP
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-04-26
    Hui Wang, Kai Li, Yu Mei, Xuemei Huang, Zhenglin Li, Qingzhu Yang, Huanjie Yang

    Specificity protein (Sp1) plays an important role in invasion-metastasis cascade. Sp1 regulation on protein coding genes have been extensively investigated, however little is known about its regulation on protein non-coding genes. In this study, miR-3178 is reported as a novel target of Sp1 in multiple cancer cell models. Sp1 functions as its transcriptional suppressor as evidenced by luciferase reporter and ChIP assays. In line with the pro-metastatic role of Sp1, miR-3178 exerts anti-metastasis function. Overexpression of miR-3178 inhibits both migration and invasion of highly metastatic prostate, lung and breast cancer cells while antagonizing miR-3178 promotes those events in their lowly metastatic counterparts. In vivo study demonstrates that miR-3178 suppresses the tail vein inoculated prostate cancer cells to form colonies in lung, lymph node and liver of BALB/c nude mice. MiR-3178 directly targets the 3' UTR of TRIOBP-1 and TRIOBP-5, the two isoforms of TRIOBP expressed in prostate, lung and breast cancer cells. Furthermore, overexpression of TRIOBP-1 could rescue miR-3178 inhibition on cell migration and invasion. Collectively, our findings reveal the regulatory axis of Sp1/miR-3178/TRIOBP in metastasis cascade. Our results suggest miR-3178 as a promising application to suppress metastasis in Sp1 overexpressed cancers.

    更新日期:2018-04-26
  • The ASH1-miR-375-YWHAZ signaling axis regulates tumor properties in hepatocellular carcinoma
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-04-25
    Juan-Feng Zhao, Qiu Zhao, Hui Hu, Jia-Zhi Liao, Ju-Sheng Lin, Chao Xia, Ying Chang, Jing Liu, An-Yuan Guo, Xing-Xing He

    Hepatocellular carcinoma (HCC) is a worldwide malignance and the underlying mechanisms of this disease are not fully elucidated. In this study, the existence and function of ASH1-miR-375-YWHAZ signaling axis in HCC were determined. Our experiments and TCGA sequencing data analyses showed that ASH1 and miR-375 were significantly down-regulated, while YWHAZ was significantly up-regulated in HCC. Furthermore, we found that ASH1 positively regulates miR-375, and miR-375 directly down-regulates its target YWHAZ. Gain- and loss-of-function study demonstrated ASH1 and miR-375 function as tumor suppressors, while YWHAZ acts as an oncogene in HCC. Animal experiment indicated that si-YWHAZ delivered by nanoliposomes could suppress the growth of hepatoma xenografts and was well tolerant by nude mice. Further studies revealed that YWHAZ was involved in several protein networks such as cell autophagy, EMT, apoptosis, cell cycle, invasion and migration. In addition, the patient group with ASH1-high-expression-miR-375-high-expression-YWHAZ-low-expression was correlated with a better clinical prognosis compared with the opposite expression group. In conclusion, we proved the existence of ASH1-miR-375-YWHAZ signaling axis and interpreted its important role in driving HCC tumor progression.

    更新日期:2018-04-25
  • miR-24 and miR-122 negatively regulate transforming growth factor-β/Smad signaling pathway in skeletal muscle fibrosis
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-04-22
    Yaying Sun, Hui Wang, Yan Li, Shaohua Liu, Jiwu Chen, Hao Ying

    Fibrosis is common after skeletal muscle injury, undermining tissue regeneration and function. The mechanism underlying skeletal muscle fibrosis remains unveiled. Transforming growth factor-β/Smad signaling pathway is supposed to play a pivotal role. However, how microRNAs interact with transforming growth factor-β/Smad related muscle fibrosis remains unclear. We showed that miR-24-3p and miR-122-5p declined in skeletal muscle fibrosis, which was a consequence of transforming growth factor-β. Up-regulating Smad4 suppressed two microRNAs, while inhibiting Smad4 elevated microRNAs. Luciferase reporter assay and chromatin immunoprecipitation confirmed that Smad4 directly inhibited two microRNAs. On the other hand, overexpression of these two miRs retarded fibrotic process. We further identified that Smad2 was a direct target of miR-24-3p, while miR-122-5p targeted transforming growth factor-β receptor-II. Both targets were important participants in transforming growth factor-β/Smad signaling. Taken together, a positive feedback loop in transforming growth factor-β/Smad4 signaling pathway in skeletal muscle fibrosis was identified. Transforming growth factor-β/Smad axis could be down-regulated by microRNAs. This effect, however, was suppressed by Smad4, the downstream of transforming growth factor-β.

    更新日期:2018-04-25
  • Antibody-antisense oligonucleotide conjugate downregulates a key gene in glioblastoma stem cells
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-04-19
    Amy E. Arnold, Elise Malek-Adamian, Phuong U. Le, Anika Meng, Saúl Martínez-Montero, Kevin Petrecca, Masad J. Damha, Molly S. Shoichet

    Glioblastoma stem cells (GSCs) are invasive, treatment-resistant brain cancer cells that express downregulated in renal cell carcinoma (DRR), also called FAM107A, a genetic driver of GSC invasion. We developed antibody-antisense oligonucleotide conjugates to target and reduce DRR/FAM107A expression. Specifically, we used antibodies against antigens expressed on the glioblastoma stem cells, such as CD44 and EphA2, conjugated to chemically modified antisense oligonucleotides (AONs) against DRR/FAM107A, which were designed as chimeras of DNA and 2ʹ-deoxy-2ʹ-fluoro-beta-D-arabinonucleic acid (FANA) for increased nuclease stability and mRNA affinity. We demonstrate that these therapeutic conjugates successfully internalize, accumulate, and reduce DRR/FAM107A expression in patient-derived GSCs. This is the first example of an antibody-antisense strategy against cancer stem cells.

    更新日期:2018-04-25
  • Promoter-operating targeted expression of gene therapy in cancer: current stage and prospect
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-04-12
    Chao Chen, Dongxu Yue, Liangyu Lei, Hairong Wang, Jia Lu, Ya Zhou, Shiming Liu, Tao Ding, Mengmeng Guo, Lin Xu

    The technique of targeted expression of interesting genes, including distinct delivery systems and specific gene promoter-operating expression, is an important strategy for gene therapy against cancers. Up to now, extensive literatures documented the efficacy of distinct delivery systems, such as liposome system, nano-partical system and PEI system, and so on, in cancer gene therapy. However, related document on the potential value of using specific gene promoter, such as tumor suppressor, in cancer gene therapy was still scare. The main obstacle might be the selection of ideal gene promoter to operate interesting gene expression in cancer gene therapy is still not fully understood. Therefore, many efforts need to be done in order to make it a real power tool for the human clinical treatment for cancer patients. The purpose of this review is to clarify the current state and some problematics on development of promoter-operating targeted expression of interesting genes and highlight its potential in cancer gene therapy.

    更新日期:2018-04-12
  • miR-199a-3p modulates MTOR and PAK4 pathways and inhibits tumor growth in a hepatocellular carcinoma transgenic mouse model
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-04-12
    Elisa Callegari, Lucilla D’Abundo, Paola Guerriero, Carolina Simioni, Bahaeldin K. Elamin, Marta Russo, Alice Cani, Cristian Bassi, Barbara Zagatti, Luciano Giacomelli, Stella Blandamura, Farzaneh Moshiri, Simona Ultimo, Antonio Frassoldati, Giuseppe Altavilla, Laura Gramantieri, Luca Maria Neri, Silvia Sabbioni, Massimo Negrini

    Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. Prognosis is poor, and therapeutic options are limited. microRNAs (miRNAs) have emerged as potential therapeutic molecules against cancer. Here, we investigated the therapeutic efficacy of miR-199a-3p, a miRNA highly expressed in normal liver and downregulated in virtually all HCCs. The therapeutic value of miR-199a-3p mimic molecules was assayed in the TG221 mouse, a transgenic model highly predisposed to the development of liver cancer. Administration of miR-199a-3p mimics in the TG221 transgenic mouse showing liver cancer led to a significant reduction of number and size of tumor nodules compared to control animals. In vivo delivery confirmed protein downregulation of the miR-199a-3p direct targets, mechanistic target of rapamycin (MTOR) and p21 activated kinase 4 (PAK4), ultimately leading to the repression of FOXM1. Remarkably, the anti-tumor activity of miR-199a-3p mimics was comparable to that obtained with sorafenib. These results suggested that miR-199a-3p may be considered a promising HCC therapeutic option.

    更新日期:2018-04-12
  • De novo synthesis of elastin by exogenous delivery of synthetic modified messenger RNA into skin and elastin deficient cells
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-30
    Mario Lescan, Regine Perl, Sonia Golombek, Martin Pilz, Ludmilla Hann, Mahua Yasmin, Andreas Behring, Timea Keller, Andrea Nolte-Karayel, Franziska Gruhn, Efrat Kochba, Yotam Levin, Christian Schlensak, Hans Peter Wendel, Meltem Avci-Adali

    Elastin is one of the most important and abundant extracellular matrix (ECM) protein that provides elasticity and resilience to tissues and organs, including vascular walls, ligaments, skin, and lung. Besides hereditary diseases, such as Williams-Beuren-Syndrome (WBS), which results in reduced elastin synthesis, injuries, aging, or acquired diseases can lead to the degradation of existing elastin fibers. Thus, the de novo synthesis of elastin is required in several medical conditions to restore the elasticity of affected tissues. Here, we applied synthetic modified messenger RNA (mRNA) encoding tropoelastin (TE) for the de novo synthesis of elastin and determined the mRNA-mediated elastin synthesis in cells as well as ex vivo in porcine skin. EA.hy926 cells, human fibroblasts, and mesenchymal stem cells (MSCs) isolated from a patient with WBS were transfected with 2.5 μg TE mRNA. After 24 h, the production of elastin was analyzed by Fastin assay and dot blot analyses. Compared to untreated cells, significantly enhanced elastin amounts were detected in TE mRNA transfected cells. The delivered synthetic TE mRNA was even able to significantly increase the elastin production in elastin deficient MSCs. In porcine skin, approximately 20% higher elastin amount was detected after the intradermal delivery of synthetic mRNA by microinjection. In this study, we demonstrated the successful applicability of synthetic TE encoding mRNA to produce elastin in elastin-deficient cells as well as in skin. Thus, this auspicious mRNA-based integration-free method has a huge potential in the field of regenerative medicine to induce de novo elastin synthesis, e.g. in skin, blood vessels, or alveoli.

    更新日期:2018-03-31
  • iRNA-3typeA: identifying 3-types of modification at RNA’s adenosine sites
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-30
    Wei Chen, Pengmian Feng, Hui Yang, Hui Ding, Hao Lin, Kuo-Chen Chou

    RNA modifications are referred to the addition of chemical groups to nucleotides, or their local structural changes. Knowledge about the occurrence sites of these modifications is essential for in-depth understanding the biological functions and mechanisms, and treating some genomic diseases as well. With the avalanche of RNA sequences generated in the post-genomic age, many computational methods have been proposed for identifying various types of RNA modifications one by one. However, so far no method whatsoever has been developed for simultaneously identifying several different types of RNA modifications. To address such a challenge, we developed a predictor called “iRNA-3typeA”, by which we can simultaneously identify the occurrence sites of the following three most frequently observed modifications in RNA: (1) N1-methyladenosine (m1A), (2) N6-methyladenosine (m6A), and (3) adenosine to inosine (A-to-I). It has been shown via rigorous cross-validations for the RNA sequences from Homo sapiens and Mus musculus transcriptomes that the success rates achieved by the powerful new predictor are quite high. For the convenience of broad experimental scientists, a user-friendly web-server for iRNA-3typeA has been established at http://lin-group.cn/server/iRNA-3typeA/. It is anticipated that iRNA-3typeA may become a useful high throughput tool for genome analysis.

    更新日期:2018-03-31
  • Targeting Nicotinamide N-Methyltransferase and miR-449a in EGFR-TKI-Resistant Non-Small Cell Lung Cancer Cells
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-29
    Duc-Hiep Bach, Donghwa Kim, Song Yi Bae, Won Kyung Kim, Ji-Young Hong, Hye-Jung Lee, Nirmal Rajasekaran, Soonbum Kwon, Yanhua Fan, Thi-Thu-Trang Luu, Young Kee Shin, Jeeyeon Lee, Sang Kook Lee
    更新日期:2018-03-29
  • Development of a reporter system to explore MMEJ in the context of replacing large genomic fragments
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-22
    Mert Yanik, Surya Prakash Goud Ponnam, Tobias Wimmer, Lennart Trimborn, Carina Müller, Isabel Gambert, Johanna Ginsberg, Annabella Janise, Janina Domicke, Wolfgang Wende, Birgit Lorenz, Knut Stieger

    Common genome editing strategies are either based on nonhomologous endjoining (NHEJ) or, in the presence of a template DNA, based on homologous recombination with long (homology directed repair HDR) or short (microhomology mediated end-joining MMEJ) homologous sequences. In the current study, we aim to develop a model system to test the activity of MMEJ post CRISPR/Cas9 mediated cleavage in cell culture. Following successful prove of concept in an episomally based reporter system, we tested template plasmids containing a promoter-less luciferase gene flanked by micro-homologous sequences (mhs) of different length (5, 10, 15, 20, 30, and 50 bp) that are complementary to the mouse RPGR-ORF15, which is under the control of a CMV promoter stably integrated into a HEK293 cell line. Luciferase signal appearance represented successful recombination events and was highest when the mhs were 5 bp long, while longer mhs revealed lower luciferase signal. In addition, presence of Csy4 RNase was shown to increase luciferase signaling. The luciferase reporter system is a valuable tool to study the input of the different DNA repair mechanisms in the replacement of large DNA sequences by microhomologous sequences.

    更新日期:2018-03-23
  • Hydrogel-assisted antisense LNA gapmer delivery for in situ gene silencing in spinal cord injury
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-20
    Pedro M.D. Moreno, Ana R. Ferreira, Daniela Salvador, Maria T. Rodrigues., Marília Torrado, Eva D. Carvalho, Ulf Tedebark, Mónica M. Sousa, Isabel F. Amaral, Jesper Wengel, Ana P. Pêgo

    After spinal cord injury (SCI) nerve regeneration is severely hampered due to the establishment of a highly inhibitory microenvironment at the injury site, through the contribution of multiple factors. The potential of antisense oligonucleotides (AONs) to modify gene expression at different levels, allowing the regulation of cell survival and cell function, together with the availability of chemically modified nucleic acids with favourable biopharmaceutical properties, make AONs an attractive tool for novel SCI therapy developments. In this work, we explored the potential of locked nucleic acid (LNA)-modified AON gapmers in combination with a fibrin hydrogel bridging material to induce gene silencing in situ at a SCI lesion site. LNA gapmers were effectively developed against two promising gene targets aiming at enhancing axonal regeneration - RhoA and GSK3β. The fibrin matrix assisted AON delivery system mediated potent RNA knockdown in vitro in a dorsal root ganglion explant culture system and in vivo at a SCI lesion site, achieving around 75% down-regulation 5 days after hydrogel injection. Our results show that local implantation of a AON gapmer loaded hydrogel matrix mediated efficient gene silencing in the lesioned spinal cord and is an innovative platform that can potentially combine gene regulation with regenerative permissive substrates aiming at SCI therapeutics and nerve regeneration.

    更新日期:2018-03-20
  • CRISPR/Cas9 mediated disruption of the Swedish APP allele as a therapeutic approach for early-onset Alzheimer’s disease
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-16
    B. György, C. Lööv, M.P. Zaborowski, S. Takeda, B.P. Kleinstiver, C. Commins, K. Kastanenka, D. Mu, A. Volak, V. Giedraitis, L. Lannfelt, C.A. Maguire, J.K. Joung, B.T. Hyman, X.O. Breakefield, M. Ingelsson

    The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer’s disease (AD) as a result of increased β-secretase cleavage of the amyloid-β (Aβ) precursor protein. This leads to abnormally high Aβ levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APPSW allele using clustered regularly interspaced short palindromic repeats (CRISPR). By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APPSW or APPWT. As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted Aβ. Next, coding sequences for the APPSW-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APPSW transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof of concept data that CRISPR/Cas9 can selectively disrupt the APPSW allele both ex vivo and in vivo - and thereby decrease pathogenic Aβ. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased Aβ.

    更新日期:2018-03-16
  • Novel DNA aptamers that bind to mutant huntingtin and modify its activity
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-16
    Baehyun Shin, Roy Jung, Hyejin Oh, Gwen E. Owens, Hyeongseok Lee, Seung Kwak, Ramee Lee, Susan L. Cotman, Jong-Min Lee, Marcy E. MacDonald, Ji-Joon Song, Ravi Vijayvargia, Ihn Sik Seong

    The CAG repeat expansion that elongates the polyglutamine tract in huntingtin is the root genetic cause of Huntington’s Disease (HD), a debilitating neurodegenerative disorder. This seemingly slight change to the primary amino acid sequence alters the physical structure of the mutant protein and alters its activity. We have identified a set of G-quadruplex-forming DNA aptamers (MS1, MS2, MS3, MS4) that bind mutant huntingtin proximal to lysines K2932/K2934 in the carboxyl-terminal CTD-II domain. Aptamer-binding to mutant huntingtin, abrogated the enhanced polycomb repressive complex 2 (PRC2) stimulatory-activity conferred by the expanded polyglutamine tract. In HD, but not normal, neuronal progenitor cells (NPC), MS3 aptamer co-localized with endogenous mutant huntingtin and was associated with significantly decreased PRC2 activity. Furthermore, MS3 transfection protected HD NPC against starvation-dependent stress with increased ATP. Therefore, DNA aptamers can preferentially target mutant huntingtin and modulate a gain of function endowed by the elongated polyglutamine segment. These mutant huntingtin binding aptamers provide novel molecular tools for delineating the effects of the HD mutation and encourage mutant huntingtin structure-based approaches to therapeutic development.

    更新日期:2018-03-16
  • Gelofusine attenuates tubulointerstitial injury induced by cRGD-conjugated siRNA via regulating the TLR3 signaling pathway
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-14
    Bohong Cen, Wenjie Liao, Zhen Wang, Linyuan Gao, Yuanyi Wei, Wen Huang, Shuai He, Wei Wang, Xiaoxia Liu, Xinghua Pan, Aimin Ji

    Integrin αvβ3, which is selectively targeted by cyclic arginine-glycine-aspartic acid (cRGD) peptides, is significantly up-regulated in tumor. Previous studies showed that small interfering RNA (siRNA) modified with cRGD (cRGD–siRNA) could significantly inhibit tumor growth through RNA interference (RNAi) of oncogene expression. However, cRGD–siRNA is partially reabsorbed and trapped in the kidneys, causing renal injury in an unpredictable manner. This study aimed to investigate the influence of Gelofusine on tubulointerstitial injury induced by cRGD–siRNA in vitro and in vivo. Besides, the effect of Gelofusine on the distribution of cRGD–siRNA in tumor-bearing nude mice and wild-type mice was also explored. We found that Gelofusine inhibited the apoptosis and activation of innate immune response of human tubular epithelial cells induced by cRGD–siRNA in vitro. In addition, co-injection of Gelofusine efficiently reduced the renal retention of cRGD–siRNA without affecting its tumor targeting in vivo. Further in vivo studies indicated that Gelofusine significantly attenuated tubulointerstitial injury induced by cRGD–siRNA through regulating Toll-like receptor (TLR)3-mediated activation of nuclear factor-kappa B (NF-κB) and caspase-3 apoptotic pathway. In conclusion, Gelofusine, acting as a novel and effective renal protective agent, could form a compound preparation with siRNA drugs for future clinical applications.

    更新日期:2018-03-14
  • Intradermal delivery of synthetic mRNA using hollow microneedles for efficient and rapid production of exogenous proteins in skin
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-14
    Sonia Golombek, Martin Pilz, Heidrun Steinle, Efrat Kochba, Yotam Levin, Dominique Lunter, Christian Schlensak, Hans Peter Wendel, Meltem Avci-Adali

    In recent years, synthetic messenger RNA (mRNA)-based applications to produce desired exogenous proteins in cells are gaining in importance. However, systemic delivery of synthetic mRNA can result in unspecific uptake into undesired cells or organs, and thereby, fail to target desired cells. Thus, the local and targeted delivery of synthetic mRNA becomes increasingly important to reach the desired cell types and tissues. In this study, the intradermal delivery of synthetic mRNA using a hollow microneedle injection-based method was evaluated. Furthermore, an ex vivo porcine skin model was established to analyze the synthetic mRNA-mediated protein expression in the skin following intradermal delivery. Using this model, highly efficient delivery of synthetic mRNA was demonstrated, which resulted in detection of high levels of secretable humanized Gaussia luciferase (hGLuc) protein encoded by the microinjected synthetic mRNA. Interestingly, synthetic mRNA injected without transfection reagent was also able to enter into the cells and resulted in protein expression. The established ex vivo porcine skin model can be used to evaluate the successful production of desired proteins after the intradermal delivery of synthetic mRNAs before starting with in vivo experiments. Furthermore, the use of microneedles enables the patient friendly, painless and efficient delivery of synthetic mRNAs into the dermis, thus this method could be applied for local treatment of different skin diseases, as well as for vaccination and immunotherapy.

    更新日期:2018-03-14
  • Linc-ROR promotes osteogenic differentiation of mesenchymal stem cell through functioning as a competing endogenous RNA for miR-138 and miR-145
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-12
    Lu Feng, Liu Shi, Ying-fei Lu, Bin Wang, Tao Tang, Wei-ming Fu, Wei He, Gang Li, Jin-fang Zhang

    Long noncoding RNAs (lncRNAs) have gained widespread attention in recent years, which serve as important and powerful regulators of various biological activities. Emerging evidences have shown that some lncRNAs play important regulatory roles in osteoblast differentiation of mesenchymal stem cell (MSCs), suggesting a potential therapeutic strategy for bone fracture. As a recently identified lncRNA, linc-ROR was reported to mediate the reprogramming ability of differentiated cells into induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs) self-renewal. However, other functions of linc-ROR remain elusive. In this study, linc-ROR was found to be upregulated during osteogenesis of human bone marrow-derived MSCs. Ectopic expression of linc-ROR significantly accelerated while knockdown of linc-ROR suppressed osteoblast differentiation. Using bioinformatic prediction and luciferase reporter assays, we demonstrated that linc-ROR functioned as a miRNA sponge for miR-138 and miR-145, both of which were negative regulators of osteogenesis. Further investigations revealed that linc-ROR antagonized the functions of these two miRNAs and led to the de-repression of their shared target ZEB2, which eventually activated Wnt/β-catenin pathway and hence potentiated osteogenesis. Taken together, linc-ROR modulated osteoblast differentiation by acting as a competing endogenous RNA (ceRNA), which may shed light on the functional characterization of lncRNAs in coordinating osteogenesis.

    更新日期:2018-03-13
  • MicroRNA-411 Downregulation Enhances Tumor Growth by Upregulating MLLT11 Expression in Human Bladder Cancer
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-10
    Honglei Jin, Wenrui Sun, Yuanmei Zhang, Huiying Yan, Huating Liufu, Shuai Wang, Caiyi Chen, Jiayan Gu, Xiaohui Hua, Lingli Zhou, Guosong Jiang, Dapang Rao, Qipeng Xie, Haishan Huang, Chuanshu Huang

    Although several previous studies have reported the implication of various microRNAs (miRNAs) in regulation of human bladder cancer (BC) development, alterations and function of many miRNAs in bladder cancer growth are not explored yet at present. Here, we screened 1900 known miRNAs and first discovered that miR-411 was one of major miRNAs, which was downregulated in n-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BCs. This miR-411 downregulation was also observed in human BC tissues and cell lines. The results from evaluating the relationship between miR-411 and patient survival in BC using the TCGA database indicated that miR-411 was positively correlated with disease-free survival (DFS). Our studies also showed that miR-411 inhibited tumor growth of human BC cells in xenograft animal model. Mechanistic studies revealed that overexpression of miR-411 repressed the expression of ALL1-fused gene from the chromosome 1q (AF1q, MLLT11) by binding to the 3′-untranslated region of MLLT11 mRNA, and in turn induced p21 expression, and caused cell cycle arrest at the G2/M phase, further inhibiting BC tumor growth. Collectively, our results improve our understanding of the role of miR-411 in BC tumor growth and suggest miR-411 and MLLT11 as potential new targets for the treatment of BC patients.

    更新日期:2018-03-11
  • Inflammatory and autophagy-related gene p62 triggers malignant transformation of mesenchymal stem cells through the cascade of CUDR-CTCF-IGFII-RAS signaling
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-09
    Xiaoru Xin, Zhuojia Lin, Yanan Lu, Qiuyu Meng, Chen Wang, Xiaonan Li, Yuxin Yang, Jie Xu, Qidi Zheng, Xin Gui, Tianming Li, Hu Pu, Wujun Xiong, Dongdong Lu

    Inflammatory and autophagy-related gene p62 is highly expressed in most human tumor tissues. Herein, we demonstrate that p62 promotes human mesenchymal stem cells malignant transformation via the cascade of P62-TNFα- CUDR- CTCF- IGFII-H-Ras signaling. Mechanistically, we reveal p62 enhances IGFII transcriptional activity through forming IGFII promoter-enhancer chromatin loop and increasing METTL3 occupancy on IGFII 3’-UTR, and enhances H-Ras overexpression by harboring inflammation related factors, e.g. TNFR1, CLYD, EGR1, NFκB, TLR4, PPARγ. Furthermore, the P62 cooperates with TNFα to promote malignant transformation of mesenchymal stem cells. These findings, for the first time , provide insight into the positive role that P62 palys in malignant transformation of mesenchymal stem cells, and reveal a novel link between P62 and the inflammation factors in mesenchymal stem cells.

    更新日期:2018-03-09
  • A Deep Learning Framework for Robust and Accurate prediction of ncRNA-Protein Interactions using Evolutionary Information
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-09
    Hai-Cheng Yi, Zhu-Hong You, De-Shuang Huang, Xiao Li, Tong-Hai Jiang, Li-Ping Li

    The interactions between non-coding RNAs (ncRNAs) and proteins play an important role in many biological processes, and their biological functions are primarily achieved by binding with a variety of proteins. High-throughput biological techniques are used to identify protein molecules bound with specific ncRNA, but they are usually expensive and time-consuming. Deep learning provides a powerful solution for computationally predict RNA-protein interactions. In this work, we proposed RPI-SAN model by using the deep-learning stacked auto-encoder network to mine the hidden high-level features from RNA and protein sequences and feed them into Random Forest (RF) model to predict ncRNA binding proteins. Stacked assembling is further used to improve the robust and accuracy of the proposed method. Four benchmark datasets including RPI2241, RPI488, RPI1807, and NPInter v2.0 was employed for the unbiased evaluation of five established prediction tools: RPI-Pred, IPMiner, RPISeq-RF, lncPro and RPI-SAN. The experimental results show that our RPI-SAN model achieves much better performance than other methods with accuracies of 90.77%, 89.7%, 96.1%, and 99.33%, respectively. It is anticipated that RPI-SAN can be used as an effective computational tool for future biomedical researches, and can accurately predict the potential non-coding RNA-protein interacted pairs, which provides reliable guidance for the biological research.

    更新日期:2018-03-09
  • CircFGFR4 Promotes Differentiation of Myoblasts via Binding miR-107 to Relieve Its Inhibition of Wnt3a
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-06
    Hui Li, Xuefeng Wei, Jiameng Yang, Dong Dong, Dan Hao, Yongzhen Huang, Xianyong Lan, Martin Plath, Chuzhao Lei, Yun Ma, Fengpeng Lin, Yueyu Bai, Hong Chen

    Muscle development is regulated under a series of complicate processes, and non-coding RNAs such as miRNAs and circRNAs have been reported to play important roles in regulating myoblast proliferation and differentiation. We found that miR-107 expression was high in skeletal muscle of Qinchuan cattle. Overexpression of miR-107 inhibited bovine myoblasts differentiation and protected cells from apoptosis. Wnt3a was identified as a target of miR-107 by luciferase activity, quantitative real-time PCR (qPCR) and western blotting assays. Knockdown of Wnt3a inhibited bovine myoblasts differentiation and proliferation, and this effect was similar to miR-107 overexpression. We also found circFGFR4 to promote myoblasts differentiation and to induce cell apoptosis. Via luciferase screening and RNA pulldown assays, circFGFR4 was observed to sponge miR-107. Overexpression of circFGFR4 increased the expression of Wnt3a, while this effect was abolished by miR-107. These results demonstrated that circFGFR4 binding miR-107 promotes cell differentiation via targeting Wnt3a in bovine primary myoblasts.

    更新日期:2018-03-07
  • Expression differentiation is not helpful in identifying prognostic genes based on TCGA datasets
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-06
    Ning An, Zhuang Yu, Xue Yang

    A routine pipeline seems very common in many cancer studies that expression differentiation might be helpful in identifying prognostic molecules. There also exists a striking unanimity that molecules up-regulated in cancer usually shorten survival, while down-regulated ones have the opposite effect. In this study, based on the transcriptional profiles of 18 malignancies, cancer and corresponding adjacent normal tissues were used to calculate differential score. Cox correlation coefficients of global genes were also calculated to denote survival association. The relationship between expression differentiation and survival association has been extensively studied in 18 malignancy types. Contradictory to our stereotypic research pattern, expression differentiation between cancer and adjacent normal tissues was proven irrelevant to corresponding survival correlation. Surprisingly, the more stringent cutoff we used in differentially expressed gene identification, the less prognostic information we would obtain from the collected gene groups. Moreover, the direction of dysregulated genes in cancer was irrelevant to the direction of corresponding survival correlation. Cancer-normal expression differentiation is irrelevant to genes’ survival correlation in multiple cancers, therefore not helpful in identifying prognostic genes. In future studies, it is more sensible to look into another alternative rather than collect differentially expressed molecules in the initial step.

    更新日期:2018-03-07
  • Hepatocellular carcinoma cell-secreted exosomal microRNA-210 promotes angiogenesis in vitro and in vivo
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-06
    Xue-Jia Lin, Jian-Hong Fang, Xiao-Jing Yang, Chong Zhang, Yunfei Yuan, Limin Zheng, Shi-Mei Zhuang

    We previously found that 19 miRNAs significantly increased in the sera of hepatocellular carcinoma (HCC) patients. Here we evaluated whether these miRNAs were secreted by HCC cells and contributed to tumor angiogenesis. High level of miR-210-3p (miR-210) was detected in the exosomes isolated from the sera of HCC patients and the conditioned media of hepatoma cells. Higher miR-210 level in serum was correlated with higher microvessel density in HCC tissues. Moreover, the HCC cell-secreted exosomes promoted in vitro tubulogenesis of endothelial cells, which was strengthened by overexpressing miR-210 in HCC cells but was attenuated by repressing miR-210 or DROSHA in HCC cells. This pro-tubulogenesis effect by HCC-exosomes was also abrogated by antagonizing miR-210 in endothelial cells. Subsequent in vivo studies revealed that Matrigel plug and subcutaneous tumor xenografts treated with HCC cell-derived exosomal miR-210 displayed much more vessels. Furthermore, exosomal miR-210 could be delivered into endothelial cells and directly inhibited the expression of SMAD4 and STAT6, resulting in enhanced angiogenesis. Collectively, HCC cell-secreted exosomal miR-210 may be transferred into endothelial cells, thereby promotes tumor angiogenesis by targeting SMAD4 and STAT6. Our findings identify a novel mechanism of HCC angiogenesis and highlight the biological importance of exosomal miR-210.

    更新日期:2018-03-07
  • Novel DNA aptamers for Parkinson’s Disease treatment inhibit α-synuclein aggregation and facilitate its degradation
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-06
    Yuan Zheng, Jing Qu, Fenqin Xue, Yan Zheng, Bo Yang, Yongchang Chang, Hui Yang, Jianliang Zhang

    Parkinson’s disease (PD) is one most prevalent form of synucleinopathies, and it is characterized neuropathologically by the presence of intracellular inclusions composed primarily of the protein α-synuclein (α-syn) in neurons. The previous immunotherapy targeting the α-syn in PD models with monoclonal antibodies has established α-syn protein as an effective target for neuronal cell death. However, due to the essential weaknesses of antibody and the unique features of aptamers, the aptamers could represent a promising alternative to the currently used antibodies in immunotherapy for PD. In this study, the purified human α-syn was used as the target for in vitro selection of aptamers using systematic evolution by exponential enrichment. This resulted in the identification of two 58-base DNA aptamers with high binding affinity and good specificity to the α-syn with Kd values in the nanomolar range. Both aptamers could effectively reduce α-syn aggregation in vitro and in cells, and target the α-syn to intracellular degradation through lysosomal pathway. These effects consequently rescued the mitochondrial dysfunction and cellular defects caused by α-syn overexpression. To our knowledge, this is the first study to employ aptamers to block the aberrant cellular effects of the overexpressed α-syn in cells.

    更新日期:2018-03-07
  • Overexpression of Mutant FKRP Associated with CMD Restores Functional Glycosylation and Improves Dystrophic Phenotype in Skeletal and Cardiac Muscle of FKRP mutant Mice
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-06
    Jason D. Tucker, Pei J. Lu, Xiao Xiao, Qi L. Lu

    Autosomal recessive homozygous or compound heterozygous mutations in FKRP result in forms of muscular dystrophy-Dystroglycanopathy varying in age of onset, clinical presentation, and disease progression, ranging from the severe Walker-Warburg, Type A,5 (MDDGA5), Muscle-Eye-Brain (MDDGB5) with or without cognitive deficit, to Limb-Girdle Type 2I (MDDGC5). Phenotypic variation indicates degrees of functionality of individual FKRP mutation which has been supported by the presence of residual expression of functionally glycosylated α-DG in muscles of both animal models and patients. However, direct evidence showing enhancement in glycosylation of α-DG by mutant FKRP is lacking. Using AAV9-mediated overexpression of mutant human FKRP bearing the P448L mutation (mhFKRP-P448L) associated with severe congenital muscular dystrophy (CMD), we demonstrate the restoration of functional glycosylation of α-DG and reduction in markers of disease progression. Expression of mhFKRP-P448L also corrects dystrophic phenotypes in the models of L276I mutation with mild disease phenotype, and causes no obvious histological or biomarker alteration in C57BL/6 normal mice. Our results confirm the existing function of mutant FKRP. The results also suggest that mutant FKRP could be an alternative approach for potential gene therapy should normal FKRP gene products be immunogenic.

    更新日期:2018-03-06
  • MicroRNA-326 upregulates B cells activity and autoantibody production in lupus disease of MRL/lpr mice
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-01
    Yuan Xia, Jinhui Tao, Xuan Fang, Nan Xiang, Xiaojuan Dai, Li Jin, Xiaomei Li, Yiping Wang, Xiangpei Li

    B cells are recognized as key participant in various autoimmune diseases, including systemic lupus erythematosus (SLE). Although sets of transcription factors and cytokines are known to regulate B cells differentiation, the roles of microRNAs are poorly understood. Our previous study has proved that microRNA-326 (miR-326) was markedly upregulated in SLE patients; however, the biological function of miR-326 during SLE pathogenesis remained unknown. In this study, we found that miR-326 overexpression in MRL/lpr mice led to B cell hyperactivity and severe SLE. Moreover, E26 transformation–specific-1 (Ets-1), a negative regulator of B cells differentiation, was identified as a target of miR-326. Therefore, a novel mechanism has been found in which the elevated miR-326 in B cells of SLE promotes plasmablasts development and antibody production through down-regulation of Ets-1.

    更新日期:2018-03-02
  • Enhancing electrotransfection efficiency through improvement in nuclear entry of plasmid DNA
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-03-01
    Lisa D. Cervia, Chun-Chi Chang, Liangli Wang, Mao Mao, Fan Yuan

    The nuclear envelope is a physiological barrier to electrogene transfer. To understand different mechanisms of the nuclear entry for electrotransfected plasmid DNA (pDNA), the current study investigated how manipulation of the mechanisms could affect electrotransfection efficiency (eTE), transgene expression level (EL), and cell viability. In the investigation, cells were first synchronized at G2-M phase prior to electrotransfection so that the nuclear envelope breakdown (NEBD) occurred before pDNA entered the cells. The NEBD significantly increased the eTE and the EL while the cell viability was not compromised. In the second experiment, the cells were treated with a nuclear pore dilating agent (i.e., trans-1,2-cyclohexanediol). The treatment could increase the EL, but had only minor effects on eTE. Furthermore, the treatment was cytotoxic, compared to the cell synchronization. In the third experiment, a nuclear targeting sequence (i.e., SV40) was incorporated into the pDNA prior to electrotransfection. The incorporation was more effective than the cell synchronization for enhancing the EL but not the eTE, and the effectiveness was cell-type dependent. Taken together, the data described above suggested that synchronization of the NEBD could be a practical approach to improving electrogene transfer in all dividing cells.

    更新日期:2018-03-02
  • Gene therapy for neuropathic pain through siRNA-IRF5 gene delivery with homing peptides to microglia
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-02-27
    Tomoya Terashima, Nobuhiro Ogawa, Yuki Nakae, Toshiyuki Sato, Miwako Katagi, Junko Okano, Hiroshi Maegawa, Hideto Kojima

    Astrocyte and microglia targeting peptides were identified and isolated using phage display technology. A series of procedures including three cycles of both in vivo and in vitro biopanning were performed separately in astrocytes and in M1 or M2 microglia, yielding 50-58 phage plaques in each cell type. Analyses of the sequences of this collection identified one candidate homing peptide targeting astrocyte (AS1[C-LNSSQPS-C]) and two candidate homing peptides targeting microglia (MG1[C-HHSSSAR-C] and MG2[C-NTGSPYE-C]). To determine peptide specificity for the target cell in vitro, each peptide was synthesized and introduced into the primary cultures of astrocytes or microglia. Those peptides could bind to the target cells and be selectively taken up by the corresponding cell, namely astrocytes, M1 microglia or M2 microglia. To confirm cell-specific gene delivery to M1 microglia, the complexes between the MG1 peptide and siRNA-interferon regulatory factor 5 were prepared and intrathecally injected into a mouse model of neuropathic pain. The complexes successfully suppressed hyperalgesia with high efficiency in this neuropathic pain model. Here, we describe a novel gene therapy for the treatment neuropathic pain, which has a high potential to be of clinical relevance. This strategy will ensure the targeted delivery of therapeutic genes while minimizing side effects to non-target tissues or cells.

    更新日期:2018-02-27
  • Liver-targeted anti-HBV single stranded oligonucleotides with Locked Nucleic Acid potently reduces HBV-gene expression in vivo
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-02-23
    Hassan Javanbakht, Henrik Mueller, Johanna Walther, Xue Zhou, Anaïs Lopez, Thushara Pattupara, Julie Blaising, Lykke Pedersen, Nanna Albæk, Malene Jackerott, Tianlai Shi, Corinne Ploix, Wouter Driessen, Robert Persson, Jacob Ravn, John A.T. Young, Søren Ottosen

    Chronic hepatitis B infection (CHB) is an area of high unmet medical need. Current standard of care therapies only rarely lead to a functional cure, defined as durable HBsAg loss following treatment. The goal for next generation CHB therapies is to achieve a higher rate of functional cure with finite treatment duration. To address this urgent need we are developing liver-targeted single-stranded oligonucleotide (SSO) therapeutics for CHB based on the Locked Nucleic Acid (LNA) platform. These LNA-SSOs target HBV transcripts for RNase H-mediated degradation. Here we describe a HBV-specific LNA-SSO that effectively reduces intracellular viral mRNAs and viral antigens (HBsAg and HBeAg) over an extended time period in cultured human hepatoma cell line that were infected with HBV with mean 50% effective concentration (EC50) values ranging from 1.19 to 1.66 μM. To achieve liver-specific targeting and minimize kidney exposure, this LNA-SSO was conjugated to a cluster of three N-acetylgalactosamine (GalNAc)-moieties that direct specific binding to the asialoglycoprotein receptor (ASGPR) expressed specifically on the surface of hepatocytes. The GalNAc-conjugated LNA-SSO showed a strikingly higher level of potency when tested in the AAV-HBV mouse model as compared with its non-conjugated counterpart. Remarkably, higher doses of GalNAc-conjugated LNA-SSO resulted in a rapid and long-lasting reduction of HBsAg to below the detection limit for quantification, i.e. by 3 log10 (ρ<0.0003). This antiviral effect depended on a close match between the sequences of the LNA-SSO and its HBV target, indicating that the antiviral effect is not due to non-specific oligonucleotide-driven immune-activation. These data support the development of LNA-SSO therapeutics for the treatment of CHB infection.

    更新日期:2018-02-23
  • Saponins as Natural Adjuvant for Antisense Morpholino Oligonucleotides Delivery in vitro and in mdx Mice
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-02-21
    Mingxing Wang, Bo Wu, Sapana N. Shah, Peijuan Lu, Qilong Lu

    Antisense oligonucleotide (AON) therapy for Duchenne muscular dystrophy has drawn great attention in preclinical and clinical trials, but its therapeutic applications are still limited due to inefficient delivery. In this study, we investigated a few Saponins for their potential to improve delivery performance of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that these Saponins, especially Digitonin and Tomatine, improve the delivery efficiency of PMO comparable to Endoporter-mediated PMO delivery in vitro. The significant enhancement of PMO targeting to dystrophin exon 23 delivery was further observed in mdx mice up to 7-fold with the Digitonin as compared to PMO alone. Cytotoxicity of the Digitonin and Glycyrrhizin was lower than Endoporter in vitro and not clearly detected in vivo under the tested concentrations. These results demonstrate that optimization of Saponins in molecular size and composition are key factors to achieve enhanced PMO exon-skipping efficiency. The higher efficiency and lower toxicity endow Saponins as gene/AON delivery enhancing agents for treating muscular dystrophy or other diseases.

    更新日期:2018-02-21
  • Repurposing Dantrolene for Long-term Combination Therapy to Potentiate Antisense-Mediated DMD Exon-Skipping in the mdx mouse
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-02-13
    Derek W. Wang, Ekaterina I. Mokhonova, Genevieve C. Kendall, Diana Becerra, Yalda B. Naeini, Rita M. Cantor, Melissa J. Spencer, Stanley F. Nelson, M. Carrie Miceli

    Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, resulting in loss of dystrophin, essential to muscle health. DMD “exon-skipping” uses anti-sense oligo-nucleotides (AON) to force specific exon exclusion during mRNA processing to restore reading frame and rescue of partially functional dystrophin protein. While exon-skipping drugs in humans show promise, levels of rescued dystrophin protein remain suboptimal. We previously identified dantrolene as a skip-booster when combined with AON in human DMD cultures and short-term mdx dystrophic mouse studies. Here we assess the effect of dantrolene/AON combination on DMD exon-23 skipping over long-term mdx treatment under conditions that better approximate potential human dosing. To evaluate dantrolene/AON combination treatment effect on dystrophin induction, we assayed three AON doses, with and without oral dantrolene, to assess multiple outcomes across different muscles. Meta-analyses of the results of statistical tests from both quadriceps and diaphragm assessing contributions of dantrolene beyond AON, across all AON treatment groups provide strong evidence that dantrolene modestly boosts exon-skipping and dystrophin rescue, while reducing muscle pathology in mdx mice (p<0.0087). These findings support trial of combination dantrolene/AON to increase exon-skipping efficacy and highlight the value of combinatorial approaches and FDA drug re-purposing for discovery of unsuspected therapeutic application and rapid translation.

    更新日期:2018-02-13
  • Immunogenicity investigations of lipidoid structures in vitro and in silico: Modulating lipidoid-mediated TLR4 activation by nanoparticle design
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-02-13
    Anne Marit de Groot, Kaushik Thanki, Monique Gangloff, Emily Falkenberg, Xianghui Zeng, Djai C.J. van Bijnen, Willem van Eden, Henrik Franzyk, Hanne M. Nielsen, Femke Broere, Nick J. Gay, Camilla Foged, Alice J.A.M. Sijts
    更新日期:2018-02-13
  • Cryptotanshinone protects cartilage against developing osteoarthritis through the miR-106a-5p/GLIS3 axis
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-02-08
    Quanbo Ji, Dengbin Qi, Xiaojie Xu, Yameng Xu, Stuart B. Goodman, Lei Kang, Qi Song, Zhongyi Fan, William J. Maloney, Yan Wang
    更新日期:2018-02-09
  • Efficient delivery and nuclear uptake is not sufficient to detect gene editing in CD34+ cells directed by a ribonucleoprotein complex
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-02-07
    Shirin R. Modarai, Dula Man, Pawel Bialk, Natalia Rivera-Torres, Kevin Bloh, Eric B. Kmiec

    CD34+ cells are a prime target for therapeutic strategies for gene editing because modified progenitor cells have the capacity to differentiate through an erythropoietic lineage. While experimental advances have been reported, by and large, the associated experimental protocols have been less than clear or robust. As such, we evaluated the relationship among cellular delivery, nuclear uptake, often viewed as the benchmark metric of successful gene editing, and single base repair. We took a combinatorial approach using single stranded oligonucleotide and a CRISPR/Cas9 ribonucleoprotein to convert wild type HBB into the Sickle Cell genotype by evaluating conditions for two common delivery strategies of gene editing tools into CD34+ cells. Confocal microscopy data show that the CRISPR/Cas9 ribonucleoprotein tends to accumulate at the outer membrane of the CD34+ cell nucleus when NEON Transfection System is employed, while the ribonucleoproteins do pass into the cell nucleus when Nucleofection is used. Yet, despite the high efficiency of cellular transformation, and the traditional view of success in efficient nuclear uptake, neither delivery methodology enabled gene editing activity. Our results indicate that more stringent criteria must be established to facilitate the clinical translation and scientific robustness of gene editing for Sickle Cell Disease.

    更新日期:2018-02-07
  • Generation of Hutat2:Fc knock-in primary human monocytes using CRISPR/Cas9
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-02-07
    Bowen Wang, Jiahui Zuo, Wenzhen Kang, Qianqi Wei, Jianhui Li, Chunfu Wang, Zhihui Liu, Yuanan Lu, Yan Zhuang, Bianli Dang, Qing Liu, Wen Kang, Yongtao Sun
    更新日期:2018-02-07
  • Supplemental treatment for Huntington’s disease (HD) with miR-132 that is deficient in HD brain
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-02-02
    Masashi Fukuoka, Masaki Takahashi, Hiromi Fujita, Tomoko Chiyo, H. Akiko Popiel, Shoko Watanabe, Hirokazu Furuya, Miho Murata, Keiji Wada, Takashi Okada, Yoshitaka Nagai, Hirohiko Hohjoh

    Huntington’s disease (HD) is an intractable neurodegenerative disorder caused by mutant Huntingtin (HTT) proteins that adversely affect various biomolecules and genes. MicroRNAs (miRNAs), which are functional small non-coding RNAs, are also affected by mutant HTT proteins. Here we show amelioration in motor function and lifespan of HD-model mice, R6/2 mice, by supplying miR-132 to HD brains using a recombinant adeno-associated virus (rAAV) miRNA expression system. MiR-132 is a miRNA related to neuronal maturation and function, but the level of miR-132 in the brain of R6/2 mice was significantly lower than that of wild-type mice. Our miR-132 supplemental treatment, i.e., supplying miR-132 to the brain, produced symptomatic improvement or retarded disease progression in R6/2 mice; and interestingly, it had little effect on disease-causing mutant HTT mRNA expression and its products. Therefore, the findings suggest that there may be a therapeutic way to treat HD without inhibiting and/or repairing disease-causing HTT genes and gene-products. Although miR-132 supplement may not be a definitive treatment for HD, it may become a therapeutic method for relieving HD symptoms and delaying HD progression.

    更新日期:2018-02-02
  • miR-182-5p and miR-183-5p act as GDNF mimics in dopaminergic midbrain neurons
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-02-02
    Anna-Elisa Roser, Lucas Caldi Gomes, Rashi Halder, Gaurav Jain, Fabian Maass, Lars Tönges, Lars Tatenhorst, Mathias Bähr, André Fischer, Paul Lingor

    Parkinson’s disease (PD) is the second most frequent neurodegenerative disorder worldwide. One major hallmark of PD is the degeneration of dopaminergic neurons in the substantia nigra. Glial cell line-derived neurotrophic factor (GDNF) potently increases dopaminergic neuron survival in models of PD; however the underlying mechanisms are incompletely understood. microRNAs (miRs) are small non-coding RNAs that are important for post-transcriptional regulation of gene expression. Using small RNA sequencing we show that GDNF specifically increases the expression of miR-182-5p and miR-183-5p in primary midbrain neurons (PMN). Transfection of synthetic miR-182-5p and miR-183-5p mimics leads to increased neurite outgrowth and mediates neuroprotection of dopaminergic neurons in vitro and in vivo, mimicking GDNF effects. This is accompanied by a decreased expression of Foxo3 and Foxo1 transcription factors and increased PI3K-Akt signaling. Inhibition of endogenous miR-182-5p or miR-183-5p in GDNF treated PMNs attenuated the pro-dopaminergic effects of GDNF. These findings unveil an unknown miR-mediated mechanism of GDNF action and suggest that targeting miRs is a new therapeutic avenue to PD phenotypes.

    更新日期:2018-02-02
  • miR-93-5p-containing exosomes treatment attenuate acute myocardial infarction induced myocardial damage
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-01-31
    Hairong Wang, Mei Jiang, Jiwen Liu, Hui Huang, Yu Zhang, Peihua Gong, Xumin Shen, Huanjun Ruan, Shengqiong Deng, Mingming Jin, Jide Lu

    Adipose-derived stromal cells (ADSCs) have been considered as an attractive therapeutic tool. Accumulating evidence indicates that the healing effects of ADSCs are mainly related to paracrine action rather than transdifferentiation. Data show that the expression of miR-93-5p has a cardio-protective effect after acute myocardial infarction (AMI). To identifield if miR-93-5p-encapsulating exosomes form ADSCs have a better cardio-protective effect, we investigated the inflammatory factors and miR-30d-5p expression in clinical levels. A rat model of AMI and in vitro model of hypoxic H9c2 cells were established to study the protective mechanism of miR-93-5p in ischemia-induced cardiac injury. The results show that the expression of inflammatory cytokines and miR-93-5p were increased following AMI in both patients and animal models. Moreover treatment with ADSC-derived miR-93-5p-containing exosomes has a greater protective effect on infarction-induced myocardial damage than simple exosome processing. Furthermore, in vitro experiments confirmed that the expression of miR-93-5p can significantly suppress hypoxia-induced autophagy and inflammatory cytokine expression by targeting Atg7 and TLR4, respectively. And confirmed with Atg7 or TLR4 overexpression. The results also show that autophagy activation can promote inflammatory cytokine expression indirectly. Taken together, these results suggest that the miR-93-5p-enhanced ADSC-derived exosomes prevent cardiac injury by inhibiting autophagy and the inflammatory response.

    更新日期:2018-02-02
  • RNA Structure Design Improves Activity and Specificity of trans-Splicing Triggered Cell Death in a Suicide Gene Therapy Approach
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-01-31
    Sushmita Poddar, Pei She Loh, Zi Hao Ooi, Farhana Osman, Joachim Eul, Volker Patzel
    更新日期:2018-02-02
  • Deletion of a pathogenic mutation-containing exon of COL7A1 allows clonal gene editing correction of RDEB patient epidermal stem cells.
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-01-31
    Ángeles Mencía, Cristina Chamorro, Jose Bonafont, Blanca Duarte, Almudena Holguin, Nuria Illera, Sara G. Llames, Maria José Escámez, Ingrid Hausser, Marcela Del Río, Fernando Larcher, Rodolfo Murillas

    Recessive dystrophic epidermolysis bullosa is a severe skin fragility disease caused by loss of functional type VII collagen at the dermal-epidermal junction. A frameshift mutation in exon 80 of COL7A1 gene, c.6527insC, is highly prevalent in the Spanish patient population. We have implemented gene editing strategies for COL7A1 frame restoration by NHEJ-induced indels in epidermal stem cells from patients carrying this mutation. TALEN nucleases designed to cut within the COL7A1 exon 80 sequence were delivered to primary patient keratinocyte cultures by non-integrating viral vectors. After genotyping a large collection of vector-transduced patient keratinocyte clones with high proliferative potential, we identified a significant percentage of clones with COL7A1 reading frame recovery and Collagen VII protein expression. Skin equivalents generated with cells from a clone lacking exon 80 entirely were able to regenerate phenotypically normal human skin upon their grafting onto immunodeficient mice. These patient-derived human skin grafts showed Collagen VII deposition at the basement membrane zone, formation of anchoring fibrils and structural integrity when analyzed twelve weeks after grafting. Our data provide a proof-of-principle for recessive dystrophic epidermolysis bullosa treatment through ex vivo gene editing based on removal of pathogenic mutation-containing, functionally expendable COL7A1 exons in patient epidermal stem cells.

    更新日期:2018-02-02
  • Antisense oligonucleotide mediated terminal intron retention of the SMN2 transcript
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-01-31
    Loren L. Flynn, Chalermchai Mitrpant, Ianthe L. Pitout, Sue Fletcher, Steve D. Wilton

    The severe childhood disease spinal muscular atrophy (SMA) arises from the homozygous loss of the survival motor neuron 1 gene (SMN1). A homologous gene potentially encoding an identical protein, SMN2 can partially compensate for the loss of SMN1, however the exclusion of a critical exon in the coding region during mRNA maturation results in insufficient levels of functional protein. The rate of transcription is known to influence the alternative splicing of gene transcripts, with a fast transcription rate correlating to an increase in alternative splicing. Conversely, a slower transcription rate is more likely to result in the inclusion of all exons in the transcript. Targeting SMN2 with antisense oligonucleotides to influence the processing of terminal exon 8 could be a way to slow transcription and induce the inclusion of exon 7. Interestingly, following oligomer treatment of SMA patient fibroblasts, we observed the inclusion of exon 7 as well as intron 7 in the transcript. Since the normal termination codon is located in exon 7, this exon/intron7-SMN2 transcript should encode the normal protein, and only carry a longer 3´ untranslated region. Further studies showed the extra 3´UTR length contained a number of regulatory motifs that modify transcript and protein regulation, leading to translational repression of SMN. While unlikely to provide therapeutic benefit for spinal muscular atrophy patients, this novel technique for gene regulation could provide another avenue for the repression of undesirable gene expression in a variety of other diseases.

    更新日期:2018-02-02
  • Association of common genetic variants in pre-microRNAs and neuroblastoma susceptibility: a two-center case-control study in Chinese children
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-01-31
    Jing He, Yan Zou, Xiaodan Liu, Jinhong Zhu, Jiao Zhang, Ruizhong Zhang, Tianyou Yang, Huimin Xia

    Neuroblastoma is a commonly occurring extracranial pediatric solid tumor without defined etiology. Polymorphisms in pre-miRNAs have been demonstrated to associate with the risk of several cancers. So far, no such polymorphism has been investigated in neuroblastoma. With this in mind, we performed a two-center case-control study to assess the association of genetic variants in pre-miRNAs and neuroblastoma susceptibility in Chinese children, including 393 cases and 812 controls. We found that miR-34b/c rs4938723 T>C polymorphism was significantly associated with decreased neuroblastoma risk [TC vs. TT: adjusted odds ratio (OR)=0.51, 95% confidence interval (CI)=0.39-0.67; TC/CC vs. TT: adjusted OR=0.62, 95% CI=0.48-0.79]. We also observed the significant association between the miR-218 rs11134527 A>G polymorphism and decreased neuroblastoma risk (AG vs. AA: adjusted OR=0.73, 95% CI=0.56-0.96). Stratified analysis further demonstrated that the protective effect of the rs4938723 T>C polymorphism remained prominent in the subgroups regardless of age, gender, and clinical stages. In term of sites of origin, this polymorphism significantly reduced the risk of tumors originating from the adrenal gland. We further validated the significant results using false-positive report probability analyses. Overall, the miR-34b/c rs4938723 T>C and miR-218 rs11134527 A>G polymorphisms displayed a protective role from neuroblastoma. These findings need further validation.

    更新日期:2018-01-31
  • miR-1266 Contributes to Pancreatic Cancer Progression and Chemoresistance by STAT3 and NF-κB Signaling Pathways
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-01-31
    Xin Zhang, Dong Ren, Xianqiu Wu, Xi Lin, Liping Ye, Chuyong Lin, Shu Wu, Jinrong Zhu, Xinsheng Peng, Libing Song

    Pancreatic cancer is characterized by chemoresistance after several cycles of chemotherapy, which is a major issue responsible for treatment failure of pancreatic cancer. Therefore, it’s necessary to explore the specific mechanism underlying chemotherapeutic resistance to overcome this issue. Here we report that miR-1266 is dramatically elevated and correlates with poor survival and chemotherapy response in pancreatic cancer patients. Upregulation of miR-1266 enhanced the chemoresistance of pancreatic cancer cells to gemcitabine in vitro and in vivo; conversely, inhibition of miR-1266 yielded an opposite effect. Importantly, silencing miR-1266 restored the sensitivity of pancreatic cancer cells to gemcitabine in a dose-dependent manner in vivo. Furthermore, our results demonstrated miR-1266 promoted the resistance of pancreatic cancer cells to gemcitabine via targeting multiple negative regulators of STAT3 and NF-κB pathways, including SOCS3, PTPN11, ITCH, and TNIP1, leading to constitutive activation of STAT3 and NF-κB signaling. Thus, our findings clarify a novel mechanism by which miR-1266 induces chemotherapeutic resistance in pancreatic cancer, indicating that miR-1266 may be used as chemotherapeutic response indicator. Antagomir-1266 as chemotherapeutic sensitizer in combination with gemcitabine may serve as a rational regimen in the treatment of chemotherapeutic resistant pancreatic cancer.

    更新日期:2018-01-31
  • Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 epi-suppressors
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-01-08
    Hong Wang, Rui Guo, Zhonghua Du, Ling Bai, Lingyu Li, Jiuwei Cui, Wei Li, Andrew R. Hoffman, Ji-Fan Hu

    The clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 system can modulate disease-causing alleles both in vivo and ex vivo, raising the possibility of therapeutic genome editing. In addition to gene targeting, epigenetic modulation by the catalytically inactive dCas9 may also be a potential form of cancer therapy. Granulin (GRN), a potent pluripotent mitogen and growth factor that promotes cancer progression by maintaining self-renewal of hepatic stem cancer cells, is upregulated in hepatoma tissues and is associated with decreased tumor survival in patients with hepatoma. We synthesized a group of dCas9 epi-suppressors to target GRN by tethering the C-terminus of dCas9 with three epigenetic suppressor genes: DNMT3a (DNA methyltransferase), EZH2 (histone 3 lysine 27 methyltransferase), and KRAB (the Krüppel-associated box transcriptional repression domain). In conjunction with guiding RNAs (gRNAs), the dCas9 epi-suppressors caused significant decreases in GRN mRNA abundance in Hep3B hepatoma cells. These dCas9 epi-suppressors initiated de novo CpG DNA methylation in the GRN promoter, and produced histone codes that favor gene suppression, including decreased H3K4 methylation, increased H3K9 methylation, and enhanced HP1a binding. Epigenetic knockdown of GRN led to inhibition of cell proliferation, decreased tumor sphere formation, and reduced cell invasion. These changes were achieved at least partially through the MMP/TIMP pathway. This study thus demonstrates the potential utility of using dCas9 epi-suppressors in the development of epigenetic targeting against tumors.

    更新日期:2018-01-09
  • Effects of aptamer to U87-EGFRvIII cells on the proliferation, radiosensitivity, and radiotherapy of glioblastoma cells
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-01-08
    Xingmei Zhang, Li Peng, Zhiman Liang, Zhewen Kou, Yue Chen, Guangwei Shi, Xiaowen Li, Yanling Liang, Fang Wang, Yusheng Shi

    Glioblastoma multiforme (GBM) is the most prevalent and lethal malignant intracranial tumor in the brain, with very poor prognosis and survival. The epidermal growth factor receptor variant III (EGFRvIII) contributes to increased oncogenicity that does not occur through binding EGFR ligands and instead occurs through constitutive activation, which enhances glioma tumorigenicity and resistance to targeted therapy. Aptamers are nucleic acids with high affinity and specificity to targets selected by Systematic Evolution of Ligands by Exponential enrichment (SELEX), and they are usually developed as antagonists of disease-associated factors. Herein, we generated a DNA aptamer U2, targeting U87-EGFRvIII cells and demonstrated that U2 alters the U87-EGFRvIII cell growth, radiosensitivity and radiotherapy of glioblastoma cells. We detected U2 and U87-EGFRvIII cells by flow cytometry and confocal microscopy to explore the binding ability of U2 to U87-EGFRvIII cells. Then, we found that aptamer U2 inhibits the proliferation, migration, invasion and downstream signaling of U87-EGFRvIII cells. Moreover, the U2 aptamer can increase the radiosensitivity of U87-EGFRvIII in vitro and have a better antitumor effect of 188Re-U2 in vivo. Therefore, the results revealed the promising potential of the U2 aptamer to be a new type of drug candidate and aptamer-targeted drug delivery system for glioblastoma therapy.

    更新日期:2018-01-09
  • Microrna-140 inhibits the epithelial-mesenchymal transition and metastasis in colorectal cancer
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2018-01-04
    Jiazhi Li, Kun Zou, Lihui Yu, Wenyue Zhao, Ying Lu, Jun Mao, Bo Wang, Lu Wang, Shujun Fan, Bo Song, Lianhong Li

    MicroRNA-140, a cartilage-specific microRNA, has recently been implicated in the cancer progression. However, the comprehensive role of miR-140 in the invasion and metastasis of colorectal cancer (CRC) is still not fully understood. In this study, we confirmed that miR-140 downregulates SMAD family member 3 (Smad3) which is a key downstream effector of TGF-β signaling pathway, at the translational level in the CRC cell lines. Ectopic expression of miR-140 inhibits the process of epithelial-mesenchymal transition (EMT) at least partially through targeting Smad3, and induces the suppression of migratory and invasive capacities of CRC cells in vitro. MiR-140 also attenuates CRC cell proliferation possibly via downregulating Samd3. Furthermore, overexpression of miR-140 inhibits the tumor formation and metastasis of CRC in vivo and silenced Smad3 has the similar effect. Additionally, miR-140 expression is decreased in the clinical primary CRC specimens and appears a progressive reduction in the metastatic specimens, whereas Smad3 is overexpressed in the CRC samples. Taken together, our findings suggest that miR-140 might be a key suppressor of CRC progression and metastasis through inhibiting EMT process by targeting Smad3. MiR-140 may represent a novel candidate for CRC treatment.

    更新日期:2018-01-04
  • Targeting EGFR/HER2/HER3 with a three-in-one aptamer-siRNA chimera confers superior antitumor activity in HER2 expressing breast cancer
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-30
    Xiaolin Yu, Sharad Ghamande, Haitao Liu, Lu Xue, Shuhua Zhao, Wenxi Tan, Lijing Zhao, Shou-Ching Tang, Daqing Wu, Hasan Korkaya, Maihle Nita, Hong Yan Liu

    HER family members are interdependent and functionally compensatory. Simultaneously targeting EGFR/HER2/HER3 by antibody combinations has demonstrated the superior treatment efficacy over targeting one HER receptor. However, antibody combinations have their limitations in high immunogenicity and high cost. In this study, we have developed a three-in-one nucleic acid aptamer-siRNA chimera, which targets on EGFR/HER2/HER3 in one molecule. This inhibitory molecule was constructed such that a single EGFR siRNA is positioned between HER2 and HER3 aptamers to create a HER2 aptamer-EGFR siRNA-HER3 aptamer chimera (H2EH3). EGFR siRNA was delivered into HER2 expressing cells by HER2/HER3 aptamers-induced internalization. HER2/HER3 aptamers act as antagonist molecules for blocking HER2 and HER3 signaling pathways and also as tumor targeting agents for siRNA delivery. H2EH3 enables down-modulation of the expression of all three receptors, thereby triggering cell apoptosis. In breast cancer xenograft models, H2EH3 is able to bind to breast tumors with high specificity, and significantly inhibit tumor growth via either systemic or intratumoral administration. Owing to low immunogenicity, ease of production, and high thermostability, H2EH3 is a promising therapeutic to supplement current single HER inhibitors and may act as a treatment for HER2 positive breast cancer with intrinsic or acquired resistance to current drugs.

    更新日期:2017-12-31
  • Endogenous Cellular microRNAs Mediate Antiviral Defense against Influenza A Virus
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-30
    Shanxin Peng, Jing Wang, Songtao Wei, Changfei Li, Kai Zhou, Jun Hu, Xin Ye, Jinghua Yan, Wenjun Liu, George F. Gao, Min Fang, Songdong Meng

    The reciprocal interaction between influenza virus and host microRNAs (miRNAs) has been implicated in the regulation of viral replication and host tropism. However, the global roles of the cellular miRNA repertoire and the mechanisms of miRNA-mediated antiviral defense await further elucidation. In this study, we systematically screened 297 cellular miRNAs from human and mouse epithelial cells and identified five inhibitory miRNAs that efficiently inhibited influenza virus replication in vitro and in vivo. Among these miRNAs, hsa-mir-127-3p, hsa-mir-486-5p, hsa-mir-593-5p, and mmu-mir-487b-5p were found to target at least one viral gene segment of both human seasonal influenza H3N2 and the attenuated PR8 (H1N1) virus. Whereas hsa-miR-1-3p inhibited viral replication by targeting a supportive host factor ATP6V1A. Moreover, the number of miRNA binding sites in viral RNA segments was positively associated with the activity of host miRNA-induced antiviral defense. Treatment with a combination of the five miRNAs through agomir delivery pronouncedly suppressed viral replication and effectively improved protection against lethal challenge with PR8 in mice. These data suggest that the highly expressed miRNAs in respiratory epithelial cells elicit effective antiviral defenses against influenza A viruses and will be useful for designing miRNA-based therapies against viral infection.

    更新日期:2017-12-31
  • Transcriptome profiling of neovascularized corneas reveals miR-204 as a multi-target biotherapy deliverable by rAAVs
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-30
    Yi Lu, Phillip W.L. Tai, Jianzhong Ai, Dominic J. Gessler, Qin Su, Xieyi Yao, Qiang Zheng, Phillip D. Zamore, Xun Xu, Guangping Gao

    Corneal neovascularization (NV) is the major sight-threatening pathology caused by angiogenic stimuli. Current drugs that directly target pro-angiogenic factors to inhibit or reverse the disease require multiple rounds of administration and have limited efficacies. Here, we identify potential anti-angiogenic corneal microRNAs (miRNAs), and demonstrate a framework that employs discovered miRNAs as biotherapies deliverable by recombinant adeno-associated viruses (rAAVs). By querying differentially expressed miRNAs in neovascularized mouse corneas induced by alkali-burn, we have revealed 39 miRNAs that are predicted to target more than 5,500 differentially expressed corneal mRNAs. Among these, we selected miR-204 and assessed its efficacy and therapeutic benefit for treating injured corneas. Our results show that delivery of miR-204 by rAAV normalizes multiple novel target genes and biological pathways to attenuate vascularization of injured mouse cornea. Importantly, this gene therapy treatment alternative is efficacious and safe for mitigating corneal NV. Overall, our work demonstrates the discovery of potential therapeutic miRNAs in corneal disorders and their translation into viable treatment alternatives.

    更新日期:2017-12-31
  • Circ-SHKBP1 regulates the angiogenesis of U87 glioma-exposed endothelial cells through miR-544a/FOXP1 and miR-379/FOXP2 pathways
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-30
    Qianru He, Lini Zhao, Yunhui Liu, Xiaobai Liu, Jian Zheng, Hai Yu, Heng Cai, Jun Ma, Libo Liu, Ping Wang, Zhen Li, Yixue Xue
    更新日期:2017-12-31
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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