A chymase inhibitory RNA aptamer improves cardiac function and survival after myocardial infarction Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-13 Denan Jin, Shinji Takai, Yosuke Nonaka, Satoko Yamazaki, Masatoshi Fujiwara, Yoshikazu Nakamura
We have reported that mast cell chymase, an angiotensin II-generating enzyme, is important in cardiovascular tissues. Recently, we developed a new chymase-specific inhibitory RNA aptamer, HA28, and evaluated the effects of HA28 on cardiac function and the mortality rate after myocardial infarction. Eechocardiographic parameters, such as left ventricular ejection fraction, fractional shortening, and the ratio of early to late ventricular filling velocities were significantly improved by treatment with HA28 after myocardial infarction. The mortality rate was significantly reduced in the HA28-treated group. Cardiac chymase activity and chymase gene expression were significantly higher in the vehicle-treated myocardial infarction group, and these were markedly suppressed in the HA28-treated myocardial infarction group. The present study provides the first evidence that a single-stranded RNA aptamer that is a chymase-specific inhibitor is very effective in the treatment of acute heart failure caused by myocardial infarction. Chymase may be a new therapeutic target in post myocardial infarction pathophysiology.
MicroRNA-29b Mediates Lung Mesenchymal-Epithelial Transition and Prevents Lung Fibrosis in the Silicosis Model Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-06 Jingping Sun, Qiuyue Li, Ximeng Lian, Zhonghui Zhu, Xiaowei Chen, Wanying Pei, Siling Li, Ali Abbas, Yan Wang, Lin Tian
A single H1 promoter can drive both guide RNA and endonuclease expression in the CRISPR-Cas9 system Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-01 Zongliang Gao, Elena Herrera-Carrillo, Ben Berkhout
The RNA-guided endonuclease Cas9 (CRISPR-Cas9) genome editing system has been widely used for biomedical research and holds great potential for therapeutic applications in eukaryotes. The conventional vector-based CRISPR-Cas9 delivery system requires two different RNA polymerase promoters for expression of the guide RNA (gRNA) and Cas9 endonuclease. The large size and relative complexity of such CRISPR transgene cassettes impede their broad implementation, especially in gene therapy applications with viral vectors that have a limited packaging capacity. Here, we report the design of a single promoter driven CRISPR-Cas9 system that employs the dual-polymerase (II and III) activity of the H1 promoter. This size reduction strategy of the vector insert provides a significant titer advantage in the lentiviral vector (LV) over the regular CRISPR system.
Overcoming the undesirable CRISPR-Cas9 expression in gene correction Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-30 Emily Xia, Rongqi Duan, Fushan Shi, Kyle E. Seigel, Hartmut Grasemann, Jim Hu
The CRISPR-Cas9 system is attractive for gene therapy as it allows for permanent genetic correction. However, as a new technology, Cas9 gene editing in clinical applications faces major challenges, such as safe delivery and gene targeting efficiency. Cas9 is a foreign protein to recipient cells, thus its expression may prompt the immune system to eliminate gene-edited cells. To overcome these challenges, we have engineered a novel delivery system based on the helper-dependent adenoviral (HD-Ad) vector, which is capable of delivering genes to airway basal stem cells in vivo. Using this system, we demonstrate the successful co-delivery of both CRISPR-Cas9/single-guide RNA and the LacZ reporter or CFTR gene as donor DNA to cultured cells. HD-Ad vector genome integrity is compromised following donor DNA integration, and because the CRISPR-Cas9/single-guide RNA and donor DNA are carried on the same vector, CRISPR-Cas9 expression is concurrently eliminated. Thus, we show the feasibility of site-specific gene targeting with limited Cas9 expression. In addition, we achieve stable CFTR expression and functional correction in cultured cells following successful gene integration.
“The AT interstrand crosslink: structure, electronic properties and influence on charge transfer in ds-DNA” Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-26 Boleslaw T. Karwowski
Circular RNA transcriptomics analysis of primary human brain microvascular endothelial cells infected with meningitic Escherichia coli Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-26 Ruicheng Yang, Bojie Xu, Bo Yang, Jiyang Fu, Lu Liu, Nouman Amjad, Aoling Cai, Chen Tan, Huanchun Chen, Xiangru Wang
With their essential regulatory roles in gene expression and high abundance in the brain, circular RNAs (circRNAs) have recently attracted considerable attention. Many studies have shown that circRNAs play important roles in the pathology of central nervous system (CNS) diseases, but whether circRNAs participate in E. coli-induced bacterial meningitis is unclear. We used high-throughput sequencing to analyze the transcriptional profiles of host circRNAs in primary brain microvascular endothelial cells in response to meningitic E. coli. A total of 308 circRNAs were significantly altered, including 140 upregulated and 168 downregulated ones (p<0.05). KEGG and Gene Ontology enrichment of the parental genes of these altered circRNAs indicated that they are likely to be involved in diverse biological processes via influencing the expression of their parental genes. Coupled with our previous mRNA and microRNA sequencing data, a competing endogenous RNA analysis was performed and the potential regulatory network was preliminary constructed and validated. By revealing the transcriptional profiles of the host circRNAs involved in E. coli meningitis, it is envisaged that the novel insight gained into the regulatory mechanisms of circRNAs in the development of bacterial meningitis will lead to better understanding of how to prevent and treat bacterial CNS infections.
Hsa-miR-202-3p regulates Sertoli cell proliferation, synthesis function and apoptosis by targeting LRP6 and Cyclin D1 of Wnt/β-catenin signaling pathway Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-25 Chao Yang, Chencheng Yao, Ruhui Tian, Zijue Zhu, Liangyu Zhao, Peng Li, Huixing Chen, Yuhua Huang, Erlei Zhi, Yuehua Gong, Yunjing Xue, Hong Wang, Qingqing Yuan, Zuping He, Zheng Li
MicroRNAs (miRNAs) play important roles in mammalian spermatogenesis which is highly dependent on Sertoli cells. However, the functions and mechanisms of miRNAs in regulating human Sertoli cells remain largely unknown. Here, we report that hsa-miR-202-3p mediates the proliferation, apoptosis and synthesis function of human Sertoli cells. MiR-202-3p was up-regulated in Sertoli cells of Sertoli-cell-only syndrome (SCOS) patients compared with obstructive azoospermia (OA) patients with normal spermatogenesis. Overexpression of miR-202-3p induced Sertoli cell apoptosis and inhibited cell proliferation and synthesis, and the effects were opposite when miR-202-3p was knockdown. LRP6 and Cyclin D1 of Wnt/β-catenin signaling pathway were identified as direct targets of miR-202-3p in Sertoli cells, which were validated by bioinformatics tools and dual luciferase reporter assay. Differentially expressed LRP6 and Cyclin D1 between OA and SCOS Sertoli cells were also verified. LRP6 siRNA inference not only mimiced the effects of miR-202-3p overexpression, but also antagonized the effects of miR-202-3p inhibition on Sertoli cells. Collectively, miR-202-3p control the proliferation, apoptosis and synthesis function of human Sertoli cells via targeting LRP6 and Cyclin D1 of Wnt/β-catenin signaling pathway. This study thus provides a novel insight into fate determinations of human Sertoli cells and niche of human testis.
Characterization of transcriptome transition associates long noncoding RNAs with glioma progression Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-24 Xiaoyu Lin, Tiantongfei Jiang, Jing Bai, Junyi Li, Tianshi Wang, Jun Xiao, Yi Tian, Xiyun Jin, Tingting Shao, Juan Xu, Lingchao Chen, Lihua Wang, Yongsheng Li
Poly-Target Selection Identifies Broad-Spectrum RNA Aptamers Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-24 Khalid K. Alam, Jonathan L. Chang, Margaret J. Lange, Phuong D.M. Nguyen, Andrew W. Sawyer, Donald H. Burke
Membrane Destabilization Induced by Lipid Species Increases Activity of Phosphorothioate-Antisense Oligonucleotides Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-24 Shiyu Wang, Nickolas Allen, Xue-hai Liang, Stanley T. Crooke
Chemically modified antisense oligonucleotides with phosphorothioate linkages (PS-ASOs) mediate site-specific cleavage of RNA by RNase H1 and are broadly used as research and therapeutic tools. PS-ASOs can enter cells via endocytic pathways and escape from membrane-enclosed endocytic organelles to reach target RNAs. We recently found that lysobisphosphatidic acid is required for release of PS-ASOs from late endosomes. Here we evaluated the effects of other lipids on PS-ASO intracellular trafficking and activities. We show that free fatty acids, ceramide, and cholesterol increase PS-ASO activities. Free fatty acids induced formation of lipid droplets without changing the intracellular localization of PS-ASOs in early or late endosomes. Ceramide and cholesterol did not obviously induce the formation of lipid droplets, but cholesterol caused enlargement of endosome size and volume. Although none of those lipids appeared to influence PS-ASO internalization or intracellular trafficking processes, all led to an increase in leakiness of late endosomes. Thus, the membrane destabilization induced by these lipids likely contributes to PS-ASO release from late endosomes, which, in turn, increases PS-ASO activity.
Circgprc5a promoted bladder oncogenesis and metastasis through Gprc5a-targeting peptide Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-18 Chaohui Gu, Naichun Zhou, Zhiyu Wang, Guanru Li, Yipping Kou, Shunli Yu, Yongjie Feng, Long Chen, Jinjian Yang, Fengyan Tian
Bladder cancer is a serious cancer in the world, especially in advanced countries. Bladder cancer stem cells (CSCs) drive bladder tumorigenesis and metastasis. Circular RNAs (CircRNAs) are involved in many biological processes, but their roles in bladder oncogenesis and bladder CSCs are unclear. Here we identified circGprc5a is up-regulated in bladder tumors and CSCs. circGpr5a knockdown impairs the self-renewal and metastasis of bladder CSCs, and its overexpression exerts an opposite role. circGpr5a has peptide-coding potential and functions through peptide-dependent manner. circGprc5a-peptide binds to Gprc5a, a surface protein highly expressed in bladder CSCs. Gprc5a knockout inhibits the bladder CSC self-renewal and metastasis. circGprc5a-peptide-Gprc5a can be utilized to target bladder cancer and bladder CSCs.
Role of long noncoding RNA 799 in the metastasis of cervical cancer through upregulation of TBL1XR1 expression Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-17 Ling-Min Liao, Feng-Hao Zhang, Gong-Ji Yao, Su-Feng Ai, Min Zheng, Long Huang
Long non-coding RNAs (lncRNAs) are closely associated with the molecular mechanisms underlying cancer development, and it would be highly useful to study their expression and mechanisms in cervical cancer too. The current study investigated lncRNA799 expression in cervical cancer in order to determine its clinical importance in the progression of cervical cancer. lncRNA799 expression was studied in 218 cervical cancer samples. Expression of lncRNA799 was significantly higher in the cervical cancer tissue than in the adjacent normal tissue. Overexpression of lncRNA799 was found to have a significant correlation with FIGO stage, SCC-Ag level and lymphatic metastasis, and it was also associated with poor survival. Ectopic expression of lncRNA799 promoted the metastasis of SiHa cells, while lncRNA799 knockdown had an inhibitory effect on metastasis. Western blot analysis demonstrated that lncRNA799 promotes the expression of transducing β-like protein 1-related protein (TBL1XR1), and that lncRNA799 and TBL1XR1 expression show strong correlation in cervical cancer. Moreover, lncRNA799 modulated the expression of TBL1XR1 by acting as a competitive endogenous RNA (ceRNA) for miR-454-3P. The results indicate that lncRNA799 could be used as a novel marker of cervical cancer prognosis. Thus, targeting the ceRNA network involving lncRNA799 could be a potential treatment strategy against cervical cancer.
Epistatic Association of CD14 and NOTCH2 Genetic Polymorphisms with Biliary Atresia in a Southern Chinese Population Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-16 Zefeng Lin, Xiaoli Xie, Huiting Lin, Ming Fu, Liang Su, Yanlu Tong, Hongjiao Chen, Hezhen Wang, Jinglu Zhao, Huimin Xia, Yan Zhang, Ruizhong Zhang
Biliary atresia (BA) is the most common cause of end-stage liver disease in infants with poor prognosis and high mortality. The etiology of BA is still unknown, but the genetic factors have been considered as an important player in BA. We investigated the association of two cis- regulated variants in CD14 (rs2569190) and NOTCH2 (rs835576) with BA susceptibility using the largest case-control cohort, totaling with 506 BA patients and 1473 healthy controls in a Southern Chinese population. Significant epistatic interaction between the two variants in our samples was observed (P= 8.1E-03, OR= 2.78, 95% CI: 1.32∼5.88). The expression of CD14 and NOTCH2 in BA group was consistently lower than that in control (CC) group (0.31±0.02 vs. 1.00±0.14, P< 0.001), which might be related to the genetic susceptibility of the genes awaiting for further validation.
Novel human miRNA-disease associations inference based on random forest Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-11 Xing Chen, Chun-Chun Wang, Jun Yin, Zhu-Hong You
Identification of novel antisense-mediated exon skipping targets in DYSF for therapeutic treatment of dysferlinopathy Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-11 Joshua J.A. Lee, Rika Maruyama, William Duddy, Hidetoshi Sakurai, Toshifumi Yokota
Dysferlinopathy is a progressive myopathy caused by mutations in the dysferlin (DYSF) gene. Dysferlin protein plays a major role in plasma membrane resealing. Some patients with DYSF deletion mutations exhibit mild symptoms, suggesting some regions of DYSF can be removed without significantly impacting protein function. Antisense-mediated exon skipping therapy uses synthetic molecules called antisense oligonucleotides to modulate splicing, allowing exons harboring or near genetic mutations to be removed and the open reading frame corrected. Previous studies have focused on DYSF exon 32 skipping as a potential therapeutic approach, based on the association of a mild phenotype with the in-frame deletion of exon 32. To date, no other DYSF exon skipping targets have been identified and the relationship between DYSF exon deletion pattern and protein function remains largely uncharacterized. In this study, we utilized a membrane wounding assay to evaluate the ability of plasmid constructs carrying mutant DYSF, as well as antisense oligonucleotides, to rescue membrane resealing in patient cells. We report that multi-exon skipping of DYSF exons 26-27 and 28-29 rescues plasma membrane resealing. Successful translation of these findings into the development of clinical antisense drugs would establish new therapeutic approaches that would be applicable to ∼5-7% (exons 26-27 skipping) and ∼8% (exons 28-29 skipping) of dysferlinopathy patients worldwide.
Long non-coding RNA PVT1 promotes cell proliferation and migration by silencing ANGPTL4 expression in cholangiocarcinoma Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-10 Yang Yu, Mingjiong Zhang, Jie Liu, Boming Xu, Jian Yang, Ni Wang, Shuai Yan, Fei Wang, Xuezhi He, Guozhong Ji, Quanpeng Li, Lin Miao
Cholangiocarcinoma (CCA) is the most common biliary tract malignancy, with a low survival rate and limited treatment options1fn1. Long non-coding RNAs (lncRNAs) have recently been verified to have significant regulatory functions in many kinds of human cancers. It was discovered in this study that the lncRNA PVT1, whose expression is significantly elevated in cholangiocarcinoma, could be a molecular marker of cholangiocarcinoma. Experiments indicated that PVT1 knockdown greatly inhibited cell migration and proliferation in vitro and in vivo. According to RNA-seq analysis, PVT1 knockdown dramatically influenced target genes associated with cell angiogenesis, cell proliferation and the apoptotic process. RIP analysis demonstrated that by binding to epigenetic modification complexes (PRC2), PVT1 could adjust the histone methylation of the promoter of ANGPTL4 (angiopoietin-like 4) and thus promote cell growth, migration and apoptosis progression. The data verified the significant functions of PVT1 in cholangiocarcinoma oncogenesis and suggested that PVT1 could be a target for cholangiocarcinoma (CCA) intervention.
Shorter Phosphorodiamidate Morpholino Splice-Switching Oligonucleotides may increase Exon Skipping Efficacy in Duchenne Muscular Dystrophy Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-10 Ugur Akpulat, Haicui Wang, Kerstin Becker, Adriana Contreras, Terence Partridge, James Novak, Sebahattin Cirak
Angpt2 induces mesangial cell apoptosis through microRNA-33-5p- SOCS5 loop in diabetic nephropathy Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-10 Yi-Chun Tsai, Po-Lin Kuo, Wei-Wen Hung, Ling-Yu Wu, Ping-Hsun Wu, Wei-An Chang, Mei-Chuan Kuo, Ya-Ling Hsu
Diabetic nephropathy (DN) is the leading cause of end stage renal disease. Mesangial cells (MCs) loss are correlated with worsening renal function in DN. Disturbance of angiopoietin (Angpt)/Tie ligand-receptor system causes inflammation and abnormal angiogenesis. This association between elevated circulating Angpt2 and poor renal outcome has been in DN patients. However, the pathogenic role of Angpt2 in the MCs remains unknown. We found serum Angpt2 levels were elevated in type 2 diabetes mellitus (DM) patients and db/db mice, which correlated with albuminuria. Angpt2 synergistically induced MC apoptosis under HG, and miR-33-5p regulated Angpt2-inducing MCs apoptosis treated with HG. Loss of miR-33-5p increased suppressor of cytokine signaling 5 (SOCS5), leading to the inhibition of Janus kinase1 and signal transducer and activator of transcription 3 signaling transduction. Elevated expression of SOCS5 was found in MCs in kidney sections of both db/db mice and type 2 DM patients. Decreased miR-33-5p levels were found in the urine of db/db mice and type 2 DM patients, and miR-33-55p levels negatively correlated with albuminuria. Angpt2 leads to MC apoptosis via miR-33-5p/SOCS5 loop in DN. miR-33-5p is predictive of kidney injury in DN. These findings may provide future applications in predicting renal dysfunction and therapeutic potential of DN.
Cationic oligospermine-oligonucleotide conjugates provide carrier-free splice switching in monolayer cells and in spheroids Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-10 Marc Nothisen, Phanélie Perche-Létuvée, Jean-Paul Behr, Jean-Serge Remy, Mitsuharu Kotera
We report the evaluation of 18-mer 2’-O-methyl-modified ribose oligonucleotides with a full-length phosphorothioate backbone chemically conjugated at the 5’-end to the oligospermine units [Sn-: n = 5, 15, 20, 25, 30 (number of spermine units)] as splice switching oligonucleotides (SSOs). These conjugates contain, in their structure, covalently linked oligocation moieties, making them capable of penetrating cells without transfection vector. In cell culture, we observed efficient cytoplasmic and nuclear delivery of fluorescein labelled S20-SSO by fluorescent microscopy. The SSO conjugates containing more than 15 spermine units induced significant carrier-free exon-skipping at nanomolar concentration in the absence and in the presence of serum. With an increasing number of spermine units, the conjugates became slightly toxic but more active. Advantages of these molecules were particularly demonstrated in 3D cell culture (MCTS: multicellular tumor spheroids) that mimics living tissues. While vector-complexed SSOs displayed a drastically reduced splice switching in MCTS compared to the assay in monolayer culture, an efficient exon-skipping without significant toxicity was observed with oligospermine grafted SSOs (S15- and S20-SSOs) transfected without vector. It was shown, by flow cytometry and confocal microscopy, that the fluorescein-labelled S20-SSO was freely diffusing and penetrating the innermost cells of MCTS while the vector-complexed SSO penetrated only the cells of the spheroid's outer layer.
LncRNA GAS5 reverses EMT and tumor stem cell-mediated gemcitabine resistance and metastasis by targeting miR-221/SOCS3 in pancreatic cancer Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-06 Bingyan Liu, Shaoqiu Wu, Jun Ma, Shuo Yan, Zhengguang Xiao, Linhuang Wan, Feng Zhang, Mingyi Shang, Aiwu Mao
Dysregulated long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) mediating chemotherapeutic drug effects and metastasis in pancreatic cancer (PC) are key reasons for the poor prognosis of this disease. LncRNA growth arrest-specific 5 (GAS5) is reported to be a tumor suppressor in multiple cancers. However, the functions of GAS5 and its related miRNAs in PC are poorly understood. This study explored the potential functions and mechanisms of GAS5 in PC gemcitabine resistance and metastasis. The results show that overexpression of GAS5 suppressed the proliferation, migration, gemcitabine resistance, stem cell-like properties, and epithelial-mesenchymal transition (EMT) of PC cells by directly binding to and suppressing miR-221 expression and enhancing SOCS3 expression. The effect of miR-221 overexpression on proliferation, migration, gemcitabine resistance, stem cell-like properties and EMT inhibition was reversed by SOCS3 overexpression in PC cells. Additionally, GAS5 promoted gemcitabine-induced tumor growth and metastasis inhibition as determined by Ki-67 staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), bioluminescence imaging, and detection of cell-like properties and EMT in vivo. Thus, lncRNA GAS5 functioned as a competing endogenous RNA for miR-221 which suppressed cell growth, metastasis, and gemcitabine resistance in PC by regulating the miR-221/SOCS3 pathway mediating EMT and tumor stem cell self-renewal.
A simplified system to express circularized inhibitors of microRNA (miRNA) (CimiR) for stable and potent suppression of miRNA functions Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-04 Yi Shu, Ke Wu, Zongyue Zeng, Shifeng Huang, Xiaojuan Ji, Chengfu Yuan, Linghuan Zhang, Wei Liu, Bo Huang, Yixiao Feng, Bo Zhang, Zhengyu Dai, Yi Shen, Wenping Luo, Xi Wang, Bo Liu, Yan Lei, Zhenyu Ye, Tong-Chuan He
MicroRNAs (miRNAs) are an evolutionarily-conserved class of small regulatory noncoding RNAs by binding to complementary target mRNAs and resulting in mRNA translational inhibition or degradation, and thus play an important role in regulating many aspects of physiologic and pathologic processes in mammalian cells. Thus, efficient manipulations of miRNA functions may be exploited as promising therapeutics for human diseases. Two commonly-used strategies to inhibit miRNA functions include direct transfection of chemically synthesized miRNA inhibitors and delivery of a gene vector that instructs intracellular transcription of miRNA inhibitors. While most miRNA inhibitors are based on antisense molecules to bind and sequester miRNAs from their natural targets, it is challenging to achieve effective and stable miRNA inhibition. Here, we develop a user-friendly system to express circular inhibitors of miRNA (or CimiR) by exploiting the noncanonical head-to-tail backsplicing mechanism for generating endogenous circular RNA sponges. In our proof-of-the-principle experiments, we demonstrate that the circular forms of hsa-miR223 binding site of ARRB1 3’-UTR sponge RNA (BUTR), the bulged antimiR223 (cirBulg223) and bulged antimiR21 (cirBulg21) exhibit more potent suppression of miRNA functions than their linear counterparts. Therefore, the engineered CimiR expression system should be a valuable tool to target miRNAs for basic and translational research.
Integrating transcriptome and experiments reveal the anti-diabetic mechanism of Cyclocarya paliurus formula Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-04 Jing Li, Qiong Zhang, Weiwei Zeng, Yuxin Wu, Mei Luo, Yanhong Zhu, An-Yuan Guo, Xiangliang Yang
Type 2 diabetes (T2D) is generally regarded as a metabolic disorder disease with various phenotypic expressions. Traditional Chinese medicine (TCM) has been widely used for preventing and treating diabetes. In our study, we demonstrated Cyclocarya paliurus formula extractum (CPE), a compound of TCM, can ameliorate diabetes in diabetic rats. Transcriptome profiles were performed to elucidate the anti-diabetic mechanisms of CPE on pancreas and liver. Pancreatic transcriptome analysis showed CPE treatment significantly inhibited gene expressions related to inflammation and apoptosis pathways, among which transcription factors (TFs) NF-κB, STAT and miR-9a/148/200 may serve as core regulators contributing to ameliorate diabetes. Biochemical studies also demonstrated CPE treatment decreased pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and reduced β cell apoptosis. In liver tissue, our transcriptome and biochemical experiments showed CPE treatment reduced lipid accumulation and liver injury, and promoted glycogen synthesis, which may be regulated by TFs Srebf1, Mlxipl and miR-122/128/192. Taken together, our findings revealed CPE could be used as a potential therapeutic agent to prevent and treat diabetes. It is the first time to combine transcriptome and regulatory network analysis to study the mechanism of CPE in preventing diabetes, which shows a demo for exploring the mechanism of TCM on complex diseases.
Circular RNAs as Potential Theranostics in the Cardiovascular System Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-02 Yihua Bei, Tingting Yang, Lijun Wang, Paul Holvoet, Saumya Das, Joost PG. Sluijter, Marta Chagas Monteiro, Yang Liu, Qiulian Zhou, Junjie Xiao
Cardiovascular diseases (CVDs) represent the largest contributor to mortality worldwide. Identification of novel therapeutic targets and biomarkers for CVDs are urgently needed. Circular RNAs (circRNAs) are endogenous, abundant, and stable non-coding RNAs formed by back-splicing events. Their role as regulator of gene expression has been increasingly reported. Notably, circRNAs mediate essential physiological and pathological processes in the cardiovascular system. Our first aim, therefore, is to summarize recent advances in the role of circRNAs in cardiac development as well as in pathogenesis of various CVDs. Because circRNAs are stable in the circulation and their dynamic changes may reflect different disease stages, they are considered as ideal biomarkers. Therefore, our second aim is to review studies that have identified circulating circRNAs as biomarkers for CVDs. Finally, we discuss the shortage of functional studies and the limitations of the available clinical studies and provide future perspectives.
Targeting Long Noncoding RNA in Glioma: A Pathway Perspective Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-02 Junyang Li, Yihao Zhu, Handong Wang, Xiangjun Ji
Long noncoding RNAs (lncRNAs) participate extensively in biological processes of various cancers. The majority of these transcripts are uniquely expressed in differentiated tissues or specific cancer types. LncRNAs are aberrantly expressed in gliomas and exert diverse functions. In this article, we provided an overview of how lncRNAs regulate cellular processes in glioma, enumerated the lncRNAs that may act as glioma biomarkers and showed their potential clinical implications.
miR-125a promotes the progression of giant cell tumors of bone by stimulating IL-17A and β-Catenin expression Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-02 Hua Jin, Dian-Wei Li, Shu-Nan Wang, Song Luo, Qing Li, Ping Huang, Jian-Min Wang, Meng Xu, Cheng-Xiong Xu
Giant cell tumors of bone (GCTB) exhibit high recurrence and aggressive bone lytic behavior. But, the mechanism of GCTB progression is largely unknown. In GCTB, we detected abundant levels of miR-125a, which were associated with tumor extension, grade and recurrence. miR-125a stimulate stromal cell tumorigenicity and growth in vivo by promotes the expression of IL-17A and β-catenin. In contrast, inhibition of miR-125a suppressed stromal cell tumorigenicity and growth. Then, we found that miR-125a stimulates IL-17A by targeting TET2 and Foxp3, and stimulates β-catenin expression by targeting APC and GSK3β in stromal cells. Furthermore, we identified that IL-17A stimulates miR-125a by activating NF-κB signaling in stromal cells. Finally, our data show that simultaneously inhibition of IL-17A signaling and miR-125a more significantly inhibits stromal cell growth than miR-125a inhibition alone. miR-125a stimulates the progression of GCTB and might represent a useful candidate marker for progression. Simultaneously blocking miR-125a and IL-17A might represent a new therapeutic strategy for GCTB.
The Bipartite Network Projection Recommended Algorithm for predicting long non-coding RNA–protein interactions Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-29 Qi Zhao, Haifan Yu, Zhong Ming, Huan Hu, Guofei Ren, Hongsheng Liu
With the development of science and biotechnology, many evidences show that ncRNAs play an important role in the development of important biological processes, especially in the chromatin modification, cell differentiation and proliferation, RNA progressing, human diseases, etc. Moreover, lncRNAs account for the majority of ncRNAs, and the functions of lncRNAs are expressed by the related RNA-binding proteins. It is well known that the experimental verification of lncRNA-protein relationships is a waste of time and expensive. So many time-saving and inexpensive computational methods are proposed to uncover potential lncRNA-protein interactions. In this work, we propose a novel computational method to predict the potential lncRNA-protein interactions with the bipartite network projection recommended algorithm (LPI-BNPRA). Our approach is a semi-supervised method based on lncRNA similarity matrix, protein similarity matrix and lncRNA-protein interaction matrix. Compared with three previous methods under the leave-one-out cross validation, our model has a more high-confidence result with the AUC value of 0.8754 and the AUPR value of 0.6283. We also do case studies by the “Mus musculus” dataset to further reflect the reliability of our approach. This suggests that LPI-BNPRA will be a reliable computational method to uncover lncRNA-protein interactions in biomedical research.
Astrocyte EV-induced lincRNA-Cox2 regulates microglial phagocytosis: Implications for morphine-mediated potentiation of neurodegeneration Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-29 Guoku Hu, Ke Liao, Fang Niu, Lu Yang, Blake W. Dallon, Shannon Callen, Changhai Tian, Jiang Shu, Juan Cui, Zhiqiang Sun, Yuri L. Lyubchenko, Minhan Ka, Xian-Ming Chen, Shilpa Buch
Impairment of microglial functions such as phagocytosis and/or dysregulation of immune responses have been implicated as underlying factors involved in the pathogenesis of various neurodegenerative disorders. Our previous studies have demonstrated that lincRNA-Cox2 expression is influenced by NF-κB signaling and serves as a coactivator of transcriptional factors to regulate the expression of a vast array of immune related genes in microglia. Extracellular vesicles (EVs) have been recognized as primary facilitators of cell-to-cell communication and cellular regulation. Herein, we show that EVs derived from astrocytes exposed to morphine can be taken up by microglial endosomes, leading, in turn, to activation of TLR7 with a subsequent upregulation of lincRNA-Cox2 expression, ultimately resulting in impaired microglial phagocytosis. This was further validated in vivo, wherein inhibition of microglial phagocytic activity was also observed in brain slices isolated from morphine administrated mice compared with control mice. Additionally, we also showed that intranasal delivery of EVs containing lincRNA-Cox2-siRNA was able to restore microglial phagocytic activity in mice administered morphine. These findings have ramifications for the development of EV-loaded RNA based therapeutics for the treatment of various disorders involving functional impairment of microglia.
Genetic Strategies for HIV Treatment and Prevention Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-29 Alexander Falkenhagen, Sadhna Joshi
Conventional HIV gene therapy approaches are based on engineering HIV target cells that are non-permissive to viral replication. However, expansion of gene-modified HIV target cells has been limited in patients. Alternative genetic strategies focus on generating gene-modified producer cells that secrete antiviral proteins (AVPs). The secreted AVPs interfere with HIV entry and therefore extend the protection against infection to unmodified HIV target cells. Since any cell type can potentially secrete AVPs, hematopoietic and non-hematopoietic cell lineages can function as producer cells. Secretion of AVPs from non-hematopoietic cells opens the possibility of using a genetic approach for HIV prevention. Another strategy aims at modifying cytotoxic T cells to selectively target and eliminate infected cells. This review provides an overview of the different genetic approaches for HIV treatment and prevention.
NS-065/NCNP-01: an Antisense Oligonucleotide for Potential Treatment of Exon 53 Skipping in Duchenne Muscular Dystrophy Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-27 Naoki Watanabe, Tetsuya Nagata, Youhei Satou, Satoru Masuda, Takashi Saito, Hidetoshi Kitagawa, Hirofumi Komaki, Kazuchika Takagaki, Shinichi Takeda
Duchenne muscular dystrophy (DMD), the most common lethal heritable childhood disease, is caused by mutations in the DMD gene that result in the absence of functional dystrophin protein. Exon skipping mediated by antisense oligonucleotides has recently emerged as an effective approach for the restoration of dystrophin, and skipping of exon 51 of DMD has received accelerated approval. Identifying antisense sequences that can provide the highest possible skipping efficiency is crucial for future clinical applications. Herein, we systematically tested two-step antisense oligonucleotide walks along human DMD exon 53 in order to define sequence-dependent effects of antisense oligonucleotide binding sites in human rhabdomyosarcoma cell lines. The first rough whole exon 53 walk enabled the identification of a target region, and a second walk of this region was used to determine an optimal antisense oligonucleotide sequence (NS-065/NCNP-01) for exon 53 skipping. This oligonucleotide strongly promoted exon 53 skipping in a dose-dependent manner during pre-mRNA splicing in rhabdomyosarcoma and DMD patient-derived cells and restored dystrophin protein levels in patient-derived cells. NS-065/NCNP-01, a phosphorodiamidate morpholino oligomer, appears to be a promising candidate for treating exon 53 skipping and is potentially applicable to 8% of patients with DMD.
Inhibition of miR-449a Promotes Cartilage Regeneration and Prevents Progression of Osteoarthritis in in vivo Rat Models Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-27 Dawoon Baek, Kyoung-Mi Lee, Ki Won Park, Jae Wan Suh, Seong Mi Choi, Kwang Hwan Park, Jin Woo Lee, Sung-Hwan Kim
Expression, Clinical Significance and Functional Prediction of MNX1 in Breast Cancer Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-27 Tian Tian, Meng Wang, Yuyao Zhu, Wenge Zhu, Tielin Yang, Hongtao Li, Shuai Lin, Cong Dai, Yujiao Deng, Dingli Song, Na Li, Zhen Zhai, Zhijun Dai
Motor neuron and pancreas homeobox 1 (MNX1) is a key developmental gene. Previous studies found it was up-regulated in several tumors, but its role in breast cancer (BC) remains unclear. In order to have a better understanding of this gene in BC, we examined the expression of MNX1 in BC tissues and normal breast tissues by qRT-RCR and by analyzing data from The Cancer Genome Atlas (TCGA) database. We also assessed the association of MNX1 expression with BC clinicopathologal features and investigated the impact of MNX1 on BC survival. Potential molecular function of MNX1 was predicted through protein–protein interactions and functional enrichment. The results showed that the expression of MNX1 was significantly increased in BC tissues, especially in the HER2-positive subtype. And MNX1 expression was associated with several clinical characteristics including menopause status, receptor status, subtypes, tumor size, lymph node metastasis and race. In addition, patients with higher MNX1 expression had poorer survival. Enrichment analysis suggested MNX1 is probably involved in biological processes and pathways related to nuclear division, cell cycle and p53 signaling. In conclusion, our study suggests that MNX1 may act as a tumor promoter in BC. We hope these findings will draw more attention to MNX1 in future cancer studies.
Aptamer-miR-34c conjugate affects cell proliferation of non-small cell lung cancer cells Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-27 Valentina Russo, Alessia Paciocco, Alessandra Affinito, Giuseppina Roscigno, Danilo Fiore, Francesco Palma, Marco Galasso, Stefano Volinia, Alfonso Fiorelli, Carla Lucia Esposito, Silvia Nuzzo, Giorgio Inghirami, Vittorio de Franciscis, Gerolama Condorelli
MicroRNAs (miRNAs) are key regulators of different human processes that represent a new promising class of cancer therapeutics or therapeutic targets. Indeed, in several tumor types, including non-small-cell lung carcinoma (NSCLC), the deregulated expression of specific miRNAs has been implicated in cell malignancy. As expression levels of the oncosuppressor miR-34c-3p are decreased in NSCLC compared to normal lung, we show that reintroduction of miR-34c-3p reduces NSCLC cell survival in vitro. Further, in order to deliver the miR-34c-based therapeutic selectively to tumor cells, we took advantage of a reported nucleic acid aptamer (GL21.T) that binds and inhibits the AXL transmembrane receptor and is rapidly internalized in the target cells. By applying methods successfully used in our laboratory, we conjugated miR-34c to the GL21.T aptamer as targeting moiety for the selective delivery to AXL-expressing NSCLC cells. We demonstrate that miR-34c-3p and the GL21.T/miR-34c chimera affect NSCLC cell proliferation and are able to overcome acquired RTK-inhibitor resistance by targeting AXL receptor. Thus, the GL21.T/miR-34c chimera exerts dual inhibition of AXL, at functional and transcriptional levels, and represent a novel therapeutic tool for the treatment of NSCLC.
Phosphorothioate Modified AP613-1 Used for Hepatocellular Carcinoma Cell Imaging Via Specifically Targeting GPC3 Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-27 Lili Dong, Hongxin Zhou, Menglong Zhao, Xinghui Gao, Yang Liu, Dongli Liu, Wei Guo, Hongwei Hu, Qian Xie, Jia Fan, Jiang Lin, Weizhong Wu
Glypican-3 (GPC3), the cellular membrane proteoglycan, has been established as a tumor biomarker for early diagnosis of hepatocellular carcinoma (HCC). GPC3 is highly expressed in more than 70% HCC tissues detected by antibody based histopathological systems. Recently, aptamers, a short single-strand DNA or RNA generated from systematic evolution of ligands by exponential enrichment (SELEX), were reported as potential alternatives in tumor targeted imaging and diagnosis. In this study, a total of 19 GPC3-bound aptamers were successfully screened by capillary electrophoresis (CE)-SELEX technology. After truncated, AP613-1 was confirmed to specifically target GPC3 with Kd of 59.85 nM. When modified with phosphorothioate linkage, APS613-1 targeted GPC3 with Kd of 15.48 nM and could be used as a specific probe in living Huh7 and PLC/PRF/5 imaging, GPC3-positive cell lines, but not in L02 or A549, two GPC3-negative cell lines. More important, Alexa Fluor 750-conjugated APS613-1 could be used as a fluorescent probe to subcutaneous HCC imaging in xenograft nude mice. Our results indicated that modified AP613-1, especially APS613-1, was a potential agent in GPC3-positive tumor imaging for HCC early diagnosis.
Anti-cancer drug response prediction using neighbor-based collaborative filtering with global effect removal Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-22 Hui Liu, Yan Zhao, Lin Zhang, Xing Chen
Synthetic circular RNA functions as a miR-21 sponge to suppress gastric carcinoma cell proliferation Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-22 Xi Liu, John M. Abraham, Yulan Cheng, Zhixiong Wang, Zhe Wang, Guanjun Zhang, Hassan Ashktorab, Duane T. Smoot, Robert N. Cole, Tatiana N. Boronina, Lauren DeVine, Conover Talbot, Zhengwen Liu, Stephen J. Meltzer
MicroRNA (miR) sponges containing miR binding sequences constitute a potentially powerful molecular therapeutic strategy. Recently, naturally-occurring circular RNAs (circRNAs) were shown to function as efficient miR sponges in cancer cells. We hypothesized that synthetic circRNA sponges could achieve therapeutic loss of function targeted against specific miRs. Linear RNA molecules containing miR-21 binding sites were transcribed in vitro; after dephosphorylation and phosphorylation, circularization was achieved using 5’-3’ end-ligation by T4 RNA ligase 1. CircRNA stability was assessed using RNase R and fetal bovine serum. Competitive inhibition of miR-21 activity by a synthetic circRNA sponge was assessed using luciferase reporter, cell proliferation and cell apoptosis assays in 3 gastric cancer cell lines. CircRNA effects on downstream proteins were also delineated by Tandem Mass Tag (TMT) labeling (data available via ProteomeXchange, identifier PXD008584), followed by Western blotting. We conclude that artificial circRNA sponges resistant to nuclease digestion can be synthesized using simple enzymatic ligation steps. These sponges inhibit cancer cell proliferation and suppress the activity of miR-21 on downstream protein targets, including the cancer protein DAXX. In summary, synthetic circRNA sponges represent a simple, effective, convenient strategy for achieving targeted loss of miR function in vitro, with potential future therapeutic application in human patients.
Adeno-associated virus-mediated RNA interference against mutant allele attenuates abnormal calvarial phenotypes in an Apert syndrome mouse model Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-22 Fengtao Luo, Yangli Xie, Zuqiang Wang, Junlan Huang, Qiaoyan Tan, Xianding Sun, Fangfang Li, Can Li, Mi Liu, Dali Zhang, Meng Xu, Nan Su, Zhenhong Ni, Wanling Jiang, Jinhong Chang, Hangang Chen, Shuai Chen, Xiaoling Xu, Lin Chen
Apert syndrome (AS), the most severe form of craniosynostoses, is caused by missense mutations including Pro253Arg(P253R) of fibroblast growth factor receptor 2 (FGFR2), which leads to enhanced FGF/FGFR2 signaling activity. Typical treatment for AS is surgical correction of the deformed skulls. Because of constant maldevelopment of sutures, the corrective surgery is often executed for several times, resulting in increased suffering of patients and complications. Biological therapies targeting the signaling of mutant FGFR2 allele, in combination with surgery, may bring better outcome. Here we screened and found a siRNA specifically targeting to Fgfr2-P253R allele and revealed that it inhibited osteoblastic differentiation and matrix mineralization by reducing the signaling of ERK1/2 and P38 in cultured primary calvarial cells and calvarial explants from Apert mice (Fgfr2+/P253R). Furthermore, AAV9 carrying shRNA (AAV9-Fgfr2-shRNA) against mutant Fgfr2 was delivered to skulls of AS mice. Results demonstrate that AAV9-Fgfr2-shRNA attenuated the premature closure of coronal suture and the decreased calvarial bone volume of AS mice. Our study provides a novel practical biological approach, which will, in combination with other therapies including surgeries, help treatment of patients with AS, while provide experimental clues for the biological therapies of other genetic skeletal diseases.
Enhanced tailored microRNA sponge activity of RNA polymerase II-transcribed Tough Decoy hairpins relative to ectopically expressed ciRS7-derived circular RNAs Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-21 Anne Kruse Hollensen, Sofie Andersen, Karina Hjorth, Rasmus O. Bak, Thomas B. Hansen, Jørgen Kjems, Lars Aagaard, Christian Kroun Damgaard, Jacob Giehm Mikkelsen
miR-370-3p is a therapeutic tool in anti-glioblastoma therapy, but is not an intratumoral or cell-free circulating biomarker Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-13 Arulraj Nadaradjane, Joséphine Briand, Gwenola Bougras-Cartron, Valentine Disdero, François M. Vallette, Jean-Sébastien Frenel, Pierre-François Cartron
In the last decade, microRNAs (miR) have been described as biomarkers and therapeutic agents. Based on this finding, our aim here is to know of 1) miRNA-370-3p can be used as a biomarker associated with a favorable survival, and if 2) miRNA-370-3p can be used as a therapeutic tool that increases the efficiency of standard anti-GBM treatment. A first approach using the data available on the “Prognostic miRNA Database”, indicated that the expression level of miRNA-370-3p in GBM (T-miR-370-3p) is not associated with a prognosis value for survival. A second approach quantifying the expression level of cell-free circulating miRNA-370-3p (cfc-miR-370-3p) also indicated that cfc-miR-370-3p is not associated with a prognosis value for survival. To investigate whether miR-370-3p can be used in vivo to increase the anti-GBM effect of TMZ, we then used the model of LN18-induced GBMs in mice. Our data indicated that the miRNA-370-3p/TMZ treatment was 2 times more efficient than the TMZ treatment for decreasing the tumor volume. In addition, our study correlated the decrease of tumor volume induced by the miRNA-370-3p/TMZ treatment with the decrease in FOXM1 and MGMT (i.e. two targets of miR-370-3p). Our data thus support the idea that miR-370-3p could be used as therapeutic tool for anti-glioblastoma therapy, but not as a biomarker.
Potential role of miRNAs as theranostic biomarkers of epilepsy Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-13 C. Cava, I. Manna, A. Gambardella, G. Bertoli, I. Castiglioni
Improving angiogenic potential of EPCs via engineering with synthetic modified mRNAs Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-13 Heidrun Steinle, Sonia Golombek, Andreas Behring, Christian Schlensak, Hans Peter Wendel, Meltem Avci-Adali
Antisense oligonucleotides against miR-21 inhibit the growth and metastasis of colorectal carcinoma via DUSP8 pathway Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-13 Tao Ding, Panpan Cui, Ya Zhou, Chao Chen, Juanjuan Zhao, Hairong Wang, Mengmeng Guo, Zhixu He, Lin Xu
Accumulating pieces of literature documented that microRNA-21 (miR-21) played an important role in the development of human colorectal carcinoma (CRC). Our recent work also showed that antisense oligonucleotides (ASOs) against miR-21 could impair the growth of CRC cells in vitro. However, the potential role of miR-21 in the gene therapy against CRC remains to be fully elucidated. Here, we further observed the effect of ASOs against miR-21 on the growth and metastasis of CRC in vivo using a xenograft model of human CRC. We found that ASOs could effectively inhibit the growth and metastasis of CRC in vivo, accompanied by downregulated expression of miR-21 and reduced transduction of AKT and ERK pathway. Mechanically, global gene expression analysis showed that the expression of DUSP8, a novel target of miR-21, was upregulated in tumor mass. Furthermore, overexpression of DUSP8 could remarkably suppress the proliferation and migration of CRC cells in vitro. Finally, downregulation of DUSP8 could abrogate the effects of ASOs against miR-21 on the proliferation and migration of CRC cells, as well as altered transduction of the AKT and ERK signaling pathway. Together, these data suggest that ASOs against miRNAs is an attractive and potential therapeutic for the treatment of human CRC and warrants further development.
Transient retrovirus-based CRISPR/Cas9 all-in-one particles for efficient, targeted gene knockout Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-13 Yvonne Knopp, Franziska K. Geis, Dirk Heckl, Stefan Horn, Thomas Neumann, Johannes Kuehle, Janine Meyer, Boris Fehse, Christopher Baum, Michael Morgan, Johann Meyer, Axel Schambach, Melanie Galla
The recently discovered CRISPR/Cas9 system is widely-used in basic research and is a useful tool for disease modeling and gene editing therapies. However, long-term expression of DNA-modifying enzymes can be associated with cytotoxicity and is particularly unwanted in clinical gene editing strategies. Since current transient expression methods may still suffer from cytotoxicity and/or low efficiency, we developed non-integrating retrovirus-based CRISPR/Cas9 all-in-one particles for targeted gene knockout. By redirecting the gammaretroviral packaging machinery, we transiently delivered Streptococcus pyogenes Cas9 (SpCas9) mRNA and single guide RNA transcripts into various (including primary) cell types. Spatiotemporal co-delivery of CRISPR/Cas9 components resulted in efficient disruption of a surrogate reporter gene as well as functional knockout of endogenous human genes CXCR4 and TP53. Remarkably, although acting in a hit-and-run fashion, knockout efficiencies of our transient particles corresponded to 52-80% to those obtained from constitutively active integrating vectors. Noteworthy, stable SpCas9 overexpression at high doses in murine NIH3T3 cells caused a substantial G0/G1 arrest accompanied by reduced cell growth and metabolic activity, which was prevented by transient SpCas9 transfer. In summary, the non-integrating retrovirus-based vector particles introduced here allow efficient and dose-controlled delivery of CRISPR/Cas9 components into target cells.
A Novel Aurora-A Inhibitor (MLN8237) Synergistically Enhances the Antitumour Activity of Sorafenib in Hepatocelluar Carcinoma In Vitro and In Vivo Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-12 Kai Zhang, Ting Wang, Hao Zhou, Bing Feng, Ying Chen, Yingru Zhi, Rui Wang
Currently, sorafenib-based therapy is the standard treatment for advanced hepatocellular carcinoma (HCC), and there is a strong rationale for investigating its use in combination with other agents to achieve better therapeutic effects. Aurora A, a member of a family of mitotic serine/threonine kinases, is frequently overexpressed in human cancers and therefore represents a target for therapy. Here, we investigated a novel Aurora-A inhibitor, MLN8237, together with sorafenib in HCC cells in vitro and in vivo and elucidated the possible molecular mechanism. Here, it was found that MLN8237 was strongly synergistic with sorafenib in inhibition of HCC progression by altering cell growth, cell-cycle regulation, apoptosis, migration, invasion and angiogenesis. Mechanism dissection suggests that the combination of MLN8237 and sorafenib led to significant inhibition of the activation of phospho-Akt (p-Akt) and phospho-p38 mitogen-activated protein kinase (p-p38 MAPK) and their downstream genes including CDK4, cyclinD1 and VEGFA. The activators of p-Akt and p-p38 MAPK signalings partially reversed the synergistic inhibitory effects of sorafenib and MLN8237 on HCC progression. Subsequent in vivo studies further confirmed the synergistic effects of sorafenib and MLN8237. Collectively, the newly developed sorafenib-MLN8237 combination may be a novel therapy to better inhibit HCC progression.
HIV Entry and Its Inhibition by Bifunctional Antiviral Proteins Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-11 Alexander Falkenhagen, Sadhna Joshi
HIV entry is a highly specific and time-sensitive process that can be divided into receptor binding, coreceptor binding, and membrane fusion. Bifunctional antiviral proteins (bAVPs) exploit the multi-step nature of the HIV entry process by binding to two different extracellular targets. They are generated by expressing a fusion protein containing two entry inhibitors with a flexible linker. The resulting fusion proteins exhibit exceptional neutralization potency and broad cross-clade inhibition. In this review, we summarize the HIV entry process and provide an overview of the design, antiviral potency, and methods of delivery of bAVPs. Additionally, we discuss the advantages and limitations of bAVPs for HIV prevention or treatment.
An efficient bivalent cyclic RGD-PIK3CB siRNA conjugate for specific targeted therapy against glioblastoma in vitro and in vivo Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-06 Bohong Cen, Yuanyi Wei, Wen Huang, Muzhou Teng, Shuai He, Jianlong Li, Wei Wang, Guolin He, Xin Bai, Xiaoxia Liu, Yawei Yuan, Xinghua Pan, Aimin Ji
The PI3K-AKT-mTOR signaling pathway is frequently activated in glioblastoma (GBM). Inhibition of PIK3CB/p110β (a PI3K catalytic isoform) by RNA interference (RNAi) substantially suppresses GBM growth with less toxicity to normal astrocytes. However, insufficient and non-specific siRNA delivery may limit the efficacy of RNAi-based therapies against GBM. Here, we prepared a novel methoxy-modified PIK3CB siRNA molecule (siPIK3CB) that was covalently conjugated to a [cyclo(Arg-Gly-Asp-D-Phe-Lys)-Ahx]2-Glu-PEG-MAL (biRGD) peptide which selectively binds to integrin αvβ3 receptors. The αvβ3-positive U87MG cell line was selected as a representative for GBM. An orthotopic GBM xenograft model based on luciferase-expressing U87MG was established and validated in vivo to investigate bio-distribution and anti-tumor efficacy of biRGD-siPIK3CB. In vitro, biRGD-siPIK3CB specifically entered and silenced PIK3CB expression in GBM cells through a αvβ3 receptor-dependent manner, thus inhibiting cell cycle progression and migration and enhancing apoptosis. In vivo, intravenously injected biRGD-siPIK3CB substantially slowed GBM growth and prolong survival by reducing tumor viability with silencing PIK3CB expression. Furthermore, biRGD-siPIK3CB led mild tubulointerstitial injury in the treatment of GMB without obvious hepatotoxicity, whereas co-infusion of Gelofusine obviously alleviated this injury without compromising its anti-tumor efficacy. These findings revealed a great translational potential of biRGD-siPIK3CB conjugate as a novel molecule for glioblastoma therapy.
Oligonucleotide Aptamer-mediated Precision Therapy of Hematological Malignancies Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-06 Shuanghui Yang, Huan Li, Ling Xu, Zhenhan Deng, Wei Han, Yanting Liu, Wenqi Jiang, Youli Zu
Precision medicine has recently emerged as a promising strategy for cancer therapy because it not only specifically targets cancer cells but it also does not have adverse effects on normal cells. Oligonucleotide aptamers are a class of small molecule ligands that can specifically bind to their targets on cell surfaces with high affinity. Aptamers have great potential in precision cancer therapy due to their unique physical, chemical, and biological properties. Therefore, aptamer technology has been widely investigated for biomedical and clinical applications. This review focuses on the potential applications of aptamer technology as a new tool for precision treatment of hematological malignancies including leukemia, lymphoma, and multiple myeloma.
Metformin protects against H2O2-induced cardiomyocytes injury by inhibiting miR-1a-3p/GRP94 pathway Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-06 Ying Zhang, Xue Liu, Lu Zhang, Xuelian Li, Zhongqiu Zhou, Lei Jiao, Yingchun Shao, Mengmeng Li, Bing Leng, Yuhong Zhou, Tianyi Liu, Qiushuang Liu, Hongli Shan, Zhimin Du
Ischemia-reperfusion (I/R) injury is a major side effect of the reperfusion treatment of the ischemic heart. Few therapies are available for the effective prevention of this injury caused by the oxidative stress-induced cardiomyocytes apoptosis. Metformin was shown to have a potential cardiac protective effect and ability to reduce cardiac events, but the exact mechanism remains unclear. Here, we aimed to confirm and investigate the mechanisms underlying potential metformin activity against I/R injury in response to oxidative stress. We determined that the expression of miR-1a-3p was significantly increased in neonatal rat ventricular cells (NRVCs) which were exposed to H2O2in vitro and in the hearts of mice which underwent the I/R injury. MiR-1a-3p was shown to target the 3′-untranslated region (3′-UTR) of GRP94, which results in the accumulation of un/misfolded proteins, leading to the endoplasmic reticulum (ER) stress. The obtained results demonstrated that C/EBP β directly induces the upregulation of miR-1a-3p by binding to its promoter. Furthermore, as a direct allosteric AMPK activator, metformin was shown to activate AMPK, and significantly reduces C/EBP β and miR-1a-3p level, compared with those in the control group. In conclusion, metformin protects cardiomyocytes against H2O2 damage through AMPK/ C/EBP β/miR-1a-3p/GRP94 pathway, which indicates that metformin may be applied for the treatment of I/R injury.
Potential regulatory roles of microRNAs and long noncoding RNAs in anticancer therapies Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-01 Mengyan Xie, Ling Ma, Tongpeng Xu, Yutian Pan, Qiang Wang, Yutian Wei, Yongqian Shu
MicroRNAs and long noncoding RNAs have long been investigated due to their roles as diagnostic and prognostic biomarkers of cancers and regulators of tumorigenesis, and the potential regulatory roles of these molecules in anticancer therapies are attracting increasing interest as more in-depth studies are performed. The major clinical therapies for cancer include chemotherapy, immunotherapy, and targeted molecular therapy. MicroRNAs and long noncoding RNAs function through various mechanisms in these approaches, and the mechanisms involve direct targeting of immune checkpoints, cooperation with exosomes in the tumor microenvironment, and alteration of drug resistance through regulation of different signaling pathways. Herein, we review the regulatory functions and significance of microRNAs and long noncoding RNAs in three anticancer therapies, especially in targeted molecular therapy, and their mechanisms.
HIT-Cas9: a CRISPR/Cas9 Genome Editing Device Under Tight and Effective Drug Control. Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-01 Chen Zhao, Yingze Zhao, Jingfang Zhang, Jia Lu, Li Chen, Yue Zhang, Yue Ying, Junjun Xu, Shixian Wei, Yu Wang
Ablation of a single N-glycosylation site in human FSTL1 induces cardiomyocyte proliferation and cardiac regeneration Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-01 Ajit Magadum, Neha Singh, Ann AnuKurian, Mohammad Tofael KabirSharkar, Elena Chepurko, Lior Zangi
Adult mammalian hearts have a very limited regeneration capacity, due largely to a lack of cardiomyocyte (CM) proliferation. It was recently reported that epicardial, but not myocardial, follistatin-like 1 (Fstl1) activates CM proliferation and cardiac regeneration after myocardial infarction (MI). Furthermore, bacterially synthesized human FSTL1 (hFSTL1) was found to induce CM proliferation, whereas hFSTL1 synthesized in mammals did not, suggesting that posttranslational modifications (e.g. glycosylation) of the hFSTL1 protein affect its regenerative activity. We used modified mRNA (modRNA) technology to investigate the possible role of specific hFSTL1 N-glycosylation sites in the induction, by hFSTL1, of CM proliferation and cardiac regeneration. We found that the mutation of a single site (N180Q) was sufficient and necessary to increase the proliferation of rat neonatal and mouse adult cardiomyocytes in vitro and after MI in vivo, respectively. A single administration of the modRNA construct encoding the N180Q mutant significantly increased cardiac function, decreased scar size and increased capillary density 28 days post MI. Overall, our data suggest that the delivery of N180Q hFSTL1 modRNA to the myocardium can mimic the beneficial effect of epicardial hFSTL1, triggering marked CM proliferation and cardiac regeneration in a mouse MI model.
miR-409 inhibits human non-small cell lung cancer progression by directly targeting SPIN1 Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-01 Qi Song, Quanbo Ji, Jingbo Xiao, Fang Li, Lingxiong Wang, Yin Chen, Yameng Xu, Shunchang Jiao
Lung cancers, the leading cause of cancer mortality in the worldwide, are characterized by a high metastatic potential. Growing evidence reveals that Spindlin 1 (SPIN1) is involved in tumour progression and carcinogenesis. However, the role of SPIN1 in non-small cell lung cancer (NSCLC) and the molecular mechanisms underlying SPIN1 in human NSCLC remain undetermined. Here, we examined the function of SPIN1 in human NSCLC and found that the expression of SPIN1 was closely correlated with the overall survival and poor prognosis of NSCLC patients. Aberrant regulation of microRNAs (miRNAs) exerts an important role in cancer progression. We revealed that miR-409 inhibits the expression of SPIN1 through binding directly to the 3’-untranslated region of SPIN1 using dual-luciferase reporter assays. Overexpression of miR-409 significantly suppressed cell migration, growth and proliferation by inhibiting SPIN1 in vitro and in vivo. SPIN1 overexpression in the miR-409-transfected NSCLC cells effectively rescued the suppression of cell migration, growth and proliferation regulated by miR-409. miR-409 regulates PI3K/AKT pathway in NSCLC. Moreover, clinical data showed that the NSCLC patients with high levels of miR-409 experienced significantly better survival. Besides, the miR-409 expression was negatively associated with the SPIN1 expression. Taken together, these findings highlight that miR-409/SPIN1 axis is a useful pleiotropic regulatory network and could early predict the metastatic potential in NSCLC patients, thus indicating the possibility that miR-409 and SPIN1 might be attractive prognostic markers for treating NSCLC patients.
Protecting Pax6 3’UTR from microRNA-7 partially restores PAX6 in islets from an aniridia mouse model. Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-09-01 Kevin Yongblah, Spencer C. Alford, Bridget C. Ryan, Robert L. Chow, Perry L. Howard
RNA polymerase II activity of type 3 Pol III promoters Mol. Ther. Nucl. Acids (IF 5.66) 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.
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 5.66) 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.
Self-amplifying Replicon RNA delivery to Dendritic Cells by Cationic Lipids Mol. Ther. Nucl. Acids (IF 5.66) 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.
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 5.66) 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.
MicroRNA-125b promotes hepatic stellate cell activation and liver fibrosis by activating RhoA signaling Mol. Ther. Nucl. Acids (IF 5.66) 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.
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 5.66) 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.
Targeting the IGF1R pathway in breast cancer using antisense lncRNA-mediated promoter cis competition Mol. Ther. Nucl. Acids (IF 5.66) 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.
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 5.66) 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.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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