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  • 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
  • LncRNA Plscr4 controls cardiac hypertrophy by regulating miR-214
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-30
    Lifang Lv, Tianyu Li, Xuelian Li, Chaoqian Xu, Qiushuang Liu, Hua Jiang, Yingnan Li, Yingqi Liu, He Yan, Qihe Huang, Yuhong Zhou, Mingyu Zhang, Hongli Shan, Haihai Liang

    Cardiac hypertrophy accompanied by maladaptive cardiac remodeling is the uppermost risk factor for the development of heart failure. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have various biological functions, and their vital role in the regulation of cardiac hypertrophy still needs to be explored. In this study, we demonstrated that lncRNA Plscr4 was upregulated in hypertrophic mice hearts and in angiotensin II (Ang II)–treated cardiomyocytes. Next, we observed that overexpression of Plscr4 attenuated Ang II-induced cardiomyocyte hypertrophy. Conversely, inhibition of Plscr4 gave rise to cardiomyocyte hypertrophy. Furthermore, overexpression of Plscr4 attenuated TAC (transverse aortic constriction)-induced cardiac hypertrophy. Finally, we demonstrated that Plscr4 acted as an endogenous sponge of miR-214, and forced expression of Plscr4 downregulated miR-214 expression to promote Mfn2 and attenuate hypertrophy. In contrast, knockdown of Plscr4 upregulated miR-214 to induce cardiomyocyte hypertrophy. Additionally, luciferase assay showed that miR-214 was the direct target of Plscr4, and overexpression of miR-214 counteracted the anti-hypertrophic effect of Plscr4. Collectively, these findings identify Plscr4 as a negative regulator of cardiac hypertrophy in vivo and in vitro due to its regulation of the miR-214-Mfn2 axis, suggesting that Plscr4 might act as a therapeutic target for the treatment of cardiac hypertrophy and heart failure.

    更新日期:2017-12-31
  • Gene Therapy via Trans-Splicing for LMNA-related Congenital Muscular Dystrophy
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-30
    Feriel Azibani, Astrid Brull, Ludovic Arandel, Maud Beuvin, Isabelle Nelson, Arnaud Jollet, Esma Ziat, Bernard Prudhon, Sofia Benkhelifa-Ziyyat, Marc Bitoun, Stéphanie Lorain, Gisèle Bonne, Anne T. Bertrand

    We assessed the potential of Lmna-mRNA repair by spliceosome-mediated RNA trans-splicing as a therapeutic approach for LMNA-related congenital muscular dystrophy. This gene therapy strategy leads to reduction of mutated transcript expression for the benefit of corresponding wild type transcripts. We developed 5’-RNA pre-trans-splicing molecules containing the 5 first exons of Lmna and targeting intron 5 of Lmna pre-mRNA. Among 9 pre-trans-splicing molecules, differing in the targeted sequence in intron 5 and tested in C2C12 myoblasts, 3 induced trans-splicing events on endogenous Lmna mRNA and confirmed at protein level. Further analyses performed in primary myotubes derived from a L-CMD mouse model led to a partial rescue of the mutant phenotype. Finally, we tested this approach in vivo using adeno-associated virus (AAV) delivery in newborn mice and showed that trans-splicing events occurred in WT mice 50 days after AAV delivery although at a low rate. Altogether, while these results provide the first evidence for reprogramming LMNA mRNA in vitro, strategies to improve the rate of trans-splicing events still need to be developed for efficient application of this therapeutic approach in vivo.

    更新日期:2017-12-31
  • Thermodynamic, anticoagulant and antiproliferative properties of thrombin binding aptamer analogues containing novel UNA derivative
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-30
    Weronika Kotkowiak, Jolanta Lisowiec-Wachnicka, Jakub Grynda, Ryszard Kierzek, Jesper Wengel, Anna Pasternak
    更新日期:2017-12-31
  • STAT3 gene silencing by Aptamer-siRNA chimera as selective therapeutic for Glioblastoma
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-30
    Carla Lucia Esposito, Silvia Nuzzo, Silvia Catuogno, Simona Romano, Filomena de Nigris, Vittorio de Franciscis

    Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults and despite advances in neuro-oncology, the prognosis for patients remains dismal. The signal transducer and activator of transcription-3 (STAT3) has been reported as key regulator of the highly aggressive mesenchymal glioblastoma subtype and its direct silencing (by RNAi oligonucleotides) has revealed a great potential as anti-cancer therapy. However, clinical use of oligonucleotide-based therapies is dependent on safer ways for tissue-specific targeting and increased membrane penetration. Objective of this study is to explore the use of nucleic acid aptamers as carriers to specifically drive a STAT3 siRNA to GBM cells in a receptor-dependent manner. Using an aptamer that binds to and antagonizes the oncogenic receptor tyrosine kinase PDGFRβ (Gint4.T), here we describe the design of a novel aptamer-siRNA chimera (Gint4.T-STAT3) to target STAT3. We demonstrate the efficient delivery and silencing of STAT3 in PDGFRβ positive GBM cells. Importantly, the conjugate reduces cell viability and migration in vitro and inhibits tumor growth and angiogenesis in vivo in a subcutaneous xenograft mouse model. Our data reveals Gint4.T-STAT3 conjugate as a novel molecule with great translational potential for GBM therapy.

    更新日期:2017-12-31
  • The antibiotic-free pFAR4 vector paired with the Sleeping Beauty transposon system mediates efficient transgene delivery in human cells
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-30
    Marie Pastor, Sandra Johnen, Nina Harmening, Mickäel Quiviger, Julie Pailloux, Martina Kropp, Peter Walter, Zoltán Ivics, Zsuzsanna Izsvák, Gabriele Thumann, Daniel Scherman, Corinne Marie

    The anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF) demonstrated a potency to control choroidal neovascularization in age-related macular degeneration (AMD) patients. The goal of the present study was the development of an efficient and safe technique to integrate ex vivo the PEDF gene into retinal pigment epithelial (RPE) cells for later transplantation to the subretinal space of AMD patients to allow a continuous PEDF secretion in the vicinity of the affected macula. Since successful gene therapy approaches require efficient gene delivery and stable gene expression, we have used the antibiotic-free pFAR4 mini-plasmid vector to deliver the hyperactive Sleeping Beauty transposon system that mediates transgene integration into the genome of host cells. In an initial study, lipofection-mediated co-transfection of HeLa cells with the SB100X transposase gene and a reporter marker delivered by pFAR4 showed a two-fold higher level of genetically modified cells than when using the pT2 vectors. Similarly, with the pFAR4 constructs, electroporation-mediated transfection of primary human RPE cells led to a 2.4-fold higher secretion of recombinant PEDF protein, which was still maintained eight months after transfection. Thus, our results show that the pFAR4 plasmid is a superior vector for the delivery and integration of transgenes into eukaryotic cells.

    更新日期:2017-12-31
  • PIWIL1/piRNA-DQ593109 regulates the permeability of the blood-tumor barrier via the MEG3/miR-330-5p/RUNX3 axis
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-30
    Shuyuan Shen, Hai Yu, Xiaobai Liu, Yunhui Liu, Jian Zheng, Ping Wang, Wei Gong, Jiajia Chen, Lini Zhao, Yixue Xue

    The blood-tumor barrier (BTB) restricts the efficient delivery of anti-glioma drugs to cranial glioma tissues. Increased BTB permeability may allow greater delivery of the therapeutic agents. Increasing evidence has revealed that PIWI proteins and PIWI-interacting RNAs (piRNAs) play important role in tumor progression. However, whether PIWI proteins and piRNAs regulate BTB permeability remains unclear. In the present study, we demonstrated that the PIWIL1/piRNA-DQ593109 (piR-DQ593109) complex was the predominant regulator of the BTB permeability. Briefly, PIWIL1 was upregulated in glioma endothelial cells (GECs). Furthermore, piR-DQ593109 was also overexpressed in GECs as revealed via piRNA microarray. Downregulation of PIWIL1 or piR-DQ593109 increased the permeability of the BTB. Moreover, PIWIL1 and piR-DQ593109, which formed a piRNA-induced silencing complex, degraded the long non-coding RNA maternally expressed 3 (MEG3) in a sequenced-dependent manner. Furthermore, restoring MEG3 released post-transcriptional inhibition of Runt related transcription factor 3 (RUNX3) by sponging miR-330-5p. In addition, RUNX3 bound to the promoter regions and reduce the promoter activities of ZO-1, occludin, and claudin-5, which significantly impaired the expression levels of ZO-1, occludin, and claudin-5. In conclusion, downregulating PIWIL1 and piR-DQ593109 increased the BTB permeability through the MEG3/miR-330-5p/RUNX3 axis. These data may provide insight into the glioma treatment.

    更新日期:2017-12-31
  • Long non-coding RNAs, novel culprits or bodyguards in neurodegenerative diseases
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-22
    Ding-Qi Wang, Peng Fu, Chengye Yao, Ling-Shuang Zhu, Tong-Yao Hou, Jian-Guo Chen, Youming Lu, Dan Liu, Ling-Qiang Zhu

    Long non-coding RNA (lncRNA) is a kind of non-coding RNA (ncRNA) with a length of 200 nt to 100 kb that lacks a significant open reading frame (ORF) encoding a protein. lncRNAs are widely implicated in various physiological and pathological processes, such as epigenetic regulation, cell cycle regulation, cell differentiation regulation, cancer, and neurodegenerative diseases, through their interactions with chromatin, protein and other RNAs. Numerous studies have suggested that lncRNAs are closely linked with the occurrence and development of a variety of diseases, especially neurodegenerative diseases, of which the etiologies are complicated and the underlying mechanisms remain elusive. Determining the roles of lncRNA in the pathogenesis of neurodegenerative diseases will not only deepen understanding of the physiological and pathological processes that occur in those diseases but also provide new ideas and solutions for their diagnosis and prevention. This review aims to highlight the progress of lncRNA research in the pathological and behavioral changes of neurodegenerative diseases. Specifically, we focus on how lncRNA dysfunctions are involved in the pathogenesis of Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis.

    更新日期:2017-12-22
  • DNA methylation-mediated down-regulation of miR-300 regulates the ubiquitination of PTEN through the CRL4BDCAF13 E3 ligase in osteosarcoma cells
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-22
    Zhi Chen, Wei Zhang, Kaibiao Jiang, Bin Chen, Kun Wang, Lifeng Lao, Canglong Hou, Fei Wang, Caiguo Zhang, Hongxing Shen

    Cullins, critical members of the cullin-RING ubiquitin ligases (CRLs), are often aberrantly expressed in different cancers. However, the underlying mechanisms regarding aberrant expression of these cullins and the specific substrates of CRLs in different cancers are mostly unknown. Here, we demonstrate that overexpressed CUL4B in human osteosarcoma cells forms an E3 complex with DDB1 and DCAF13. In vitro and in vivo analyses indicated that the CRL4BDCAF13 E3 ligase specifically recognized the tumor suppressor PTEN for degradation, and disruption of this E3 ligase resulted in PTEN accumulation. Further analyses indicated that miR-300 directly targeted the 3’-UTR of CUL4B, and DNA hypermethylation of a CpG island in the miR-300 promoter region contributed to the down-regulation of miR-300. Interestingly, ectopic expression of miR-300 or treatment with 5-AZA-2’-deoxycytidine, a DNA methylation inhibitor, decreased the stability of CRL4BDCAF13 E3 ligase and reduced PTEN ubiquitination. By applying in vitro screening to identify small molecules that specifically inhibit CUL4B-DDB1 interaction, we found that TSC01131 could greatly inhibit osteosarcoma cell growth and could disrupt the stability of the CRL4BDCAF13 E3 ligase. Collectively, our findings shed new light on the molecular mechanism of CUL4B function and might also provide a new avenue for osteosarcoma therapy.

    更新日期:2017-12-22
  • RNAseq analysis of an antisense sequence harbored by a U7snRNA optimized for exon skipping in Duchenne patients reveals no apparent off-target effect
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-21
    Claire Domenger, Marine Allais, Virginie François, Adrien Léger, Emilie Lecomte, Marie Montus, Laurent Servais, Thomas Voit, Philippe Moullier, Yann Audic, Caroline Le Guiner

    Non-coding uridine-rich small nuclear RNAs have emerged in recent years as effective tools for exon skipping for the treatment of Duchenne Muscular Dystrophy (DMD), a degenerative muscular genetic disorder. We recently showed the high capacity of a recombinant AAV-U7snRNA vector to restore the reading frame of the DMD messenger RNA in the muscles of DMD dogs. We are now moving toward a phase I/II clinical trial with a rAAV-U7snRNA-E53, carrying an antisense sequence designed to hybridize exon 53 of the human DMD messenger. As observed for genome editing tools, antisense sequences present a risk of off-target effects, reflecting partial hybridization onto unintended transcripts. To characterize the clinical antisense sequence, we studied its expression and explored the occurrence of its off-target effects in human in vitro models of skeletal muscle and liver. We presented a comprehensive methodology combining RNA sequencing and in silico filtering to analyze off-targets. We showed that U7snRNA-E53 induced the effective exon skipping of the DMD transcript without inducing the notable deregulation of transcripts in human cells, neither at gene expression nor at the mRNA splicing level. Altogether, these results suggest that the use of the rAAV-U7snRNA-E53 vector for exon skipping could be safe in eligible DMD patients.

    更新日期:2017-12-21
  • Glucagon-like peptide 2 promotes directed-differentiation from osteosarcoma cells to osteoblast and inhibits growth of osteosarcoma cells
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-21
    Yi Lu, Dongdong Lu, Yu Hu

    Glucagon-like peptide 2 (GLP2) is a proglucagon-derived peptide which is involved in the regulation of energy absorption and exerts beneficial effects on glucose metabolism. However, the exact mechanisms underlying the GLP2 during osteogenic differentiation has not been illustrated. Herein, we indicated that GLP2 was demonstrated to result in positive action during the osteogenic differentiation of human osteosarcoma cells. Our findings demonstrate that GLP2 inhibis the growth of osteosarcoma cells in vivo and in vitro. Mechanistic investigations reveal GLP2 inhibits the expression and activity of NFκB , triggering the decrease of C-Myc,PKM2,CyclinD1 in osteosarcoma cells. In particular, rescued NFκB abrogates the functions of GLP2 in osteosarcoma cells. Strikingly, overexpression of GLP2 significantly increased the expression of osteogenesis-associated genes (e.g. Ocn and PICP ) dependent on C-Fos-BMP signaling which promotes directed-differentiation from osteosarcoma cells to osteoblast with higher alkaline phosphatase activity. Taken together, our results suggested that GLP2 could be a valuable drug to promote directed-differentiation from osteosarcoma cells to osteoblast, which may provide potential therapeutic targets for the treatment of osteosarcoma.

    更新日期:2017-12-21
  • CircHLA-C Plays an Important Role in Lupus Nephritis by Sponging miR-150
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-20
    Junjun Luan, Congcong Jiao, Weiwei Kong, Jingqi Fu, Wei Qu, Ying Chen, Xinwang Zhu, Yu Zeng, Guangyin Guo, Huimeng Qi, Li Yao, Jingbo Pi, Lining Wang, Hua Zhou

    Circular RNAs (circRNAs) participate in the pathogenesis of various diseases by sponging microRNAs (miRs). However, the roles of circRNAs remain unreported in glomerular diseases. We previously reported that miR-150 positively correlated with renal chronicity index in patients with lupus nephritis (LN). We aimed to investigate renal circRNA profiling and the interaction between circRNAs and miR-150 in LN patients. Six renal biopsies from untreated female patients with LN class IV and five normal kidney tissues from urology patients were used for circRNA sequencing. 171 circRNAs with 2-fold differential expression were identified in LN compared with normal control. Ten selected circRNAs were validated by qPCR, and seven circRNAs showed same significant increases as the sequencing results. CircHLA-C positively correlated with proteinuria (r=0.92, p<0.01), serum creatinine (r=0.76, p=0.08), renal activity index (r=0.88, p<0.05), and crescentic glomeruli (r=0.93, p<0.01). Renal circHLA-C increased 2.72 folds and miR-150 decreased 66% in LN compared with normal control (p<0.05). Bio-informatic analysis predicted miR-150 was regulated by circHLA-C and displayed one perfect match seed between circHLA-C and miR-150. The renal miR-150 showed a tendency of negative correlation with circHLA-C in LN patients. In conclusion, circHLA-C may play an important role in the pathogenesis of lupus nephritis by sponging miR-150.

    更新日期:2017-12-20
  • Circular siRNAs for reducing off-target effects and enhancing long-term gene silencing in cells and mice
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-19
    Liangliang Zhang, Duanwei Liang, Changmai Chen, Yuan Wang, Gubu Amu, Jiali Yang, Lijia Yu, Ivan J. Dmochowski, Xinjing Tang
    更新日期:2017-12-19
  • Prevention of Asthma Exacerbation by Simultaneous Inhibition of NFκB and STAT6 Activation Using a Chimeric Decoy Strategy in a Mouse Model
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-14
    Tetsuo Miyake, Takashi Miyake, Makoto Sakaguchi, Hirokazu Nankai, Takahiro Nakazawa, Ryuichi Morishita
    更新日期:2017-12-14
  • Downregulation of microRNA-455-3p links to proliferation and drug resistance of pancreatic cancer cells via targeting TAZ
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-09
    Ting Zhan, Xiaodong Huang, Xia Tian, Xiaoli Chen, Yu Ding, Hesheng Luo, Yadong Zhang

    Drug resistance is a major cause of treatment failure in pancreatic cancer. The limited evidence indicates the involvement of miR-455-3p in chemotherapy resistance of cancer. Herein, we observed that miR-455-3p was significantly decreased in pancreatic cancer tissues and cell lines by quantitative PCR. We then confirmed that inhibition of miR-455-3p increased cell proliferation and gemcitabine resistance of pancreatic cancer, whereas forced overexpression of miR-455-3p had the opposite effect. Furthermore, we demonstrated that TAZ, which is associated with drug resistance of pancreatic cancer, is a new direct downstream target of miR-455-3p. Our present study suggests that miR-455-3p contributes to cell proliferation and drug resistance in pancreatic cancer cells via targeting TAZ.

    更新日期:2017-12-14
  • Selection and identification of skeletal muscle-targeted RNA aptamers
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-09
    Styliana Philippou, Nikolaos P. Mastroyiannopoulos, Neoklis Makrides, Carsten W. Lederer, Marina Kleanthous, Leonidas A. Phylactou
    更新日期:2017-12-14
  • In Vivo SELEX of an Inhibitory NSCLC-Specific RNA Aptamer from PEGylated RNA Library
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-09
    Hanlu Wang, Yibang Zhang, Haiping Yang, Meng Qin, Xinxin Ding, Rihe Liu, Yongping Jiang

    Aptamers are widely used in numerous biochemical, bioanalytical and biological studies. Most aptamers are developed through an in vitro selection process called SELEX against either purified targets or living cells expressing targets of interest. We report here an in vivo SELEX in mice using PEGylated RNA library for the identification of a 2’-F RNA aptamer (RA16) that specifically binds to NCI-H460 non-small cell lung cancer cells with an affinity (Kd) at 9 ± 2 nM. Interestingly, RA16 potently inhibited cancer cell proliferation in a dose-dependent manner with an IC50 of 116.7 nM. When tested in vivo in xenografted mice, RA16 showed gradual migration toward tumor and accumulation at tumor site over time. In vivo anti-cancer study showed that the average inhibition rate for mouse tumors in RA16-treated group was 54.26 ± 5.87% on day 16 versus the control group. The aptamer RA16 adducted with epirubicin (RA16-epirubicin) showed significantly higher toxicity against targeted NCI-H460 cells and low toxicity against non-targeted tumor cells. Furthermore, RA16-epirubicin adduct exhibited in vivo anticancer efficacy, with an inhibition rate of 64.38 ± 7.92% when administrated in H460 xenograft mouse model. In summary, a specific bi-functional RNA aptamer RA16 was selected targeting and inhibiting towards NCI-H460 in vitro and in vivo.

    更新日期:2017-12-14
  • Inhibition of TDP43-mediated SNHG12-miR-195-SOX5 feedback loop impeded malignant biological behaviors of glioma cells
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-08
    Xiaobai Liu, Jian Zheng, Yixue Xue, Chengbin Qu, Jiajia Chen, Zhenhua Wang, Zhen Li, Lei Zhang, Yunhui Liu

    Long non-coding RNAs (lncRNAs) dysregualtion is involved in tumorigenesis and regulation of diverse cellular process in gliomas. LncRNA SNHG12 is up-regulated and promotes cell growth in human osteosarcoma cells.TAR-DNA binding protein 43 (TDP43) functions as an oncogene in various tumors by modulating RNAs expression. Down-regulation of TDP43 or SNHG12 significantly inhibited malignant biological behaviors of glioma cells. MiR-195, down-regulated in glioma tissues and cells, significantly impaired the malignant progression of glioma cells. TDP43 upregulated miR-195 in a SNHG12-dependent manner. We further revealed that, SNHG12 and miR-195 were in a RNA-induced silencing complex(RISC). Inhibition of SNHG12 combined with restoration of miR-195 robustly reduced tumor growth in vivo. SOX5 was over-expressed in glioma tissues and cells. MiR-195 targeted SOX5 3′-UTR in a sequence-specific manner. Gelsolin was activated by SOX5. More importantly, SOX5 activated SNHG12 promoter and up-regulated its expression, forming a feedback loop. Dysregulation of SNHG12, miR-195 and SOX5predicted poor prognosis of glioma patients. The present study demonstrated that SNHG12-miR-195-SOX5 feedback loop exerted a crucial role in the regulation of glioma cells’ malignant progression.

    更新日期:2017-12-14
  • Targeted Delivery of Auristatin Modified Toxins to Pancreatic Cancer Using Aptamers
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-01
    Christina Kratschmer, Matthew Levy

    Pancreatic cancer is one of the most lethal malignancies. Treatment with the first-line agent, gemcitabine, is often unsuccessful because it, like other traditional chemotherapeutic agents, is non-specific, resulting in off-target effects that necessitate administration of sub-curative doses. Alternatively, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) are highly toxic small molecules that require ligand targeted delivery. MMAE has already received FDA approval as a component of an anti-CD30 antibody drug conjugate, brentuximab vedotin. However, in contrast to antibodies, aptamers have distinct advantages. They are chemicals, which allows them to be produced synthetically and facilitates the rapid development of diagnostics and therapeutics with clinical applicability. Additionally, their small size allows for enhanced tissue distribution and rapid systemic clearance. Here, we assayed the toxicity of MMAE and MMAF conjugated to an anti-transferrin receptor aptamer, Waz, and an anti-epidermal growth factor receptor aptamer, E07, on the pancreatic cancer cell lines Panc-1, MIA PaCa-2, and BxPC3. In vitro, our results indicate that these aptamers are a viable option for the targeted delivery of toxic payloads to pancreatic cancer cells.

    更新日期:2017-12-14
  • Exosomes in cancer liquid biopsy: a focus on breast cancer
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-01
    Sina Halvaei, Shiva Daryani, Zahra Eslami-S, Tannaz Samadi, Narges Jafarbeik-Iravani, Tayebeh Oghabi Bakhshayesh, Keivan Majidzadeh-A, Rezvan Esmaeili

    The important challenge about cancer is diagnosis in primary stages and proper treatment. Although classical clinico-pathological features of the tumor have major prognostic value, the advances in diagnosis and treatment are indebted to discovery of molecular biomarkers and control of cancer in the pre-invasive state. Moreover, the efficiency of available therapeutic options is highly diminished and chemotherapy is still the main treatment due to lack of enough specific targets. Accordingly, finding the new non-invasive biomarkers for cancer is still an important clinical challenge which is not achieved yet. There are current technologies in order to screen, diagnose, prognose and treat cancer, but the limitations of these implements and procedures are undeniable. Liquid biopsy as a noninvasive method has promising futures in the field of cancer, and exosomes as one of the recent areas have drawn much attention. In this review, the potential capability of exosomes are summarized in cancer with the special focus on breast cancer, as the second cause of cancer mortality in women all around the world. It discusses reasons to choose exosomes for liquid biopsy and the studies related to different potential biomarkers found in the exosomes. Moreover, exosome studies on milk as a specific biofluid are also discussed. At last, since choosing the method for exosome studies is very challenging, the summary of different techniques is provided.

    更新日期:2017-12-14
  • Transplantation of Gene-edited Hepatocyte-like Cells Modestly Improves Survival of Arginase-1 Deficient Mice
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-01
    Yuan Yan Sin, Laurel L. Ballantyne, Christopher R. Richmond, Colin D. Funk

    Progress in gene editing research has been accelerated by utilizing engineered nucleases in combination with induced pluripotent stem cell (iPSC) technology. Here, we report transcription activator-like effector nuclease (TALEN)-mediated reincorporation of Arg1 exons 7 and 8 in iPSCs derived from arginase-1 deficient mice possessing Arg1Δ alleles lacking these terminal exons. The edited cells could be induced to differentiate into hepatocyte-like cells (iHLCs) in vitro and were subsequently used for transplantation into our previously described (Sin et al., PLoS ONE 2013) tamoxifen-inducible Arg1-Cre arginase-1 deficient mouse model. While successful gene targeted repair was achieved in iPSCs containing Arg1Δ alleles, only minimal restoration of urea cycle function could be observed in the iHLC-transplanted mice compared to control mice and survival in this lethal model was extended by up to a week in some mice. The partially rescued phenotype may be due to inadequate regenerative capacity of arginase-1 expressing cells in the correct metabolic zones. Technical hurdles exist, and will need to be overcome for gene-edited iPSC to iHLC rescue of arginase-1 deficiency, a rare urea cycle disorder.

    更新日期:2017-12-14
  • Long non-coding RNA LINC00339 stimulates glioma vasculogenic mimicry formation by regulating miR-539-5p/ TWIST1/MMPs axis
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-11-26
    Junqing Guo, Heng Cai, Xiaobai Liu, Jian Zheng, Yunhui Liu, Wei Gong, Jiajia Chen, Zhuo Xi, Yixue Xue

    Glioma is recognized as a highly angiogenic malignant brain tumor. Vasculogenic mimicry (VM) greatly restricts the therapeutic effect of antiangiogenic tumor therapy for glioma patients. However, the molecular mechanisms of VM formation in glioma remain unclear. Here, we demonstrated that LINC00339 was upregulated in glioma tissue as well as in glioma cell lines. The expression of LINC00339 in glioma tissues was positively correlated with glioma VM formation. Knockdown of LINC00339 inhibited glioma cell proliferation, migration, invasion and tube formation, meanwhile downregulating the expression of VM-related molecular MMP-2 and MMP-14. Furthermore, knockdown of LINC00339 significantly increased the expression of miR-539-5p. Both bioinformatics and luciferase reporter assay revealed that LINC00339 regulated the above effects via binding to miR-539-5p. Besides, over-expression of miR-539-5p resulted in decreased expression of TWIST1, a transcription factor known to play an oncogenic role in glioma and identified as a direct target of miR-539-5p. TWIST1 upregulated the promoter activities of MMP-2 and MMP-14. The in vivo study showed that nude mice carrying tumors with knockdown of LINC00339 and over-expression of miR-539-5p exhibited the smallest tumor volume through inhibiting VM formation. In conclusion, LINC00339 may be used as a novel therapeutic target for VM formation in glioma.

    更新日期:2017-12-14
  • MiR-185 inhibits fibrogenic activation of hepatic stellate cells and prevents liver fibrosis
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-11-24
    Li Zhou, Shunai Liu, Ming Han, Yanhua Ma, Shenghu Feng, Jing Zhao, Hongping Lu, Xiaoxue Yuan, Jun Cheng

    Recent studies have shown the effect of microRNAs on HSCs activation and transformation, which is essential for the pathogenesis of liver fibrosis. In our study, we explored the role of miR-185 in liver fibrosis. Plasma miR-185 was detected in hepatitis B virus-related liver fibrosis patients (S2/3, n = 10) by Illumina HiSeq sequencing, and healthy volunteers were selected (n = 8) as the control group. We found that the plasma miR-185 level in fibrosis patients was significantly downregulated. CCl4-induced fibrosis tissues in mice liver and TGF-β1–activated HSCs also presented downregulated miR-185 concomitant with an increased expression of RHEB and RICTOR. To explore the correlations, LX-2 cells were transiently transfected with miR-185 mimics. The expression levels of α-SMA, collagen I, and collagen III were decreased as well as RHEB and RICTOR. Inhibition of endogenous miR-185 increases fibrogenic activity. Furthermore, dual-luciferase reporter assays indicated that miR-185 inhibited the expression of RHEB and RICTOR by directly targeting their 3ʹ-UTRs regions. Moreover, silencing RHEB and RICTOR suppressed α-SMA, collagen expression levels. In conclusion, miR-185 prevents liver fibrogenesis by inhibiting HSC activation by inhibiting RHEB and RICTOR. These results provide new insights into the mechanisms behind the anti-fibrotic effect of miR-185.

    更新日期:2017-12-14
  • Recombinant Adeno-Associated Virus Mediated Delivery of MicroRNA-21-3p Lowers Hypertension
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-11-24
    Feng Wang, Qin Fang, Chen Chen, Ling Zhou, Huaping Li, Zhongwei Yin, Yan Wang, Chun Xia Zhao, Xiao Xiao, Dao Wen Wang

    Hypertension is the most important risk factor for cardiovascular diseases worldwide. However, the underlying molecular mechanisms of hypertension are complex and remain largely elusive. Here, we described a novel, microRNA-dependent therapeutic strategy for hypertension. First, we found that plasma microRNA-21-3p (miR-21-3p) levels were significantly reduced both in hypertensive patients and spontaneously hypertensive rats (SHRs) when compared with the normal controls. In a series of experiments to dissect the role of miR-21-3p in hypertension, we showed that intravenous delivery of recombinant adeno-associated virus (rAAV)-mediated miR-21-3p expression induced a persistent attenuation of hypertension with marked amelioration of target organ damages, including cardiac hypertrophy and fibrosis, artery and kidney fibrosis in SHRs, whereas miR-21-3p tough decoys (TuDs) counteracted the above effects. Computational prediction coupled with biochemical experiments revealed that miR-21-3p mediated-hypotensive reduction effect was accomplished by regulating phenotypic switch of vascular smooth muscle cell (VSMC) via suppression of ADRA2B in arteries. Furthermore, we observed that activation of transcription factor NF-κB and SRF significantly increased the expression of miR-21-3p in VSMC. In summary, our study is the first to identify a novel role and mechanism of miR-21-3p in blood pressure control and provides a possible strategy for hypertension therapy using rAAV-miR-21-3p.

    更新日期:2017-12-14
  • RNA nanotherapeutics for the amelioration of astroglial reactivity
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-11-24
    Jayden A. Smith, Alice Braga, Jeroen Verheyen, Silvia Basilico, Sara Bandiera, Clara Alfaro-Cervello, Luca Peruzzotti-Jametti, Dan Shu, Farzin Haque, Peixuan Guo, Stefano Pluchino
    更新日期:2017-12-14
  • miR-208a-3p suppresses osteoblast differentiation and inhibits bone formation by targeting ACVR1
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-11-24
    Yasir Arfat, Muhammad Asim R. Basra, Muhammad Shahzad, Kashif Majeed, Nasir Mahmood, Hina Munir

    Emerging evidence indicates that many microRNAs (miRNAs) are indispensable regulators of osteoblast differentiation and bone formation. However, role of miRNAs in mechanotransduction of osteoblasts remains to be elucidated. This study aimed to identify a mechanosensitive miRNA that regulates Activin A receptor type I (ACVR1)-induced osteogenic differentiation. After four weeks of hind limb unloading (HLU) suspension of six-month-old male C57BL/6J mice, femurs and tibias were harvested to extract total bone RNAs. Elevated levels of miR-208a-3p correlated with a lower degree of bone formation in whole bone samples of HLU mice. However, in vitro overexpression of miR-208a-3p inhibited osteoblasts differentiation whereas silencing of miR-208a-3p by antagomiR-208a-3p promoted expression of osteoblast activity, bone formation marker genes and matrix mineralization under mechanical unloading condition. Bioinformatics analysis and a luciferase assay revealed that ACVR1 is a target gene of miR-208a-3p that negatively regulates osteoblast differentiation under mechanical unloading environment. Further, this study also demonstrates that in vivo pre-treatment with antagomiR-208a-3p led to an increase in bone formation, trabecular microarchitecture and partly rescued the bone loss caused by mechanical unloading. Collectively these results suggest that in vivo, inhibition of miRNA-208a-3p by antagomiR-208a-3p may be a potential therapeutic strategy for ameliorating bone loss.

    更新日期:2017-12-14
  • Selection and identification of skeletal muscle-targeted RNA aptamers
    Mol. Ther. Nucl. Acids (IF 6.392) Pub Date : 2017-12-09
    Styliana Philippou, Nikolaos P. Mastroyiannopoulos, Neoklis Makrides, Carsten W. Lederer, Marina Kleanthous, Leonidas A. Phylactou

    Oligonucleotide gene therapy has shown great promise for the treatment of muscular dystrophies. Nevertheless, the selective delivery to affected muscles has shown to be challenging due to their high representation in the body and the high complexity of their cell membranes. Current trials show loss of therapeutic molecules to non-target tissues leading to lower target efficacy. Therefore, strategies that increase uptake efficiency would be particularly compelling. To address this need, we applied a cell-internalization SELEX approach and identified a skeletal muscle-specific RNA aptamer. A01B RNA aptamer preferentially internalizes in skeletal muscle cells and exhibits decreased affinity for off-target cells. Moreover, this in vitro selected aptamer retained its functionality in vivo, suggesting a potential new approach for targeting skeletal muscles. Ultimately, this will aid in the development of targeted oligonucleotide therapies against muscular dystrophies.

    更新日期:2017-12-12
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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