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Sample records for gene sirna protects

  1. Intracranial administration of P gene siRNA protects mice from lethal Chandipura virus encephalitis.

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    Satyendra Kumar

    Full Text Available BACKGROUND: In parts of India, Chandipura Virus (CHPV has emerged as an encephalitis causing pathogen in both epidemic and sporadic forms. This pediatric disease follows rapid course leading to 55-75% mortality. In the absence of specific treatment, effectiveness of RNA interference (RNAi was evaluated. METHODS AND FINDINGS: Efficacy of synthetic short interfering RNA (siRNA or short hairpin RNA (shRNA in protecting mice from CHPV infection was assessed. The target genes were P and M genes primarily because important role of the former in viral replication and lethal nature of the latter. Real time one step RT-PCR and plaque assay were used for the assessment of gene silencing. Using pAcGFP1N1-CHPV-P, we showed that P-2 siRNA was most efficient in reducing the expression of P gene in-vitro. Both quantitative assays documented 2 logs reduction in the virus titer when P-2, M-5 or M-6 siRNAs were transfected 2 hr post infection (PI. Use of these siRNAs in combination did not result in enhanced efficiency. P-2 siRNA was found to tolerate four mismatches in the center. As compared to five different shRNAs, P-2 siRNA was most effective in inhibiting CHPV replication. An extended survival was noted when mice infected intracranially with 100 LD50 CHPV were treated with cationic lipid complexed 5 microg P-2 siRNA simultaneously. Infection with 10LD50 and treatment with two doses of siRNA first, simultaneously and second 24 hr PI, resulted in 70% survival. Surviving mice showed 4 logs less CHPV titers in brain without histopathological changes or antibody response. Gene expression profiles of P-2 siRNA treated mice showed no interferon response. First dose of siRNA at 2 hr or 4 hr PI with second dose at 24hr resulted in 40% and 20% survival respectively suggesting potential application in therapy. CONCLUSIONS: The results highlight therapeutic potential of siRNA in treating rapid and fatal Chandipura encephalitis.

  2. Silencing of Bcl-2 gene expression by siRNA transfection inhibits the protective effect of fluvastatin against cell apoptosis in human aortic endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Wenwen Zhong; Yang Liu; Jian Li; Hui Tian

    2008-01-01

    Objective To study the protective effect of fluvastatin,one of the HMG-CoA reductase inhibitors (statins),against oxygen radical-induced oxidative damages in human aortic endothelial cell,and the role of Bcl-2 in this protection.Methods Human aortic endothelial cells with or without Bcl-2 siRNA transfection were subjected to 1-100 nM of fluvastatin and 100 la hydrogen peroxide for 24 hours.Bcl-2 mRNA and protein expression were measured by Taqman quantitative PCR and Western blotting.Cell apoptosis was measured by normal and fluorescent microscopy and Cell Death Detection ELISA.Results In the Bcl-2-expressed cells,fluvastatin significantly reversed hydrogen peroxide-induced microscopic apoptosis and apoptotic DNA fragmentation,which were accompanied by a markedly upregulation of Bcl-2 expression by fluvastatin.However,the endothelial protection by fluvastatin was completely lost in Bcl-2 siRNA transfected cells.Conclusion Fluvastatin protects human endothelial cells against oxygen radical-induced cell apoptosis in vitro,and this protection seemed to be mediated in a Bcl-2 dependent pathway.(J Geriatr Cardil 12008;5:33-38)

  3. Bioreducible polymers for efficient gene and siRNA delivery

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    Jere, Dhananjay; Arote, Rohidas; Jiang Hulin; Kim, You-Kyoung; Cho, Chong-Su [Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921 (Korea, Republic of); Cho, Myung-Haing, E-mail: chocs@plaza.snu.ac.k [College of Veterinary Medicines, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2009-04-15

    Bioreducible disulfide linkage-employing drug conjugate has already been approved for drug delivery application, and also has shown immense potential in gene and siRNA transfection. This paper will focus on the recent developments in bioreducible polymeric systems for gene and siRNA delivery application, and will discuss the advantages and challenges associated with reducible polymeric carriers.

  4. Dendrimeric siRNA for Efficient Gene Silencing.

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    Hong, Cheol Am; Eltoukhy, Ahmed A; Lee, Hyukjin; Langer, Robert; Anderson, Daniel G; Nam, Yoon Sung

    2015-06-01

    Programmable molecular self-assembly of siRNA molecules provides precisely controlled generation of dendrimeric siRNA nanostructures. The second-generation dendrimers of siRNA can be effectively complexed with a low-molecular-weight, cationic polymer (poly(β-amino ester), PBAE) to generate stable nanostructures about 160 nm in diameter via strong electrostatic interactions. Condensation and gene silencing efficiencies increase with the increased generation of siRNA dendrimers due to a high charge density and structural flexibility. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Gene silencing by chemically modified siRNAs.

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    Engels, Joachim W

    2013-03-25

    RNA interference (RNAi) has not only already risen as a gold standard for validating gene function in basic science studies, but also holds great promise as a new therapeutic paradigm. Advantages of RNAi-based therapeutics include relatively fast initial screening and the ability to target proteins not yet addressable by traditional drug design strategies. In this review we describe the development of chemically modified small inhibiting siRNAs and their application as potential therapeutics during the past decade. Focus is on proper siRNA design, choice of chemical modification and how to circumvent immunogenicity as well as off-target effects.

  6. Disulfide-Based Poly(amido amine)s for siRNA Delivery: Effects of Structure on siRNA Complexation, Cellular Uptake, Gene Silencing and Toxicity

    NARCIS (Netherlands)

    Vader, Pieter; Aa, van der Leonardus J.; Engbersen, Johan F.J.; Storm, Gert; Schiffelers, Raymond M.

    2011-01-01

    Purpose RNA interference (RNAi) is a process by which small interfering RNAs (siRNA) induce sequence-specific gene silencing. Therefore, siRNA is an emerging promise as a novel therapeutic. In order to realize the high expectations for therapeutic applications, efficient delivery systems for siRNA

  7. LNA-antisense rivals siRNA for gene silencing

    DEFF Research Database (Denmark)

    Jepsen, Jan Stenvang; Wengel, Jesper; Stenvang, Jan

    2004-01-01

    Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing unprecedented binding affinity toward complementary DNA and RNA while obeying the Watson-Crick base-pairing rules. For efficient gene silencing in vitro and in vivo, fully modified or chimeric LNA oligonucleotides have been a...... or phosphorothioate-DNA segment flanked by LNA gaps, rivals siRNA as the technology of choice for target validation and therapeutic applications....... applied. LNA oligonucleotides are commercially available, can be transfected using standard techniques, are non-toxic, lead to increased target accessibility, can be designed to activate RNase H, and function in steric block approaches. LNA-Antisense, including gapmer LNA containing a central DNA...

  8. Designing siRNA that distinguish between genes that differ by a single nucleotide.

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    Schwarz, Dianne S; Ding, Hongliu; Kennington, Lori; Moore, Jessica T; Schelter, Janell; Burchard, Julja; Linsley, Peter S; Aronin, Neil; Xu, Zuoshang; Zamore, Phillip D

    2006-09-01

    Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1) gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT) gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the "seed" sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3' to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide.

  9. Designing siRNA that distinguish between genes that differ by a single nucleotide.

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    Dianne S Schwarz

    2006-09-01

    Full Text Available Small interfering RNAs (siRNAs, the guides that direct RNA interference (RNAi, provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1 gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the "seed" sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3' to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide.

  10. Multifunctional triblock Nanocarrier (PAMAM-PEG-PLL) for the efficient intracellular siRNA delivery and gene silencing.

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    Patil, Mahesh L; Zhang, Min; Minko, Tamara

    2011-03-22

    A novel triblock poly(amido amine)-poly(ethylene glycol)-poly-l-lysine (PAMAM-PEG-PLL) nanocarrier was designed, synthesized, and evaluated for the delivery of siRNA. The design of the nanocarrier is unique and provides a solution to most of the common problems associated with the delivery and therapeutic applications of siRNA. Every component in the triblock nanocarrier plays a significant role and performs multiple functions: (1) tertiary amine groups in the PAMAM dendrimer work as a proton sponge and play a vital role in the endosomal escape and cytoplasmic delivery of siRNA; (2) PEG, a linker connecting PLL and PAMAM dendrimers renders nuclease stability and protects siRNA in human plasma; (3) PLL provides primary amines to form polyplexes with siRNA through electrostatic interaction and also acts as penetration enhancer; and (4) conjugation to PEG and PAMAM reduced toxicity of PLL and the entire triblock nanocarrier PAMAM-PEG-PLL. The data obtained show that the polyplexes resulted from the conjugation of siRNA, and the proposed nanocarriers were effectively taken up by cancer cells and induced the knock down of the target BCL2 gene. In addition, triblock nanocarrier/siRNA polyplexes showed excellent stability in human plasma.

  11. Serum-stabilized naked caspase-3 siRNA protects autotransplant kidneys in a porcine model.

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    Yang, Cheng; Zhao, Tian; Zhao, Zitong; Jia, Yichen; Li, Long; Zhang, Yufang; Song, Mangen; Rong, Ruiming; Xu, Ming; Nicholson, Michael L; Zhu, Tongyu; Yang, Bin

    2014-10-01

    The naked small interfering RNA (siRNA) of caspase-3, a key player in ischemia reperfusion injury, was effective in cold preserved and hemoreperfused kidneys, but not autotransplanted kidneys in our porcine models. Here, chemically modified serum stabilized caspase-3 siRNAs were further evaluated. The left kidney was retrieved and infused by University of Wisconsin solution with/without 0.3 mg caspase-3 or negative siRNA into the renal artery for 24-hour cold storage (CS). After an intravenous injection of 0.9 mg siRNA and right-uninephrectomy, the left kidney was autotransplanted for 2 weeks. The effectiveness of caspase-3 siRNA was confirmed by caspase-3 knockdown in the post-CS and/or post-transplant kidneys with reduced apoptosis and inflammation, while the functional caspase-3 siRNA in vivo was proved by detected caspase-3 mRNA degradation intermediates. HMGB1 protein was also decreased in the post-transplanted kidneys; correlated positively with renal IL-1β mRNA, but negatively with serum IL-10 or IL-4. The minimal off-target effects of caspase-3 siRNA were seen with favorable systemic responses. More importantly, renal function, associated with active caspase-3, HMGB1, apoptosis, inflammation, and tubulointerstitial damage, was improved by caspase-3 siRNA. Taken together, the 2-week autotransplanted kidneys were protected when caspase-3 siRNA administrated locally and systemically, which provides important evidence for future clinical trials.

  12. Functional characterization of endogenous siRNA target genes in Caenorhabditis elegans

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    Heikkinen Liisa

    2008-06-01

    Full Text Available Abstract Background Small interfering RNA (siRNA molecules mediate sequence specific silencing in RNA interference (RNAi, a gene regulatory phenomenon observed in almost all organisms. Large scale sequencing of small RNA libraries obtained from C. elegans has revealed that a broad spectrum of siRNAs is endogenously transcribed from genomic sequences. The biological role and molecular diversity of C. elegans endogenous siRNA (endo-siRNA molecules, nonetheless, remain poorly understood. In order to gain insight into their biological function, we annotated two large libraries of endo-siRNA sequences, identified their cognate targets, and performed gene ontology analysis to identify enriched functional categories. Results Systematic trends in categorization of target genes according to the specific length of siRNA sequences were observed: 18- to 22-mer siRNAs were associated with genes required for embryonic development; 23-mers were associated uniquely with post-embryonic development; 24–26-mers were associated with phosphorus metabolism or protein modification. Moreover, we observe that some argonaute related genes associate with siRNAs with multiple reads. Sequence frequency graphs suggest that different lengths of siRNAs share similarities in overall sequence structure: the 5' end begins with G, while the body predominates with U and C. Conclusion These results suggest that the lengths of endogenous siRNA molecules are consequential to their biological functions since the gene ontology categories for their cognate mRNA targets vary depending upon their lengths.

  13. Low cytotoxicity fluorescent PAMAM dendrimer as gene carriers for monitoring the delivery of siRNA

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    Guan, Lingmei [Sichuan University, State Key Laboratory of Bio-resources and Eco-environment, The Ministry of Education, College of Life Sciences (China); Huang, Saipeng [Chinese Academy of Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center for Molecular Sciences, Institute of Chemistry (China); Chen, Zhao [Xi’an Jiaotong University, School of Science (China); Li, Yanchao [Chinese Academy of Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center for Molecular Sciences, Institute of Chemistry (China); Liu, Ke [Sichuan University, State Key Laboratory of Bio-resources and Eco-environment, The Ministry of Education, College of Life Sciences (China); Liu, Yang, E-mail: yliu@iccas.ac.cn; Du, Libo, E-mail: dulibo@iccas.ac.cn [Chinese Academy of Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center for Molecular Sciences, Institute of Chemistry (China)

    2015-09-15

    Visual detection of gene vectors has attracted a great deal of attention due to the application of these vectors in monitoring and evaluating the effect of gene carriers in living cells. A non-viral vector, the fluorescent PAMAM dendrimer (F-PAMAM), was synthesized through conjugation of PAMAM dendrimers and fluorescein. In vitro and ex vivo experiments show that F-PAMAM exhibits superphotostability, low cytotoxicity and facilitates endocytosis by A549 cells. The vector has a high siRNA binding affinity and it increases the efficiency of cy5-siRNA delivery in A549 cells, in comparison with a cy5-siRNA monomer. Our results provide a new method for simultaneously monitoring the delivery of siRNA and its non-viral carriers in living cells.

  14. Multifunctional nanocarrier based on clay nanotubes for efficient intracellular siRNA delivery and gene silencing.

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    Wu, Hui; Shi, Yinfeng; Huang, Chusen; Zhang, Yang; Wu, Jiahui; Shen, Hebai; Jia, Nengqin

    2014-04-01

    RNA interference-mediated gene silencing relating to disease has recently emerged as a powerful method in gene therapy. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. Halloysites are cheap and naturally available aluminosilicate clay nanotubes with high mechanical strength and biocompatibility. In this study, a novel multifunctional nanocarrier based on functionalized halloysite nanotubes (f-HNTs) has been developed via electrostatic layer-by-layer assembling approach for loading and intracellular delivery of therapeutic antisurvivin siRNA and simultaneously tracking their intracellular transport, in which PEI-modified HNTs are used as gene vector, antisurvivin siRNA as gene therapeutic agent, and mercaptoacetic acid-capped CdSe quantum dots as fluorescent labeling probes. The successful assembly of the f-HNTs-siRNA complexes was systematically characterized by transmission electron microscopy (TEM), UV-visible spectrophotometry, Zeta potential measurement, fluorescence spectrophotometry, and electrochemical impedance spectroscopy. Confocal microscopy, biological TEM, and flow cytometry studies revealed that the complexes enabled the efficient intracellular delivery of siRNA for cell-specific gene silencing. MTT assays exhibited that the complexes can enhance antitumor activity. Furthermore, Western blot analysis showed that f-HNTs-mediated siRNA delivery effectively knocked down gene expression of survivin and thereby decreased the levels of target proteins of PANC-1 cells. Therefore, this study suggested that the synthesized f-HNTs were a new effective drug delivery system for potential application in cancer gene therapy.

  15. Knockdown of antiapoptotic genes in breast cancer cells by siRNA loaded into hybrid nanoparticles

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    João de Mello, Leônidas, Jr.; Rosa Souza, Gabriela Regina; Winter, Evelyn; Silva, Adny Henrique; Pittella, Frederico; Creczynski-Pasa, Tânia Beatriz

    2017-04-01

    Tumorigenesis is related to an imbalance in controlling mechanisms of apoptosis. Expression of the genes BCL-2 and BCL-xL results in the promotion of cell survival by inhibiting apoptosis. Thus, a novel approach to suppress antiapoptotic genes is the use of small interfering RNA (siRNA) in cancer cells. However, there are some limitations for the application of siRNA such as the need for vectors to pass the cell membrane and deliver the nucleic acid. In this study CaP-siRNA-PEG-polyanion hybrid nanoparticles were developed to promote siRNA delivery to cultured human breast cancer cells (MCF-7) in order to evaluate whether the silencing of antiapoptotic genes BCL-2 and BCL-xL by siRNA would increase cancer cell death. After 48 h of incubation the expression of BCL-2 and BCL-xL genes decreased to 49% and 23%, respectively. The siRNA sequence used induced cancer cell death at a concentration of 200 nM siRNA after 72 h of incubation. As the targeted proteins are related to the resistance to chemotherapeutic drugs, the nanocarriers systems were also tested in the presence of doxorubicin (DOX). The results showed a significant reduction in the CC50 of the DOX, after silencing the antiapoptotic genes. In addition, an increase in apoptotic cell counts for both incubations conditions was observed as well. In conclusion, silencing antiapoptotic genes such as BCL-2 and BCL-xL through the use of siRNA carried by hybrid nanoparticles showed to be effective in vitro, and presents a promising strategy for pre-clinical analysis, especially when combined with DOX against breast cancer.

  16. siRNA Versus miRNA as Therapeutics for Gene Silencing.

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    Lam, Jenny K W; Chow, Michael Y T; Zhang, Yu; Leung, Susan W S

    2015-09-15

    Discovered a little over two decades ago, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are noncoding RNAs with important roles in gene regulation. They have recently been investigated as novel classes of therapeutic agents for the treatment of a wide range of disorders including cancers and infections. Clinical trials of siRNA- and miRNA-based drugs have already been initiated. siRNAs and miRNAs share many similarities, both are short duplex RNA molecules that exert gene silencing effects at the post-transcriptional level by targeting messenger RNA (mRNA), yet their mechanisms of action and clinical applications are distinct. The major difference between siRNAs and miRNAs is that the former are highly specific with only one mRNA target, whereas the latter have multiple targets. The therapeutic approaches of siRNAs and miRNAs are therefore very different. Hence, this review provides a comparison between therapeutic siRNAs and miRNAs in terms of their mechanisms of action, physicochemical properties, delivery, and clinical applications. Moreover, the challenges in developing both classes of RNA as therapeutics are also discussed.

  17. [Downregulation of Human Adenovirus DNA Polymerase Gene by Modified siRNAs].

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    Nikitenko, N A; Speiseder, T; Chernolovskaya, E L; Zenkova, M A; Dobner, T; Prassolov, V S

    2016-01-01

    Human adenoviruses, in particular D8, D19, and D37, cause ocular infections. Currently, there is no available causally directed treatment, which efficiently counteracts adenoviral infectious diseases. In our previous work, we showed that gene silencing by means of RNA interference is an effective approach for downregulation of human species D adenoviruses replication. In this study, we compared the biological activity of siRNAs and their modified analogs targeting human species D adenoviruses DNA polymerase. We found that one of selectively 2'-O-methyl modified siRNAs mediates stable and long-lasting suppression of the target gene (12 days post transfection). We suppose that this siRNA can be used as a potential therapeutic agent against human species D adenoviruses.

  18. [Experimental study of MAT1 gene silencing mediated by siRNA in pancreatic cancer].

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    Liu, Jian-ping; Yuan, Shi-zhen; Zhang, Shi-neng

    2007-10-16

    To investigate the inhibitory effect of gene silencing mediated by MAT1-siRNA constructed in vitro transcription for pancreatic cancer in vivo and in vitro. 21-nt double strand siRNA targeting MAT1 gene was constructed and labeled with Cy3 fluorescent labeling reagent. Human pancreatic cancer cells of the line BxPC3 were cultured and divided into 4 groups: MAT1-siRNA transfected group, negative siRNA control group, lipid control group, and blank control group. The rate of cell duplication was determined by counting the cells for three consecutive days. Cell cycle profiles were determined by flow cytometry. Semi-quantitative analysis of the level of MAT1-mRNA expression was performed using the RT-PCR technique. The level of MAT1 protein expression was analyzed by Western-blotting. 18 nude mice were injected subcutaneously with BxPC3 cells to establish mouse tumor models, and then divided randomly into 3 equal groups: MAT1-siRNA group undergoing injection of MAT1-siRNA directly into the tumors 2 times a week for 4 weeks, blank control group, and negative MAT1-siRNA group. 4 weeks later the mice were killed to observe the weight and size of tumor and to calculate the tumor inhibition rate. Two of the 4 designed MAT1-siRNAs significantly suppressed the growth of the BxPC3 cells. 72 h after transfection the cell duplication was inhibited by 34.9% in the MAT1-siRNA transfection group. The cell cycle profile showed 83.9% of the MAT1-siRNA transfected cells were in the G0/G1 phase, a rate significantly higher than that in the blank control group (59.86%, P < 0.01). 48 h later the content of MAT1-mRNA of the MAT1-siRNA transfected cells was significantly reduced by 80.12%, and 72 h after the transfection the content of MAT1 protein was reduced by 50.12%, a rate significantly higher than those of the 2 control groups (both P < 0.01). The weight and volume of the transplant tumors in the MAT1-siRNA injected nude mice were significantly reduced compare with the negative siRNA

  19. Changes of osteosarcoma cell biological behavior afterId1 gene silencing by siRNA

    Institute of Scientific and Technical Information of China (English)

    De-Li Xu

    2016-01-01

    Objective:To study the changes of osteosarcoma cell biological behaviors proliferation, migration and invasion afterId1gene silencing by siRNA.Methods:Osteosarcoma HEK293 cell lines were cultured and transfected with Id1-siRNA and NC-siRNA respectively, cell proliferation, migration and invasion were detected after 24 h and 48 h of transfection, and the expression levels of proliferation-promoting genes, proliferation-inhibiting genes as well as migration and invasion-related genes in cells were detected after 48 h of transfection. Results:After 24 h and 48 h of transfection, cell OD value and the number of invasive cells of Id1-siRNA group were significantly lower than those of NC-siRNA group, and scratch area was significantly larger than that of NC-siRNA group; after 48 h of transfection, hnRNP A2, hnRNP B1, S100A6, RUNX2, Aurora-A, Grb2, Gab2 and Rap2a expression levels of Id1-siRNA group were significantly lower than those of NC-siRNA group, and p53, TAp73 and IGFBP5 were significantly higher than those of NC-siRNA group.Conclusions:Id1 gene silencing by siRNA can inhibit osteosarcoma cell proliferation, migration and invasion.

  20. Chitosan Hydrogel as siRNA vector for prolonged gene silencing

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    2014-01-01

    Background The periodontitis is one of the most prevalent diseases with alveolar resorption in adult people and is the main cause of the tooth loss. To investigate the possibility for protecting the loss of alveolar bone in periodontal diseases, a RNAi-based therapeutic strategy is applied for silencing RANK signaling using thermosensitive chitosan hydrogel as siRNA reservoir and vector. Results The thermosensitive chitosan hydrogel was formed from solution (PH = 7.2, at 4°C) at 37°C within 8 minutes. The degradation rates of hydrogel were ~50% and 5% (W remaining/W beginning) in the presence and absence of lysozyme, respectively, over a period of 20 days. The concurrent cumulative in vitro release of Cy3-labeled siRNA from the hydrogel was 50% and 17% over 14 days, with or without lysozyme digestion, respectively. High cell viability (>88%) was maintained for cells treated with hydrogel loaded with RANK specific siRNA and RANK knockdown was prolonged for up to 9 days when cells were incubated with siRNA/hydrogel complex. In vivo release of siRNA was investigated in a subcutaneous delivery setup in mice. The fluorescent signal from siRNA within hydrogel was remained for up to 14 days compared to less than one day for siRNA alone. Conclusions Chitosan hydrogel can potentially serve as a suitable reservoir and vector for local sustained delivery of siRNA in potential therapy. PMID:24946934

  1. SiRNAs conjugated with aromatic compounds induce RISC-mediated antisense strand selection and strong gene-silencing activity

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    Kubo, Takanori, E-mail: kubo-t@yasuda-u.ac.jp [Faculty of Pharmacy, Yasuda Women' s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153 (Japan); Yanagihara, Kazuyoshi [Faculty of Pharmacy, Yasuda Women' s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153 (Japan); Division of Genetics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Takei, Yoshifumi [Department of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumi-cho, Showa-ku, Nagoya 466-8550 (Japan); Mihara, Keichiro [Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553 (Japan); Sato, Yuichiro; Seyama, Toshio [Faculty of Pharmacy, Yasuda Women' s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153 (Japan)

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer SiRNAs conjugated with aromatic compounds (Ar-siRNAs) at 5 Prime -sense strand were synthesized. Black-Right-Pointing-Pointer Ar-siRNAs increased resistance against nuclease degradation. Black-Right-Pointing-Pointer Ar-siRNAs were thermodynamically stable compared with the unmodified siRNA. Black-Right-Pointing-Pointer High levels of cellular uptake and cytoplasmic localization were found. Black-Right-Pointing-Pointer Strong gene-silencing efficacy was exhibited in the Ar-siRNAs. -- Abstract: Short interference RNA (siRNA) is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with aromatic compounds in order to improve the potency of RNAi and thus to overcome several problems with siRNAs, such as cellular delivery and nuclease stability. The siRNAs conjugated with phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives showed strong resistance to nuclease degradation, and were thermodynamically stable compared with unmodified siRNA. A high level of membrane permeability in HeLa cells was also observed. Moreover, these siRNAs exhibited enhanced RNAi efficacy, which exceeded that of locked nucleic acid (LNA)-modified siRNAs, against exogenous Renilla luciferase in HeLa cells. In particular, abundant cytoplasmic localization and strong gene-silencing efficacy were found in the siRNAs conjugated with phenyl and hydroxyphenyl derivatives. The novel siRNAs conjugated with aromatic compounds are promising candidates for a new generation of modified siRNAs that can solve many of the problems associated with RNAi technology.

  2. Comparing Gene Silencing and Physiochemical Properties in siRNA Bound Cationic Star-Polymer Complexes.

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    Dearnley, Megan; Reynolds, Nicholas P; Cass, Peter; Wei, Xiaohu; Shi, Shuning; Mohammed, A Aalam; Le, Tam; Gunatillake, Pathiraja; Tizard, Mark L; Thang, San H; Hinton, Tracey M

    2016-11-14

    The translation of siRNA into clinical therapies has been significantly delayed by issues surrounding the delivery of naked siRNA to target cells. Here we investigate siRNA delivery by cationic acrylic polymers developed by Reversible Addition-Fragmentation chain Transfer (RAFT) mediated free radical polymerization. We investigated cell uptake and gene silencing of a series of siRNA-star polymer complexes both in the presence and absence of a protein "corona". Using a multidisciplinary approach including quantitative nanoscale mechanical-atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis we have characterized the nanoscale morphology, stiffness, and surface charge of the complexes with and without the protein corona. This is one of the first examples of a comprehensive physiochemical analysis of siRNA-polymer complexes being performed alongside in vitro biological assays, allowing us to describe a set of desirable physical features of cationic polymer complexes that promote gene silencing. Multifaceted studies such as this will improve our understanding of structure-function relationships in nanotherapeutics, facilitating the rational design of polymer-mediated siRNA delivery systems for novel treatment strategies.

  3. Stable gene transfer of CCR5 and CXCR4 siRNAs by sleeping beauty transposon system to confer HIV-1 resistance

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    Akkina Ramesh

    2008-07-01

    Full Text Available Abstract Background Thus far gene therapy strategies for HIV/AIDS have used either conventional retroviral vectors or lentiviral vectors for gene transfer. Although highly efficient, their use poses a certain degree of risk in terms of viral mediated oncogenesis. Sleeping Beauty (SB transposon system offers a non-viral method of gene transfer to avoid this possible risk. With respect to conferring HIV resistance, stable knock down of HIV-1 coreceptors CCR5 and CXCR4 by the use of lentiviral vector delivered siRNAs has proved to be a promising strategy to protect cells from HIV-1 infection. In the current studies our aim is to evaluate the utility of SB system for stable gene transfer of CCR5 and CXCR4 siRNA genes to derive HIV resistant cells as a first step towards using this system for gene therapy. Results Two well characterized siRNAs against the HIV-1 coreceptors CCR5 and CXCR4 were chosen based on their previous efficacy for the SB transposon gene delivery. The siRNA transgenes were incorporated individually into a modified SB transfer plasmid containing a FACS sortable red fluorescence protein (RFP reporter and a drug selectable neomycin resistance gene. Gene transfer was achieved by co-delivery with a construct expressing a hyperactive transposase (HSB5 into the GHOST-R3/X4/R5 cell line, which expresses the major HIV receptor CD4 and and the co-receptors CCR5 and CXCR4. SB constructs expressing CCR5 or CXCR4 siRNAs were also transfected into MAGI-CCR5 or MAGI-CXCR4 cell lines, respectively. Near complete downregulation of CCR5 and CXCR4 surface expression was observed in transfected cells. During viral challenge with X4-tropic (NL4.3 or R5-tropic (BaL HIV-1 strains, the respective transposed cells showed marked viral resistance. Conclusion SB transposon system can be used to deliver siRNA genes for stable gene transfer. The siRNA genes against HIV-1 coreceptors CCR5 and CXCR4 are able to downregulate the respective cell surface proteins

  4. Optimizations of siRNA design for the activation of gene transcription by targeting the TATA-box motif.

    Directory of Open Access Journals (Sweden)

    Miaomiao Fan

    Full Text Available Small interfering RNAs (siRNAs are widely used to repress gene expression by targeting mRNAs. Some reports reveal that siRNAs can also activate or inhibit gene expression through targeting the gene promoters. Our group has found that microRNAs (miRNAs could activate gene transcription via interaction with the TATA-box motif in gene promoters. To investigate whether siRNA targeting the same region could upregulate the promoter activity, we test the activating efficiency of siRNAs targeting the TATA-box motif of 16 genes and perform a systematic analysis to identify the common features of the functional siRNAs for effective activation of gene promoters. Further, we try various modifications to improve the activating efficiency of siRNAs and find that it is quite useful to design the promoter-targeting activating siRNA by following several rules such as (a complementary to the TATA-box-centered region; (b UA usage at the first two bases of the antisense strand; (c twenty-three nucleotides (nts in length; (d 2'-O-Methyl (2'-OMe modification at the 3' terminus of the antisense strand; (e avoiding mismatches at the 3' end of the antisense strand. The optimized activating siRNAs potently enhance the expression of interleukin-2 (IL-2 gene in human and mouse primary CD4+ T cells with a long-time effect. Taken together, our study provides a guideline for rational design the promoter-targeting siRNA to sequence-specifically enhance gene expression.

  5. Polyethyleneimine (PEI mediated siRNA gene silencing in the Schistosoma mansoni snail host, Biomphalaria glabrata.

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    Matty Knight

    2011-07-01

    Full Text Available An in vivo, non-invasive technique for gene silencing by RNA interference (RNAi in the snail, Biomphalaria glabrata, has been developed using cationic polymer polyethyleneimine (PEI mediated delivery of long double-stranded (ds and small interfering (si RNA. Cellular delivery was evaluated and optimized by using a 'mock' fluorescent siRNA. Subsequently, we used the method to suppress expression of Cathepsin B (CathB with either the corresponding siRNA or dsRNA of this transcript. In addition, the knockdown of peroxiredoxin (Prx at both RNA and protein levels was achieved with the PEI-mediated soaking method. B. glabrata is an important snail host for the transmission of the parasitic digenean platyhelminth, Schistosoma mansoni that causes schistosomiasis in the neotropics. Progress is being made to realize the genome sequence of the snail and to uncover gene expression profiles and cellular pathways that enable the snail to either prevent or sustain an infection. Using PEI complexes, a convenient soaking method has been developed, enabling functional gene knockdown studies with either dsRNA or siRNA. The protocol developed offers a first whole organism method for host-parasite gene function studies needed to identify key mechanisms required for parasite development in the snail host, which ultimately are needed as points for disrupting this parasite mediated disease.

  6. Inhibition of core gene of HCV 3a genotype using synthetic and vector derived siRNAs

    Directory of Open Access Journals (Sweden)

    Samreen Baila

    2010-11-01

    Full Text Available Abstract Background Hepatitis C virus (HCV is a major causative agent of liver associated diseases throughout the world, with genotype 3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current therapy, RNA interference (RNAi a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option. Results The current study was purposed to assess and explore the possibility of RNAi to silence the HCV-3a Core gene expression, which play complex role in regulation of cell growth and host genes expression essential for infectivity and disease progression. To identify the potent siRNA target sites, 5 small interfering RNAs (siRNAs against Core gene were designed and in vitro transcribed after consensus sequence analysis of different HCV-3a isolates. Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells. For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs. Furthermore, the efficacy of cell culture tested siRNA and shRNA, were evaluated for inhibition of HCV replication in HCV infected serum inoculated Huh-7 cells and a significant decrease in HCV viral copy number was observed. Conclusions Our results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype.

  7. siRNA mediated gene silencing in Fusarium sp. HKF15 for overproduction of bikaverin.

    Science.gov (United States)

    Deshmukh, Radhika; Purohit, Hemant J

    2014-04-01

    Fusarium sp. HKF15 is an isolate from effluent treatment plant which produces bikaverin. Bikaverin is a polyketide having antitumor and antibiotic potential. Acetyl coenzyme A is a common precursor for bikaverin as well as carotenoids and gibberellins. A polyketide synthase gene bik1 is responsible for bikaverin production whereas, hydroxymethyl glutaryl coenzyme A reductase (hmgR) and farnesyl pyrophosphate synthase (fpps) are carotenoid and gibberellin pathway genes. Aim of this study was assessing siRNA mediated gene silencing for bikaverin overproduction with down-regulation of carotenoid and gibberellin pathway. HKF15 protoplasts derived from glucose grown culture were treated with 200pmolml(-1)hmgR and fpps siRNAs separately. Along with down-regulation of target genes, there was 2.4-fold increase in bik1 gene expression. The silencing was effective till 48h with a 41% increase in bikaverin production. The study proposes a strategy for manipulation of physiology towards desired secondary metabolite overproduction.

  8. Efficient gene silencing in metastatic tumor by siRNA formulated in surface-modified nanoparticles.

    Science.gov (United States)

    Li, Shyh-Dar; Chono, Sumio; Huang, Leaf

    2008-02-18

    We have developed a nanoparticle (NP) formulation for systemically delivering siRNA into metastatic tumors. The NP, composed of nucleic acids, a polycationic peptide and cationic liposome, was prepared in a self-assembling process. The NP was then modified by PEG-lipid containing a targeting ligand, anisamide, and thus was decorated for targeting sigma receptor expressing B16F10 tumor. The activity of the targeted NP was compared with the naked NP (no PEGylation) and non-targeted NP (no ligand). The delivery efficiency of the targeted NP was 4-fold higher than the non-targeted NP and could be competed by excess free ligand. Luciferase siRNA was used to evaluate the gene silencing activity in the B16F10 cells, which were stably transduced with a luciferase gene. The gene silencing activity of the targeted NP was significantly higher than the other formulations and lasted for 4 days. While confocal microscopy showed that the naked NP provided no tissue selectivity and non-targeted NP was ineffective for tumor uptake, the targeted NP effectively penetrated the lung metastasis, but not the liver. It resulted in 70-80% gene silencing in the metastasis model after a single i.v. injection (150 microg siRNA/kg). This effective formulation also showed very little immunotoxicity.

  9. INHIBITION OF ACTIVATED K-RAS GENE BY SIRNA EXPRESSION CASSETTES IN HUMAN PANCREATIC CARCINOMA CELL LINE MIAPACA-2

    Institute of Scientific and Technical Information of China (English)

    WANG Wei; WANG Chun-you; DONG Ji-hua; CHEN Xiong; ZHANG Min; ZHAO Gang

    2005-01-01

    Objective: To construct the small interfering RNA(siRNA) expression cassettes (SECs) targeting activated K-ras gene sequence and investigate the effects of SECs on K-ras gene in human pancreatic cancer cell line MIAPaCa-2. Methods: Three different sites of SECs were constructed by PCR. The K1/siRNA, K2/siRNA and K3/siRNA were located at the site 194, 491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of site 194,491, we observed the cytopathic effect of confluent MiaPaCa-2 cells after they were incubated for 48 hours, and detected the apoptosis in cells by FACS, then we tested the alternation of K-ras gene in confluent MiaPaCa-2 cells by RT-PCR,immunofluorescence and western blot, respectively. Results: Introductions of the K1/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells led to cytopathic effect, slower proliferation and increased apoptosis, while the appearances of control MiaPaCa-2 cells remained well. The number of apoptotic cells increased compared with control cells. RT-PCR,immunofluorescence and western blot showed the effects of inhibited expression of activated K-ras gene by RNA interference in the K1/siRNA and K2/siRNA groups. We also found that the introduction of K3/siRNA had no effect on MiaPaCa-2 cells. Conclusion: K1/siRNA and K2/siRNA can inhibit the expression of activated K-ras and decrease the growth of MiaPaCa-2 cells, while K3/siRNA has no such effect, demonstrating that the suppression of tumor growth by siRNA is sequence-specific. We conclude that K-ras is involved in maintenance of tumor growth of human pancreatic cancer, and SECs against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.

  10. Varying the nucleic acid composition of siRNA molecules dramatically varies the duration and degree of gene silencing.

    Science.gov (United States)

    Lamberton, Janelle S; Christian, Allen T

    2003-06-01

    The utility of short interfering RNA (siRNA) as a means of gene silencing depends on several factors. These include the degree to which a gene can be silenced, the length of time for which the gene remains silenced, the degree of recovery of gene function, and the effects of the silencing process on general cell functions. We hypothesized that changing the nucleic acid composition of the siRNA constructs used for silencing would affect these parameters. With siRNA gene silencing of the glucose-6-phosphate dehydrogenase gene as a baseline, we found that siDNA molecules have an effect that is similar in duration but lesser in degree, whereas hybrid DNA:RNA molecules have an effect that is enormously greater in both duration and degree.

  11. Cheminformatics Approach to Gene Silencing: Z Descriptors of Nucleotides and SVM Regression Afford Predictive Models for siRNA Potency.

    Science.gov (United States)

    Ebalunode, Jerry O; Zheng, Weifan

    2010-12-17

    Short interfering RNA mediated gene silencing technology has been through tremendous development over the past decade, and has found broad applications in both basic biomedical research and pharmaceutical development. Critical to the effective use of this technology is the development of reliable algorithms to predict the potency and selectivity of siRNAs under study. Existing algorithms are mostly built upon sequence information of siRNAs and then employ statistical pattern recognition or machine learning techniques to derive rules or models. However, sequence-based features have limited ability to characterize siRNAs, especially chemically modified ones. In this study, we proposed a cheminformatics approach to describe siRNAs. Principal component scores (z1, z2, z3, z4) have been derived for each of the 5 nucleotides (A, U, G, C, T) from the descriptor matrix computed by the MOE program. Descriptors of a given siRNA sequence are simply the concatenation of the z values of its composing nucleotides. Thus, for each of the 2431 siRNA sequences in the Huesken dataset, 76 descriptors were generated for the 19-NT representation, and 84 descriptors were generated for the 21-NT representation of siRNAs. Support Vector Machine regression (SVMR) was employed to develop predictive models. In all cases, the models achieved Pearson correlation coefficient r and R about 0.84 and 0.65 for the training sets and test sets, respectively. A minimum of 25 % of the whole dataset was needed to obtain predictive models that could accurately predict 75 % of the remaining siRNAs. Thus, for the first time, a cheminformatics approach has been developed to successfully model the structure-potency relationship in siRNA-based gene silencing data, which has laid a solid foundation for quantitative modeling of chemically modified siRNAs.

  12. Construction and expression of SET gene and siRNA recombinant adenovirus vectors

    Institute of Scientific and Technical Information of China (English)

    Xu Bo-qun; Lu Pin-hong; Li Ying; Xue Kai; Li Mei; Ma Xiang; Diao Fei-yan; Cui Yu-gui; Liu Jia-yin

    2010-01-01

    Objective: To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA (SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels.Methods: The cDNA sequence of SET was cloned by reverse transcriptive polymerase chain reaction (RT-PCR) and the SET gene fragment was subcloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-SET. The shuttle plasmid pAdtrack-SET was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant Ad-CMV-SET and the recombinant Ad-CMV-SET was packaged and amplified in the AD293 cells. The expression of SET in AD293 cells was detected by Western blot. In addition, we constructed SET gene SiRNA recombinant adenovirus vector (Ad-H1-SiRNA/SET) and its efficacy of knockdown of SET protein was detected in infected GC-2spd(ts) cells by Western blot. Results: The recombinant adenovirus vectors, both SET gene recombinant adenovirus vector Ad-CMV-SET and SET gene SiRNA recombinant adenovirus vector Ad-H1-SiRNA/SET, were proven to be constructed successfully by the evidence of endonulease digestion and sequencing. AD293 cells infected with either recombinant adenovirus vector of Ad-CMV-SET or Ad-H1-SiRNA/SET were observed to express GFP. The expression of SET protein was up-regulated significantly in AD293 cells infected with SET gene recombinant adenovirus vector. On the contrast, SET protein was significantly down-regulated in the GC-2spd(ts) cells infected with Ad-H1-SiRNA/SET (P<0.05) and the knockdown efficiency was approximately 50%-70%. Conclusion: The recombinant adenovirus vector Ad-CMV-SET and Ad-H1-SiRNA/SET were successfully constructed and effectively expressed in germ cells and somatic cells. It provides an experimental tool for further study of SET gene in the physiological and pathophysiological mechanism of reproduction-related diseases.

  13. Establishment of a Screening System for Selection of siRNA Target Sites Directed against Hepatitis B Virus Surface Gene

    Institute of Scientific and Technical Information of China (English)

    Xiu-Min ZHOU; Ju-Sheng LIN; Yi SHI; De-An TIAN; Huan-Jun HUANG; He-Jun ZHOU; You-Xin JIN

    2005-01-01

    To study the inhibitory effects of plasmid-derived small interfering RNA (siRNA) and synthetic siRNA on the expression of the hepatitis B virus surface (HBs) gene, three plasmid-derived siRNAs and one synthetic siRNA that complement the coding region of the HBs gene were prepared. The HBs expression plasmid pHBs-EGFP was also constructed. HeLa cells were co-transfected with pHBs-EGFP and the above siRNAs. The HBs mRNA quantities were measured by reverse-transcription PCR, and the level of HBs-EGFP fusion protein was quantified by fluorescent microscope. The concentrations of the hepatitis B virus surface antigen (HBsAg) derived from the culture supernatant of transfected HepG2.2.15cells were measured by an enzyme-linked immunosorbent assay (ELISA) kit. The results showed that the three plasmid-derived siRNAs and the synthetic siRNA can effectively reduce the quantities of HBs mRNA and protein. The plasmid-derived siRNA psiRNA1 was found to be the most effective inhibitor of HBs expression. It can inhibit HBs-EGFP expression by 63.3% and suppress HBs mRNA by 75.6%. To further substantiate the above observations, psiRNA1 was transfected into HepG2.2.15 cells (an HBV secreting cell line). The transfections resulted in almost complete blockage of HBsAg production, whereas control vector transfected cells secreted high levels of HBsAg 7 days post-transfection. In conclusion, our data suggests that RNA interference (RNAi) is an efficient approach for reducing the level of HBs transcripts and proteins and for suppressing HBsAg production.

  14. Gene silencing by siRNAs and antisense oligonucleotides in the laboratory and the clinic

    Science.gov (United States)

    Watts, Jonathan K.; Corey, David R.

    2014-01-01

    Synthetic nucleic acids are commonly used laboratory tools for modulating gene expression and have the potential to be widely used in the clinic. Progress towards nucleic acid drugs, however, has been slow and many challenges remain to be overcome before their full impact on patient care can be understood. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are the two most widely used strategies for silencing gene expression. We first describe these two approaches and contrast their relative strengths and weaknesses for laboratory applications. We then review the choices faced during development of clinical candidates and the current state of clinical trials. Attitudes towards clinical development of nucleic acid silencing strategies have repeatedly swung from optimism to depression during the past twenty years. Our goal is to provide the information needed to design robust studies with oligonucleotides, making use of the strengths of each oligonucleotide technology. PMID:22069063

  15. Primary and secondary siRNAs in geminivirus-induced gene silencing.

    Directory of Open Access Journals (Sweden)

    Michael Aregger

    2012-09-01

    Full Text Available In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL proteins producing short interfering (siRNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV, four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR. However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a GFP transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the GFP stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the GFP 3'-untranslated region (UTR induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust GFP silencing, except when viral siRNAs targeted GFP 5'-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause GFP silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs.

  16. Promoter-targeted siRNAs induce gene silencing of simian immunodeficiency virus (SIV) infection in vitro.

    Science.gov (United States)

    Lim, Heidi G W; Suzuki, Kazuo; Cooper, David A; Kelleher, Anthony D

    2008-03-01

    RNA interference is a conserved process by which sequence-specific double-stranded RNA is converted into small interfering double-stranded RNAs (siRNAs) that can induce gene silencing via two pathways: post-transcriptional gene silencing and transcriptional gene silencing (TGS). We previously reported TGS of human immunodeficiency virus-1 (HIV-1) could be induced by siRNAs targeting regions within its 5'-long-terminal repeat (5'LTR) promoter region. Here we show that promoter-targeted siRNAs can also induce silencing of simian immunodeficiency virus (SIV) replication by similar mechanisms. Suppression of productive infection was achieved in two different cell lines: a CD4, CCR5, CXCR4 expressing HeLa cell line (MAGIC-5) and in a human lymphoid cell line (CEMx174). HpaII digestion demonstrated induction of methylation at a CpG site within the SIV promoter region following siRNA-induced suppression. Both 5-azacytidine (5-AzaC) and trichostatin A (TSA), inhibitors of DNA methyltransferases (DNMTs) and histone deacetylation, respectively, partially reversed the silencing effect. Furthermore, using chromatin immunoprecipitation (ChIP) assays we found enrichment in the region of the LTR of heterochromatin markers dimethylated histone 3 lysine 9 (H3K9) and trimethylated histone 3 lysine 27 (H3K27) in the siRNA silenced cultures. Together, these results strongly suggest certain siRNAs targeting the promoter region of SIV can effect viral silencing through the induction of epigenetic changes.

  17. Effect of combined siRNA of HCV E2 gene and HCV receptors against HCV

    Directory of Open Access Journals (Sweden)

    Ashfaq Usman Alli A

    2011-06-01

    Full Text Available Abstract Background/Aim Hepatitis C virus (HCV is a major threat as almost 3% of the world's population (350 million individual and 10% of the Pakistani population is chronically infected with this virus. RNA interference (RNAi, a sequence-specific degradation process of RNA, has potential to be used as a powerful alternative molecular therapeutic approach in spite of the current therapy of interferon-α and ribavirin against HCV which has limited efficiency. HCV structural gene E2 is mainly involved in viral cell entry via attachment with the host cell surface receptors i.e., CD81 tetraspanin, low density lipoprotein receptor (LDLR, scavenger receptor class B type 1 (SR-B1, and Claudin1 (CLDN1. Considering the importance of HCV E2 gene and cellular receptors in virus infection and silencing effects of RNAi, the current study was designed to target the cellular and viral factors as new therapeutic options in limiting HCV infection. Results In this study the potential of siRNAs to inhibit HCV-3a replication in serum-infected Huh-7 cells was investigated by combined treatment of siRNAs against the HCV E2 gene and HCV cellular receptors (CD81 and LDLR, which resulted in a significant decrease in HCV viral copy number. Conclusion From the current study it is concluded that the combined RNAi-mediated silencing of HCV E2 and HCV receptors is important for the development of effective siRNA-based therapeutic option against HCV-3a.

  18. Elucidating the role of free polycations in gene knockdown by siRNA polyplexes

    DEFF Research Database (Denmark)

    Klauber, Thomas Christopher Bogh; Søndergaard, Rikke Vicki; Sawant, Rupa R.;

    2016-01-01

    to conventional PEIs. Interestingly, strong knockdown using bPEI 25. kDa is dependent on the presence of a free vector fraction which does not increase siRNA uptake. Finally, we have investigated the effect on lysosomal pH induced by these vectors to elucidate the differences in the proton sponge effect between...... lipid conjugated PEI and conventional PEI: Neither DOPE-PEI nor bPEI 25. kDa affected lysosomal pH as a function of time, underlining that the possible proton sponge effect is not associated with changes in lysosomal pH. Statement of Significance: Gene silencing therapy has the potential to treat...... evaluate how lipid conjugation of PEI influences formulation, cytotoxicity and polymer interaction with cells and cargo as well as the proton sponge capabilities of the vectors....

  19. The intracellular pharmacodynamics of siRNA is responsible for the low gene silencing activity of siRNA-loaded nanoparticles in dendritic cells.

    Science.gov (United States)

    Nakamura, Takashi; Fujiwara, Yuki; Warashina, Shota; Harashima, Hideyoshi

    2015-10-15

    The delivery of small interfering RNA (siRNA) to dendritic cells (DCs) is a challenging issue for siRNA-loaded lipid nanoparticles. The cause of this difficulty is unknown. The findings reported herein indicate that the rate-limiting step in gene silencing using siRNA-loaded lipid nanoparticles in DCs, as evidenced by a quantitative analysis of each process in siRNA delivery between mouse bone marrow derived DC (BMDC) and other cell lines, was not associated with the actual delivery of siRNA. A gene silencing of only 50% was observed in BMDC, even when a high dose was used. Contrary to our expectation, the interval between cellular uptake and the delivery of siRNA to the cytosol was not responsible for the low gene silencing. Meanwhile, a drastic difference was found in the relationship between the efficiency of gene silencing and the amount of intracellular intact siRNA. This fact indicates that the processes after cytosolic delivery of siRNA, namely the intracellular pharmacodynamics (PD) of siRNA, appear to be the rate-limiting step in gene silencing in BMDC. The findings reported here demonstrate the importance of the intracellular PD of siRNA delivered to cytosol in the development of siRNA delivery systems for gene silencing in DCs.

  20. Inhibition of MMP-2 Expression with siRNA Increases Baseline Cardiomyocyte Contractility and Protects against Simulated Ischemic Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Han-Bin Lin

    2014-01-01

    Full Text Available Matrix metalloproteinases (MMPs significantly contribute to ischemia reperfusion (I/R injury, namely, by the degradation of contractile proteins. However, due to the experimental models adopted and lack of isoform specificity of MMP inhibitors, the cellular source and identity of the MMP(s involved in I/R injury remain to be elucidated. Using isolated adult rat cardiomyocytes, subjected to chemically induced I/R-like injury, we show that specific inhibition of MMP-2 expression and activity using MMP-2 siRNA significantly protected cardiomyocyte contractility from I/R-like injury. This was also associated with increased expression of myosin light chains 1 and 2 (MLC1/2 in comparison to scramble siRNA transfection. Moreover, the positive effect of MMP-2 siRNA transfection on cardiomyocyte contractility and MLC1/2 expression levels was also observed under control conditions, suggesting an important additional role for MMP-2 in physiological sarcomeric protein turnover. This study clearly demonstrates that intracellular expression of MMP-2 plays a significant role in sarcomeric protein turnover, such as MLC1 and MLC2, under aerobic (physiological conditions. In addition, this study identifies intracellular/autocrine, cardiomyocyte-produced MMP-2, rather than paracrine/extracellular, as responsible for the degradation of MLC1/2 and consequent contractile dysfunction in cardiomyocytes subjected to I/R injury.

  1. Cytosolic mRNA Target and Bioavailability of Nanoparticulate siRNA delivery systems for gene silencing.

    Science.gov (United States)

    Leucuta, Sorin Emilian

    2017-03-22

    Recent research in medical and pharmaceutical sciences has benefited from advances in molecular biology and genetics, which made possible a diagnosis at the molecular level in more and more diseases. This implies the drug treatment at the molecular level. The interest in Ribonucleic acid interference (RNAi) is based on the mechanism operates by eliminating the messenger RNAs (mRNAs) coding for multiple proteins, which open solutions for treating many types of diseases. Small (short) interfering RNA (siRNA) has quickly been established as an effective gene-silencing strategy in animal models, and more recently in human clinical trials, as a potential therapeutic approach. Various nanoparticulate drug delivery systems for siRNA delivery have been explored extensively. However, there are many more barriers and challenges that need to be addressed and overcome to achieve the ideal formulation in terms of selectivity, efficacy and safety. One of the major causes of the drawback of these treatments is the difficulty to transport the nucleic acids in the cytosol and organelles. These delivery systems will favorably alter the pharmacokinetics and biodistribution of siRNAs, should be biocompatible and genocompatible to avoid immune stimulation and off-target gene effects. These properties are essential for systemic use, as they prolong siRNA half-lives in blood and increase intracellular bioavailability of siRNA. Future research needs drug delivery systems with more effective design, enhanced biological stability, subcellular bioavailability, and efficient targeted delivery in vivo for improved targeting and specificity of siRNA molecules for any given clinical condition. The paper shows how to overcome physiological barriers to achieve the target, and examples in which significant results were obtained in therapeutic in vitro and in vivo research including nanoparticulate systems.To day, only a few nanoparticle-based siRNA delivery systems have been approved by the Food

  2. Both strands of siRNA have potential to guide posttranscriptional gene silencing in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Jun-Xia Wei

    Full Text Available Despite the widespread application of RNA interference (RNAi as a research tool for diverse purposes, the key step of strand selection of siRNAs during the formation of RNA-induced silencing complex (RISC remains poorly understood. Here, using siRNAs targeted to the complementary region of Survivin and the effector protease receptor 1 (EPR-1, we show that both strands of the siRNA duplex can find their target mRNA and are equally eligible for assembly into Argonaute 2 (Ago2 of RISC in HEK293 cells. Transfection of the synthetic siRNA duplexes with different thermodynamic profiles or short hairpin RNA (shRNA vectors that generate double-stranded RNAs (dsRNAs, permitting processing specifically from either the 5' or 3' end of the incipient siRNA, results in the degradation of the respective target mRNAs of either strand of the siRNA duplex with comparable efficiencies. Thus, while most RNAi reactions may follow the thermodynamic asymmetry rule in strand selection, our study suggests an exceptional mode for certain siRNAs in which both strands of the duplex are competent in sponsoring RNAi, and implies additional factors that might dictate the RNAi targets.

  3. Biodegradable Nanoparticles of mPEG-PLGA-PLL Triblock Copolymers as Novel Non-Viral Vectors for Improving siRNA Delivery and Gene Silencing

    Directory of Open Access Journals (Sweden)

    Qiu-Sheng Shi

    2012-01-01

    Full Text Available Degradation of mRNA by RNA interference is one of the most powerful and specific mechanisms for gene silencing. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of biodegradable nanoparticles (NPs made from monomethoxypoly(ethylene glycol-poly(lactic-co-glycolic acid-poly-l-lysine (mPEG-PLGA-PLL triblock copolymers. Various physicochemical properties of mPEG-PLGA-PLL NPs, including morphology, size, surface charge, siRNA encapsulation efficiency, and in vitro release profile of siRNA from NPs, were characterized by scanning electron microscope, particle size and zeta potential analyzer, and high performance liquid chromatography. The levels of siRNA uptake and targeted gene inhibition were detected in human lung cancer SPC-A1-GFP cells stably expressing green fluorescent protein. Examination of the cultured SPC-A1-GFP cells with fluorescent microscope and flow cytometry showed NPs loading Cy3-labeled siRNA had much higher intracellular siRNA delivery efficiencies than siRNA alone and Lipofectamine-siRNA complexes. The gene silencing efficiency of mPEG-PLGA-PLL NPs was higher than that of commercially available transfecting agent Lipofectamine while showing no cytotoxicity. Thus, the current study demonstrates that biodegradable NPs of mPEG-PLGA-PLL triblock copolymers can be potentially applied as novel non-viral vectors for improving siRNA delivery and gene silencing.

  4. Biodegradable nanoparticles of mPEG-PLGA-PLL triblock copolymers as novel non-viral vectors for improving siRNA delivery and gene silencing.

    Science.gov (United States)

    Du, Jing; Sun, Ying; Shi, Qiu-Sheng; Liu, Pei-Feng; Zhu, Ming-Jie; Wang, Chun-Hui; Du, Lian-Fang; Duan, You-Rong

    2012-01-01

    Degradation of mRNA by RNA interference is one of the most powerful and specific mechanisms for gene silencing. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of biodegradable nanoparticles (NPs) made from monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-l-lysine (mPEG-PLGA-PLL) triblock copolymers. Various physicochemical properties of mPEG-PLGA-PLL NPs, including morphology, size, surface charge, siRNA encapsulation efficiency, and in vitro release profile of siRNA from NPs, were characterized by scanning electron microscope, particle size and zeta potential analyzer, and high performance liquid chromatography. The levels of siRNA uptake and targeted gene inhibition were detected in human lung cancer SPC-A1-GFP cells stably expressing green fluorescent protein. Examination of the cultured SPC-A1-GFP cells with fluorescent microscope and flow cytometry showed NPs loading Cy3-labeled siRNA had much higher intracellular siRNA delivery efficiencies than siRNA alone and Lipofectamine-siRNA complexes. The gene silencing efficiency of mPEG-PLGA-PLL NPs was higher than that of commercially available transfecting agent Lipofectamine while showing no cytotoxicity. Thus, the current study demonstrates that biodegradable NPs of mPEG-PLGA-PLL triblock copolymers can be potentially applied as novel non-viral vectors for improving siRNA delivery and gene silencing.

  5. Growth inhibition of breast cancer cell line MCF-7 by siRNA silencing of Wilms tumor 1 gene.

    Science.gov (United States)

    Navakanit, Ruxapon; Graidist, Potchanapond; Leeanansaksiri, Wilairat; Dechsukum, Chavaboon

    2007-11-01

    RNA interference (RNAi) is sequence-specific inhibition of gene expression induced by double-stranded RNA. Define the role of Wilms tumor 1 gene (WT1) in breast cancer oncogenesis using RNAi. MCF-7 breast cancer cells, which express a high level of WT1, were transfected with synthetic small interfering RNA (siRNA) targeting WT1 (siRNA(WT1)) resulting in inhibition of WTI expression, as well as growth, in a dose- and time-dependent manner. The minimum concentration of siRNA(WT1) for growth inhibition and WT1 silencing was 25 nM and 50 nM respectively. WT1 expression was completely abolished at 200 nM siRNA,. These data suggest that WTI1is indispensable for the survival of breast cancer MCF-7 cell line.

  6. The ERI-6/7 helicase acts at the first stage of an siRNA amplification pathway that targets recent gene duplications.

    Directory of Open Access Journals (Sweden)

    Sylvia E J Fischer

    2011-11-01

    Full Text Available Endogenous small interfering RNAs (siRNAs are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs of the null mutant. eri-6/7 encodes a helicase homologous to small RNA factors Armitage in Drosophila, SDE3 in Arabidopsis, and Mov10 in humans. Here we show that eri-6/7 mutations cause the loss of 26-nucleotide (nt endogenous siRNAs derived from genes and pseudogenes in oocytes and embryos, as well as deficiencies in somatic 22-nucleotide secondary siRNAs corresponding to the same loci. About 80 genes are eri-6/7 targets that generate the embryonic endogenous siRNAs that silence the corresponding mRNAs. These 80 genes share extensive nucleotide sequence homology and are poorly conserved, suggesting a role for these endogenous siRNAs in silencing of and thereby directing the fate of recently acquired, duplicated genes. Unlike most endogenous siRNAs in C. elegans, eri-6/7-dependent siRNAs require Dicer. We identify that the eri-6/7-dependent siRNAs have a passenger strand that is ∼19 nt and is inset by ∼3-4 nts from both ends of the 26 nt guide siRNA, suggesting non-canonical Dicer processing. Mutations in the Argonaute ERGO-1, which associates with eri-6/7-dependent 26 nt siRNAs, cause passenger strand stabilization, indicating that ERGO-1 is required to separate the siRNA duplex, presumably through endonucleolytic cleavage of the passenger strand. Thus, like several other siRNA-associated Argonautes with a conserved RNaseH motif, ERGO-1 appears to be required for siRNA maturation.

  7. PLGA microspheres encapsulating siRNA.

    Science.gov (United States)

    De Rosa, Giuseppe; Salzano, Giuseppina

    2015-01-01

    The therapeutic use of small interfering RNA (siRNA) represents a new and powerful approach to suppress the expression of pathologically genes. However, biopharmaceutical drawbacks, such as short half-life, poor cellular uptake, and unspecific distribution into the body, hamper the development of siRNA-based therapeutics. Poly(lactide-co-glycolide), (PLGA) microspheres can be a useful tool to overcome these issues. siRNA can be encapsulated into the PLGA microspheres, which protects the loaded nucleic acid against the enzymatic degradation. Moreover, PLGA microspheres can be injected directly into the action site, where the siRNA can be released in controlled manner, thus avoiding the need of frequent invasive administrations. The complete biodegradability of PLGA to monomers easily metabolized by the body, and its approval by FDA and EMA for parenteral administration, assure the safety of this copolymer and do not require the removal of the device after the complete drug release. In chapter, a basic protocol for the preparation of PLGA microspheres encapsulating siRNA is described. This protocol is based on a double emulsion/solvent evaporation technique, a well known and easy to reproduce method. This specific protocol has been developed to encapsulate a siRNA anti-TNFα in PLGA microspheres, and it has been designed and optimized to achieve high siRNA encapsulation efficiency and slow siRNA release in vitro. However, it can be extended also to other siRNA as well as other RNA or DNA-based oligonucleotides (miRNA, antisense, decoy, etc.). Depending on the applications, chemical modifications of the backbone and site-specific modification within the siRNA sequences could be required.

  8. Protection and systemic translocation of siRNA following oral administration of chitosan/siRNA nanoparticles

    DEFF Research Database (Denmark)

    Gonzalez, Borja Ballarin; Dagnæs-Hansen, Frederik; Fenton, Robert A.

    2013-01-01

    , gastrointestinal (GI) deposition, and translocation into peripheral tissue of nonmodified siRNA after oral gavage of chitosan/siRNA nanoparticles in mice. In contrast to naked siRNA, retained structural integrity and deposition in the stomach, proximal and distal small intestine, and colon was observed at 1 and 5...... hours for siRNA within nanoparticles. Furthermore, histological detection of fluorescent siRNA at the apical regions of the intestinal epithelium suggests mucoadhesion provided by chitosan. Detection of intact siRNA in the liver, spleen, and kidney was observed 1 hour after oral gavage, with an organ...

  9. Tumor-targeting multifunctional nanoparticles for siRNA delivery: recent advances in cancer therapy.

    Science.gov (United States)

    Ku, Sook Hee; Kim, Kwangmeyung; Choi, Kuiwon; Kim, Sun Hwa; Kwon, Ick Chan

    2014-08-01

    RNA interference (RNAi) is a naturally occurring regulatory process that controls posttranscriptional gene expression. Small interfering RNA (siRNA), a common form of RNAi-based therapeutics, offers new opportunities for cancer therapy via silencing specific genes, which are associated to cancer progress. However, clinical applications of RNAi-based therapy are still limited due to the easy degradation of siRNA during body circulation and the difficulty in the delivery of siRNA to desired tissues and cells. Thus, there have been many efforts to develop efficient siRNA delivery systems, which protect siRNA from serum nucleases and deliver siRNA to the intracellular region of target cells. Here, the recent advances in siRNA nanocarriers, which possess tumor-targeting ability are reviewed; various nanoparticle systems and their antitumor effects are summarized. The development of multifunctional nanocarriers for theranostics or combinatorial therapy is also discussed.

  10. Construction and Expression of Eukaryotic Expression Vector and Plasmid Expressing siRNA of Human Protection of Telomeres 1

    Institute of Scientific and Technical Information of China (English)

    Di-Nan HUANG; Ying-Hua JIANG; Hou GAN

    2005-01-01

    @@ 1 Introduction The POT1 (protection of telomeres 1 ) protein binds the single-stranded overhang at the ends of chromosomes in diverse eukaryocytes. It is essential for chromosome end-protection in the fission yeast Schizosaccharomyces pombe, and it is involved in regulation of telomere lengthin human cells. Human POT1 had been identified in 2001year. Its amino terminal is highly conservative in eukaryocytes. Since Pot1 can bind internal loops and directly adjacent DNA-binding sites, it is likely to fully coat and protect both G-strand overhangs and the displaced G strand of a T-loop. It also participates in the regulation of telomere maintenance by telomerase or of chromosome by other enzymes. But its biological function and mechanism are still unknown. hPOT eukaryotic expression vector(pcDNA3- hPOT1 ) and 3 kinds of siRNA expression vectors(pmU6-shDNA1, pmU6-shDNA2 and pmU6-shDNA3)had been constructed. And there were overexpression and RNA silence in HeLa cells.

  11. The monitoring of gene functions on a cell-defined siRNA microarray in human bone marrow stromal and U2OS cells

    Directory of Open Access Journals (Sweden)

    Hi Chul Kim

    2016-06-01

    Full Text Available Here, we developed a cell defined siRNA microarray (CDSM for human bone marrow stromal cells (hBMSCs designed to control the culture of cells inside the spot area without reducing the efficiency of siRNA silencing, “Development of a cell-defined siRNA microarray for analysis of gene functionin human bone marrow stromal cells” (Kim et al., 2016 [1]. First, we confirmed that p65 protein inhibition efficiency was maintained when hBMSCs were culture for 7 days on the siRNA spot, and siRNA spot activity remained in spite of long term storage (10 days and 2 months. Additionally, we confirmed p65 protein inhibition in U2OS cells after 48 h reverse transfection.

  12. Novel lipoproteoplex delivers Keap1 siRNA based gene therapy to accelerate diabetic wound healing.

    Science.gov (United States)

    Rabbani, Piul S; Zhou, Anna; Borab, Zachary M; Frezzo, Joseph A; Srivastava, Nikita; More, Haresh T; Rifkin, William J; David, Joshua A; Berens, Samuel J; Chen, Raymond; Hameedi, Sophia; Junejo, Muhammad H; Kim, Camille; Sartor, Rita A; Liu, Che F; Saadeh, Pierre B; Montclare, Jin K; Ceradini, Daniel J

    2017-04-03

    Therapeutics utilizing siRNA are currently limited by the availability of safe and effective delivery systems. Cutaneous diseases, specifically ones with significant genetic components are ideal candidates for topical siRNA based therapy but the anatomical structure of skin presents a considerable hurdle. Here, we optimized a novel liposome and protein hybrid nanoparticle delivery system for the topical treatment of diabetic wounds with severe oxidative stress. We utilized a cationic lipid nanoparticle (CLN) composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the edge activator sodium cholate (NaChol), in a 6:1 ratio of DOTAP:NaChol (DNC). Addition of a cationic engineered supercharged coiled-coil protein (CSP) in a 10:1:1 ratio of DNC:CSP:siRNA produced a stable lipoproteoplex (LPP) nanoparticle, with optimal siRNA complexation, minimal cytotoxicity, and increased transfection efficacy. In a humanized murine diabetic wound healing model, our optimized LPP formulation successfully delivered siRNA targeted against Keap1, key repressor of Nrf2 which is a central regulator of redox mechanisms. Application of LPP complexing siKeap1 restored Nrf2 antioxidant function, accelerated diabetic tissue regeneration, and augmented reduction-oxidation homeostasis in the wound environment. Our topical LPP delivery system can readily be translated into clinical use for the treatment of diabetic wounds and can be extended to other cutaneous diseases with genetic components.

  13. The guanidinium group as a key part of water-soluble polymer carriers for siRNA complexation and protection against degradation.

    Science.gov (United States)

    Tabujew, Ilja; Freidel, Christoph; Krieg, Bettina; Helm, Mark; Koynov, Kaloian; Müllen, Klaus; Peneva, Kalina

    2014-07-01

    Here, the preparation of a novel block copolymer consisting of a statistical copolymer N-(2-hydroxypropyl) methacrylamide-s-N-(3-aminopropyl) methacrylamide and a short terminal 3-guanidinopropyl methacrylamide block is reported. This polymer structure forms neutral but water-soluble nanosized complexes with siRNA. The siRNA block copolymer complexes are first analyzed using agarose gel electrophoresis and their size is determined with fluorescence correlation spectroscopy. The protective properties of the polymer against RNA degradation are investigated by treating the siRNA block copolymer complexes with RNase V1. Heparin competition assays confirm the efficient release of the cargo in vitro. In addition, the utilization of microscale thermophoresis is demonstrated for the determination of the binding strength between a fluorescently labeled polyanion and a polymer molecule.

  14. Investigation of siRNA Nanoparticle Formation Using Mono-Cationic Detergents and Its Use in Gene Silencing in Human HeLa Cells

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Yuma; Suzuki, Ryosuke; Harashima, Hideyoshi, E-mail: harashima@pharm.hokudai.ac.jp [Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812 (Japan)

    2013-11-01

    The focus of recent research has been on the development of siRNA vectors to achieve an innovative gene therapy. Most of the conventional vectors are siRNA nanoparticles complexed with cationic polymers and liposomes, making it difficult to release siRNA. In this study, we report on the use of MCD, a quaternary ammonium salt detergent containing a long aliphatic chain (L-chain) as an siRNA complexation agent using human HeLa cells (a model cancer cell). We prepared siRNA nanoparticles using various MCDs, and measured the diameters and zeta-potentials of the particles. The use of an MCD with a long L-chain resulted in the formation of a positively charged nanoparticle. In contrast, a negatively charged nanoparticle was formed when a MCD with a short L-chain was used. We next evaluated the gene silencing efficiency of the nanoparticles using HeLa cells expressing the luciferase protein. The results showed that the siRNA/MCD nanoparticles showed a higher gene silencing efficiency than Lipofectamine 2000. We also found that the efficiency of gene silencing is a function of the length of the alkyl chain in MCD and zeta-potential of the siRNA/MCD nanoparticles. Such information provides another viewpoint for designing siRNA vectors.

  15. Investigation of siRNA Nanoparticle Formation Using Mono-Cationic Detergents and Its Use in Gene Silencing in Human HeLa Cells

    Directory of Open Access Journals (Sweden)

    Hideyoshi Harashima

    2013-11-01

    Full Text Available The focus of recent research has been on the development of siRNA vectors to achieve an innovative gene therapy. Most of the conventional vectors are siRNA nanoparticles complexed with cationic polymers and liposomes, making it difficult to release siRNA. In this study, we report on the use of MCD, a quaternary ammonium salt detergent containing a long aliphatic chain (L-chain as an siRNA complexation agent using human HeLa cells (a model cancer cell. We prepared siRNA nanoparticles using various MCDs, and measured the diameters and zeta-potentials of the particles. The use of an MCD with a long L-chain resulted in the formation of a positively charged nanoparticle. In contrast, a negatively charged nanoparticle was formed when a MCD with a short L-chain was used. We next evaluated the gene silencing efficiency of the nanoparticles using HeLa cells expressing the luciferase protein. The results showed that the siRNA/MCD nanoparticles showed a higher gene silencing efficiency than Lipofectamine 2000. We also found that the efficiency of gene silencing is a function of the length of the alkyl chain in MCD and zeta-potential of the siRNA/MCD nanoparticles. Such information provides another viewpoint for designing siRNA vectors.

  16. Influence of Cationic Lipid Composition on Gene Silencing Properties of Lipid Nanoparticle Formulations of siRNA in Antigen-Presenting Cells

    Science.gov (United States)

    Basha, Genc; Novobrantseva, Tatiana I; Rosin, Nicole; Tam, Yuen Yi C; Hafez, Ismail M; Wong, Matthew K; Sugo, Tsukasa; Ruda, Vera M; Qin, June; Klebanov, Boris; Ciufolini, Marco; Akinc, Akin; Tam, Ying K; Hope, Michael J; Cullis, Pieter R

    2011-01-01

    Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases. PMID:21971424

  17. Inhibition of TMV multiplication by siRNA constructs against TOM1 and TOM3 genes of Capsicum annuum.

    Science.gov (United States)

    Kumar, Sunil; Dubey, Ashvini Kumar; Karmakar, Ruma; Kini, Kukkundoor Ramachandra; Mathew, Mathew Kuriyan; Prakash, Harischandra Sripathy

    2012-12-01

    The host proteins TOM1 and TOM3 associated with tonoplast membrane are shown to be required for efficient multiplication of Tobamoviruses. In this study, homologous of TOM1 and TOM3 genes were identified in pepper (Capsicum annuum) using specific primers. Their gene sequences have similarity to Nicotiana tabacum NtTOM1 and NtTOM3. Sequence alignment showed that CaTOM1 and CaTOM3 are closely related to TOM1 and TOM3 of N. tabacum and Solanum lycopersicum with 90% and 70% nucleotide sequence identities, respectively. RNA interference approach was used to suppress the TOM1 and TOM3 gene expression which in turn prevented Tobacco mosaic virus replication in tobacco. Nicotiana plants agro-infiltrated with siRNA constructs of TOM1 or TOM3 showed no mosaic or necrotic infection symptoms upon inoculation with TMV. The results indicated that silencing of TOM1 and TOM3 of pepper using the siRNA constructs is an efficient method for generating TMV-resistant plants. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

  18. Anti-HBV efficacy of combined siRNAs targeting viral gene and heat shock cognate 70

    Directory of Open Access Journals (Sweden)

    Bian Zhongqi

    2012-11-01

    Full Text Available Abstract Background Hepatitis B virus (HBV infection is a major health concern with more than two billion individuals currently infected worldwide. Because of the limited effectiveness of existing vaccines and drugs, development of novel antiviral strategies is urgently needed. Heat stress cognate 70 (Hsc70 is an ATP-binding protein of the heat stress protein 70 family. Hsc70 has been found to be required for HBV DNA replication. Here we report, for the first time, that combined siRNAs targeting viral gene and siHsc70 are highly effective in suppressing ongoing HBV expression and replication. Methods We constructed two plasmids (S1 and S2 expressing short hairpin RNAs (shRNAs targeting surface open reading frame of HBV(HBVS and one plasmid expressing shRNA targeting Hsc70 (siHsc70, and we used the EGFP-specific siRNA plasmid (siEGFP as we had previously described. First, we evaluated the gene-silencing efficacy of both shRNAs using an enhanced green fluorescent protein (EGFP reporter system and flow cytometry in HEK293 and T98G cells. Then, the antiviral potencies of HBV-specific siRNA (siHBV in combination with siHsc70 in HepG2.2.15 cells were investigated. Moreover, type I IFN and TNF-α induction were measured by quantitative real-time PCR and ELISA. Results Cotransfection of either S1 or S2 with an EGFP plasmid produced an 80%–90% reduction in EGFP signal relative to the control. This combinational RNAi effectively and specifically inhibited HBV protein, mRNA and HBV DNA, resulting in up to a 3.36 log10 reduction in HBV load in the HepG2.2.15 cell culture supernatants. The combined siRNAs were more potent than siHBV or siHsc70 used separately, and this approach can enhance potency in suppressing ongoing viral gene expression and replication in HepG2.2.15 cells while forestalling escape by mutant HBV. The antiviral synergy of siHBV used in combination with siHsc70 produced no cytotoxicity and induced no production of IFN-α, IFN-β and TNF

  19. Hydrophobic interactions between polymeric carrier and palmitic acid-conjugated siRNA improve PEGylated polyplex stability and enhance in vivo pharmacokinetics and tumor gene silencing.

    Science.gov (United States)

    Sarett, Samantha M; Werfel, Thomas A; Chandra, Irene; Jackson, Meredith A; Kavanaugh, Taylor E; Hattaway, Madison E; Giorgio, Todd D; Duvall, Craig L

    2016-08-01

    Formation of stable, long-circulating siRNA polyplexes is a significant challenge in translation of intravenously-delivered, polymeric RNAi cancer therapies. Here, we report that siRNA hydrophobization through conjugation to palmitic acid (siPA) improves stability, in vivo pharmacokinetics, and tumor gene silencing of PEGylated nanopolyplexes (siPA-NPs) with balanced cationic and hydrophobic content in the core relative to the analogous polyplexes formed with unmodified siRNA, si-NPs. Hydrophobized siPA loaded into the NPs at a lower charge ratio (N(+):P(-)) relative to unmodified siRNA, and siPA-NPs had superior resistance to siRNA cargo unpackaging in comparison to si-NPs upon exposure to the competing polyanion heparin and serum. In vitro, siPA-NPs increased uptake in MDA-MB-231 breast cancer cells (100% positive cells vs. 60% positive cells) but exhibited equivalent silencing of the model gene luciferase relative to si-NPs. In vivo in a murine model, the circulation half-life of intravenously-injected siPA-NPs was double that of si-NPs, resulting in a >2-fold increase in siRNA biodistribution to orthotopic MDA-MB-231 mammary tumors. The increased circulation half-life of siPA-NPs was dependent upon the hydrophobic interactions of the siRNA and the NP core component and not just siRNA hydrophobization, as siPA did not contribute to improved circulation time relative to unmodified siRNA when delivered using polyplexes with a fully cationic core. Intravenous delivery of siPA-NPs also achieved significant silencing of the model gene luciferase in vivo (∼40% at 24 h after one treatment and ∼60% at 48 h after two treatments) in the murine MDA-MB-231 tumor model, while si-NPs only produced a significant silencing effect after two treatments. These data suggest that stabilization of PEGylated siRNA polyplexes through a combination of hydrophobic and electrostatic interactions between siRNA cargo and the polymeric carrier improves in vivo pharmacokinetics and

  20. Cystathionine-γ-lyase gene silencing with siRNA in monocytes/ macrophages attenuates inflammation in cecal ligation and puncture-induced sepsis in the mouse

    Indian Academy of Sciences (India)

    A Badiei; ST Chambers; RR Gaddam; M Bhatia

    2016-03-01

    Hydrogen sulphide is an endogenous inflammatory mediator produced by cystathionine-γ-lyase (CSE) in macrophages. To determine the role of H2S and macrophages in sepsis, we used small interference RNA (siRNA) to target the CSE gene and investigated its effect in a mouse model of sepsis. Cecal ligation puncture (CLP)-induced sepsis is characterized by increased levels of myeloperoxidase (MPO) activity, morphological changes in liver and pro-inflammatory cytokines and chemokines in the liver and lung. SiRNA treatment attenuated inflammation in the liver and lungs of mice following CLP-induced sepsis. Liver MPO activity increased in CLP-induced sepsis and treatment with siRNA significantly reduced this. Similarly, lung MPO activity increased following induction of sepsis with CLP while siRNA treatment significantly reduced MPO activity. Liver and lung cytokine and chemokine levels in CLP-induced sepsis reduced following treatment with siRNA. These findings show a crucial pro-inflammatory role for H2S synthesized by CSE in macrophages in sepsis and suggest CSE gene silencing with siRNA as a potential therapeutic approach for this condition.

  1. beta-catenin siRNA regulation of apoptosis- and angiogenesis-related gene expression in hepatocellular carcinoma cells: potential uses for gene therapy.

    Science.gov (United States)

    Wang, Xin-Hong; Sun, Xun; Meng, Xiang-Wei; Lv, Zhi-Wu; Du, Ya-Ju; Zhu, Yan; Chen, Jing; Kong, De-Xia; Jin, Shi-Zhu

    2010-10-01

    The molecular mechanism responsible for hepatocellular carcinoma (HCC) development remains to be defined although a number of gene pathways have been shown to play an active role, such as Wnt/beta-catenin signaling. In this study, beta-catenin small interfering RNA (siRNA) was designed, synthesized, and transfected into HCC HepG2 cells. RT-PCR and western blot assays were performed to detect expression of altered genes and proteins, and the MTT assay was used to detect cell viability. Our data showed that beta-catenin mRNA and protein expression levels were effectively knocked down by beta-catenin siRNA and subsequently, tumor cell proliferation was significantly suppressed. Flow cytometry assay showed that tumor cells were arrested at the G0/G1 phase of the cell cycles. Molecularly, expression of Smad3, p-caspase-3, and Grp78 protein were upregulated after 72 h of beta-catenin siRNA transfection, whereas expression of TERT, caspase-3, XIAP, MMP-2, MMP-9, VEGF-A, VEGF-c, and bFGF protein were reduced. However, there was no change between the expression of STAT3 and the HSP27 protein following transfection. The results from the current study demonstrated the importance of the Wnt/beta-catenin signaling pathway in regulation of gene expression in HCC. Further studies are required to investigate the role of this pathway in HCC development and targeting of this pathway to control HCC.

  2. Gene silencing with siRNA targeting E6/E7 as a therapeutic intervention in a mouse model of cervical cancer.

    Science.gov (United States)

    Jonson, Amy L; Rogers, Lisa M; Ramakrishnan, Sundaram; Downs, Levi S

    2008-11-01

    Selective silencing of HPV oncogenes using short interfering RNA (siRNA) blocks E6/E7 expression and restores normal p53 and Rb function. Our objective was to determine if siRNA targeting E6/E7 would inhibit the growth of established tumors in a mouse model of cervical cancer. In vitro studies were performed using unique siRNA sequences to confirm their ability to target and reduce E6/E7 mRNA and restore functioning p53. Next, siRNA targeting lamin was injected daily for three days into tumors established from HPV 16 positive CaSki human cervical cancer cells. Immunohistochemistry and branched DNA gene quantification were used to determine distribution and duration of activity of these siRNA. For our therapeutic studies tumors were directly injected with siRNA targeting E6/E7, non-targeting control siRNA, or saline. In preliminary experiments injections were daily or every three days for a total of three doses. A second therapeutic experiment utilized every three day dosing for 35 days. Tumor volume, growth curves and E7 mRNA levels were assessed. The two most active siRNA sequences resulted in a 67% and 71% reduction in E6/E7 mRNA. Fluorescent lamin siRNA was visualized up to 120 h after the initial tumor injection and was evenly distributed throughout the tumors. IHC showed lamin expression to be inhibited by 68% and 75% when compared to controls at 54 and 120 h respectively. In our preliminary therapeutic intervention experiments there was no significant difference in tumor growth between the treatment groups when mice were treated with three daily injections (p=0.41). However, when treated every third day for three injections final tumor volume was less in animals injected with siRNA sequences A (78% reduction; pE6/E7 mRNA. Extended treatment with siRNA completely or nearly eradicated tumors in 70% of the animals. Therapeutic siRNA targeting E6/E7 significantly inhibits tumor growth in this mouse model of cervical cancer. Further investigation is needed to

  3. Gene Knockdown in Human Rhinovirus 1B Using 2'-OMe-modified siRNAs Results in the Reactivation of the Interferon Response.

    Science.gov (United States)

    Xie, Guang Cheng; Zhang, Qing; Pang, Li Li; Li, Dan Di; Jin, Miao; Li, Hui Ying; Xu, Zi Qian; Kong, Xiang Yu; Wang, Hong; Lu, Shan; Duan, Zhao Jun

    2016-02-01

    The aim of this study was to investigate the knockdown efficiency of 2'-O-methylated (2'-OMe)-modified small interfering RNAs (siRNAs) on human rhinovirus 1B (HRV1B) replication and the interferon response. Thus, 24 2'-OMe-modified siRNAs were designed to target HRV1B. The RNA levels of HRV1B, Toll-like receptor 3, melanoma differentiation-associated gene 5, retinoic acid inducible gene-I, and interferons were determined in HRV1B-infected HeLa and BEAS-2B epithelial cells transfected with 2'-OMe-modified siRNAs. The results revealed that all 2'-OMe-modified siRNAs interfered with the replication of HRV1B in a cell-specific and transfection efficiency-dependent manner. Viral activation of Toll-like receptor 3, melanoma differentiation-associated gene 5, retinoic acid inducible gene-I, and the interferon response was detected. In conclusion, the 2'-OMe-modified siRNAs used in this study could interfere with HRV1B replication, possibly leading to the reactivation of the interferon response.

  4. Enhancement of allele discrimination by introduction of nucleotide mismatches into siRNA in allele-specific gene silencing by RNAi.

    Directory of Open Access Journals (Sweden)

    Yusuke Ohnishi

    Full Text Available Allele-specific gene silencing by RNA interference (RNAi is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi, the design and assessment of small interfering RNA (siRNA duplexes conferring ASP-RNAi is vital; however, it is also difficult. In a previous study, we developed an assay system to assess ASP-RNAi with mutant and wild-type reporter alleles encoding the Photinus and Renilla luciferase genes. In line with experiments using the system, we realized that it is necessary and important to enhance allele discrimination between mutant and corresponding wild-type alleles. Here, we describe the improvement of ASP-RNAi against mutant alleles carrying single nucleotide variations by introducing base substitutions into siRNA sequences, where original variations are present in the central position. Artificially mismatched siRNAs or short-hairpin RNAs (shRNAs against mutant alleles of the human Prion Protein (PRNP gene, which appear to be associated with susceptibility to prion diseases, were examined using this assessment system. The data indicates that introduction of a one-base mismatch into the siRNAs and shRNAs was able to enhance discrimination between the mutant and wild-type alleles. Interestingly, the introduced mismatches that conferred marked improvement in ASP-RNAi, appeared to be largely present in the guide siRNA elements, corresponding to the 'seed region' of microRNAs. Due to the essential role of the 'seed region' of microRNAs in their association with target RNAs, it is conceivable that disruption of the base-pairing interactions in the corresponding seed region, as well as the central position (involved in cleavage of target RNAs, of guide siRNA elements could influence allele discrimination. In addition, we also suggest that nucleotide mismatches at the 3'-ends of sense

  5. PSD95 Gene Specific siRNAs Attenuate Neuropathic Pain through Modulating Neuron Sensibility and Postsynaptic CaMKⅡα Phosphorylation

    Institute of Scientific and Technical Information of China (English)

    Le Shen; Xu Li; Nen Chen; Li Xu; Wei Liu; Xue-rong Yu; Yu-guang Huang

    2011-01-01

    Objective To observe the effects of PSD95 gene specific siRNAs on neuropathic pain relief,neuron viability,and postsynaptic calcium/calmodulin-dependent protein kinase Ⅱα (CaMKⅡα) phosphorylation in vitro and in vivo.Methods Gene-specific siRNAs of rat PSD95 were synthesized chemically for transfection.Adult male Sprague-Dawley (SD) rats were randomly divided into 3 groups:naive group (n=6),sham group (n=6),and sciatic nerve chronic constriction injury (CCI) group (n=24).The CCI group was further divided into 4 groups (n=6 in each group),which were pretreated with normal saline,transfection vehicle,negative control siRNAs,and PSD95 gene specific siRNAs respectively.All the subgroups received corresponding agents intrathecally for 3 days,started one day before the CCI of sciatic nerve.Both mechanical allodynia and thermal hyperalgesia were measured on post-operative day 3 and 7.PSD95 gene silenced NG108-15 cells were further stimulated by glutamate,with the cell viability and the expression/phosphorylation of CaMKⅡα measured by MTT cell proliferation assay andWestern blot,respectively.Results The siRNAs decreased PSD95 mRNA level significantly both in vivo and in vitro.Neuropathic pain rats pretreated with PSD95 gene specific siRNAs exhibited significant elevation in the mechanical withdrawal threshold and paw withdrawal thermal latency,without affecting the baseline nociception.PSD95 gene silencing enhanced neuronal tolerance against the glutamate excitotoxicity,meanwhile the phosphorylation of CaMKⅡαThr286 was attenuated.Conclusion Pre-emptive administration of PSD95 gene specific siRNAs may attenuate the central sensitization CaMKⅡα-related signaling cascades,leading to the relief of neuropathic pain.

  6. Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro - A quantitative study

    NARCIS (Netherlands)

    Asgeirsdottir, Sigridur A.; Talman, Eduard G.; de Graaf, Inge A.; Kamps, Jan A. A. M.; Satchell, Simon C.; Mathieson, Peter W.; Ruiters, Marcel H. J.; Molema, Grietje

    2010-01-01

    Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amph

  7. Preparation and characterization of polymeric nanoparticles for siRNA delivery to down-regulate the expressions of exogenous and endogenous target genes.

    Science.gov (United States)

    Huang, Wei; Lv, Ming; Gao, Zhong-Gao; Jin, Ming-Ji; Xu, Yuan-Ji; Yu, Xiao-Dan; Jin, Zhe-Hu; Yin, Xue-Zhe

    2012-08-01

    Gene silencing induced by RNA interference using small interfering RNA (siRNA) provides a promising therapeutic approach for cancers. However, the lack of siRNA delivery vector has limited the development of siRNA therapy. The purpose of this study was to use the novel copolymer (mPEG5k-PCL1.2k)1.4-g-PEl10k to prepare siRNA-loaded nanoparticles for siRNA delivery. The results suggested that (mPEG5k-PCL1.2k)1.4-g-PEl10k could load siRNA to form nanoparticles with particle size less than 200 nm in a narrow distribution. Moreover, a certain density of positive charge existed onto the surfaces of nanoparticles. MTT assay results demonstrated that (mPEG5k-PCL1.2k)1.4-g-PEl10k/siRNA nanoparticles showed very low cytotoxicity. The gene silencing efficiency of (mPEG5k-PCL1.2k)1.4-g-PEl10k/siRNA nanoparticles was investigated through luciferase reporter gene assays. The expression of exogenous luciferase gene was significantly downregulated at a range of N/P ratio from 50 to 125, and was maximally inhibited at the N/P ratio of 125 with 54% and 59% reduction in MCF-7 and HepG2 cells, respectively. In the 4T1-luc cell line expressing luciferase stably, the silencing of endogenous luciferase gene also has a similar overall profile with maximal 54% reduction of luciferase expression. These results suggested that (mPEG5k-PCL1.2k)1.4-g-PEI10k/SiRNA nanoparticles could serve as a kind of highly efficient siRNA delivery system for down-regulating the expression of exogenous and endogenous target genes.

  8. Cationic Lipid-Nucleic Acid Complexes for Gene Delivery And Silencing: Pathways And Mechanisms for Plasmid Dna And Sirna

    Energy Technology Data Exchange (ETDEWEB)

    Ewert, K.K.; Zidovska, A.; Ahmad, A.; Bouxsein, N.F.; Evans, H.M.; McAllister, C.S.; Samuel, C.E.; Safinya, C.R.; /SLAC

    2012-07-17

    Motivated by the promises of gene therapy, there is great interest in developing non-viral lipid-based vectors for therapeutic applications due to their low immunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. In fact, cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids (NAs) currently used in gene therapy clinical trials worldwide. These vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. This reflects the currently poor understanding of transfection-related mechanisms at the molecular and self-assembled levels, including a lack of knowledge about interactions between membranes and double stranded NAs and between CL-NA complexes and cellular components. In this review we describe our recent efforts to improve the mechanistic understanding of transfection by CL-NA complexes, which will help to design optimal lipid-based carriers of DNA and siRNA for therapeutic gene delivery and gene silencing.

  9. Highly potent and specific siRNAs against E6 or E7 genes of HPV16- or HPV18-infected cervical cancers.

    Science.gov (United States)

    Chang, J T-C; Kuo, T-F; Chen, Y-J; Chiu, C-C; Lu, Y-C; Li, H-F; Shen, C-R; Cheng, A-J

    2010-12-01

    Infection with high-risk types (type 16 or type 18) of human papillomaviruses (HPVs) increases a patient's risk of cervical cancer. Given the importance of the cervix and the severe side effects resulting from traditional cancer therapies, this study aimed to achieve targeted inhibition of viral oncogenes in tumor cells using small interfering RNAs (siRNA). To accomplish this, we developed nine siRNAs against either the E6 or E7 genes of HPV-16 or HPV-18 in several combinations, yielding siRNAs targeting 16E6, 16E7, 18E6 and 18E7. We measured the effectiveness of the siRNAs by examining E6 or E7 mRNA expression after transfection of the siRNAs into HPV-positive CaSki (HPV-16) or HeLa (HPV-18) cell lines. We found that the HPV-siRNAs significantly reduced cell growth and colony formation in both cell lines. Flow cytometry analysis revealed a significant increase in apoptosis. The siRNAs had no effect on cell growth, colony formation or apoptosis in HPV-negative C33A cells, demonstrating a lack of off-target effects. In addition, an in vivo xenograft study showed that intra-tumoral injection of the siRNAs reduced tumor growth in BALB/c nude mice. In conclusion, we have developed highly specific and potent HPV-siRNAs that successfully suppress tumor growth and induce apoptosis in HPV-positive cervical cancer cells. siRNA treatment has potential for further development as an adjuvant therapy for cervical cancer.

  10. Gene silencing with siRNA targeting E6/E7 as a therapeutic intervention against head and neck cancer-containing HPV16 cell lines.

    Science.gov (United States)

    Adhim, Zainal; Otsuki, Naoki; Kitamoto, Junko; Morishita, Naoya; Kawabata, Masato; Shirakawa, Toshiro; Nibu, Ken-Ichi

    2013-07-01

    Our results indicate that siRNA E6 and/or E7 may have potential as a gene-specific therapy for human papillomavirus (HPV) type 16 (HPV16)-related squamous cell carcinoma of the head and neck (HNSCC). To evaluate the effectiveness of siRNA targeting E6 and/or E7 on the in vitro and in vivo growth suppression of HPV16-related HNSCC. HPV16-related HNSCC (UM-SCC47) cell lines were used for the present study. Expression of HPV viral oncogenes E6 and/or E7 and their cellular targets, p53 and pRb, was evaluated by real-time PCR, Western blotting, and immunofluorescence staining. To study the effect of siRNA on tumor growth in vivo, we developed animal models. Representative tumors harvested from each group were processed for apoptosis analyses (TUNEL assay) and immunofluorescence staining for p53 and pRb. E6 and E7 oncogenes of HPV16 were down-regulated by E6 and/or E7 targeting siRNAs, respectively. The expression of p53 and pRb proteins in both the E6 siRNA group and E7 siRNA group was up-regulated compared with those of control groups. The cellular proliferation and apoptosis indexes of E6 and/or E7 siRNA groups were higher than those of controls. In vivo studies showed significant inhibitory effect of E6 and/or E7 siRNA compared with those of control groups, which was consistent with in vitro studies.

  11. Therapeutic potential of inhibiting ABCE1 and eRF3 genes via siRNA strategy using chitosan nanoparticles in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cengiz, Bagdat Burcu; Asik, Mehmet Dogan [Hacettepe University, Nanotechnology and Nanomedicine Division (Turkey); Kara, Goknur [Hacettepe University, Biochemistry Division, Chemistry Department (Turkey); Turk, Mustafa [Kirikkale University, Bioengineering Department (Turkey); Denkbas, Emir Baki, E-mail: denkbas@hacettepe.edu.tr [Hacettepe University, Biochemistry Division, Chemistry Department (Turkey)

    2015-04-15

    In recent years, targeted cancer therapy strategies have begun to take the place of the conventional treatments. Inhibition of the specific genes, involved in cancer progress, via small interfering RNA (siRNA) has become one of the promising therapeutic approaches for cancer therapy. However, due to rapid nuclease degradation and poor cellular uptake of siRNA, a suitable carrier for siRNA penetration inside the cells is required. We used chitosan nanoparticles (CS-NPs) to efficiently deliver ATP-binding casette E1 (ABCE1) and eukaryotic release factor 3 (eRF3)-targeting siRNAs, individually and together, to reduce the proliferation and induce the apoptosis of breast cancer cells. The CS-NPs were generated by ionic gelation method using tripolyphosphate (TPP) as a crosslinker. Nanoparticles (NPs) were obtained with diameters ranging between 110 and 230 nm and the zeta potential of approximately 27 mV optimizing the solution pH to 4.5 and CS/TPP mass ratio to 3:1. Loading efficiencies of 98.69 % ± 0.051 and 98.83 % ± 0.047 were achieved when ABCE1 siRNA and eRF3 siRNA were entrapped into the NPs, respectively. Cell proliferation assay demonstrated that siRNA-loaded CS-NPs were more effective on cancer cells when compared to siRNAs without CS-NPs. Parallel results were also obtained by apoptosis/necrosis, double-staining analysis. Within our study, the potency of ABCE1 and eRF3 siRNAs were shown for the first time with this kind of polymeric delivery system. The results also indicated that ABCE1 and eRF3, important molecules in protein synthesis, could serve as effective targets to inhibit the cancer cells.

  12. Inhibition of vascular endothelial growth factor gene expression by T7-siRNAs in cultured human retinal pigment epithelial cells

    Institute of Scientific and Technical Information of China (English)

    LI Guang-yu; FAN Bin; WU Ya-zhen; WANG Xin-rui; WANG Yao-hui; WU Jia-xiang

    2005-01-01

    Background Retinal pigment epithelial (RPE) cells play an important role in the occurrence of choroidal neovascularization (CNV). Vascular endothelial growth factor (VEGF) as a positive regulatory growth factor is produced by the RPE in an autocrine or paracrine manner, promoting CNV development. Duplexes of 21 nt RNAs, known as short interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference when introduced into mammalian cells. We searched for an efficient siRNA to interfere with VEGF expression in RPE cells and shed light on the treatment of CNV.Methods Human primary RPE (hRPE) cells were cultured and identified. Three pairs of siRNAs were designed according to the sequence of VEGF 1-5 extrons and synthesized by T7 RNA polymerase transcription in vitro. To evaluate the inhibitory activity of T7-siRNAs, hRPE cells were transfected via siPORT Amine. The interfering effect of T7-siRNAs in hRPE cells was examined by semiquantitative reverse transcription-polymerase chain reaction and immunofluorescence. Results Three pairs of T7-siRNAs synthesized by in vitro transcription with T7 RNA polymerase suppressed VEGF gene expression with efficiency from 65% to 90%. T7-siRNA (B), targeted region at 207 nt to 228 nt and double stranded for 21 nt with 2 nt UU 3' overhangs, was the most effective sequence tested for inhibition of VEGF expression in hRPE cells. Compared with nontransfected cells, the mean fluorescence in hRPE cells transfected with T7-sRNAs was significantly less (P<0.01). siRNA with a single-base mismatch and ssRNA(+) did not show suppressing effect. Furthermore, it was found that siRNAs had a dose dependent inhibitory effect (5 to 10 pmol).Conclusion T7-siRNA can effectively and specifically suppress VEGF expression in hRPE cells and may be a new way to treat CNV.

  13. Porous silicon microparticles for delivery of siRNA therapeutics.

    Science.gov (United States)

    Shen, Jianliang; Wu, Xiaoyan; Lee, Yeonju; Wolfram, Joy; Yang, Zhizhou; Mao, Zong-Wan; Ferrari, Mauro; Shen, Haifa

    2015-01-15

    Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

  14. Chitosan hydrogel as siRNA vector for prolonged gene silencing

    National Research Council Canada - National Science Library

    Ma, Zhiwei; Yang, Chuanxu; Song, Wen; Wang, Qintao; Kjems, Jørgen; Gao, Shan

    2014-01-01

    .... To investigate the possibility for protecting the loss of alveolar bone in periodontal diseases, a RNAi-based therapeutic strategy is applied for silencing RANK signaling using thermosensitive...

  15. Unique Gene-Silencing and Structural Properties of 2;#8242;-Fluoro-Modified siRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Manoharan, Muthiah; Akinc, Akin; Pandey, Rajendra K.; Qin, June; Hadwiger, Philipp; John, Matthias; Mills, Kathy; Charisse, Klaus; Maier, Martin A.; Nechev, Lubomir; Greene, Emily M.; Pallan, Pradeep S.; Rozners, Eriks; Rajeev, Kallanthottathil G.; Egli, Martin (Binghamton); (Alnylam Pharm.); (Vanderbilt)

    2015-10-15

    With little or no negative impact on the activity of small interfering RNAs (siRNAs), regardless of the number of modifications or the positions within the strand, the 2'-deoxy-2'-fluoro (2'-F) modification is unique. Furthermore, the 2'-F-modified siRNA (see crystal structure) was thermodynamically more stable and more nuclease-resistant than the parent siRNA, and produced no immunostimulatory response.

  16. Construction of Ang2-siRNA chitosan magnetic nanoparticles and the effect on Ang2 gene expression in human malignant melanoma cells

    Science.gov (United States)

    LIU, ZHAO-LIANG; YOU, CAI-LIAN; WANG, BIAO; LIN, JIAN-HONG; HU, XUE-FENG; SHAN, XIU-YING; WANG, MEI-SHUI; ZHENG, HOU-BING; ZHANG, YAN-DING

    2016-01-01

    The aim of the present study was to construct angiopoietin-2 (Ang2)-small interfering (si)RNA chitosan magnetic nanoparticles and to observe the interference effects of the nanoparticles on the expression of the Ang2 gene in human malignant melanoma cells. Ang2-siRNA chitosan magnetic nanoparticles were constructed and transfected into human malignant melanoma cells in vitro. Red fluorescent protein expression was observed, and the transfection efficiency was analyzed. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to assess the inhibition efficiency of Ang2 gene expression. Ang2-siRNA chitosan magnetic nanoparticles were successfully constructed, and at a mass ratio of plasmid to magnetic chitosan nanoparticles of 1:100, the transfection efficiency into human malignant melanoma cells was the highest of the ratios assessed, reaching 61.17%. RT-qPCR analysis showed that the magnetic chitosan nanoparticles effectively inhibited Ang2 gene expression in cells, and the inhibition efficiency reached 59.56% (P<0.05). Ang2-siRNA chitosan magnetic nanoparticles were successfully constructed. The in vitro studies showed that the nanoparticles inhibited Ang2 gene expression in human malignant melanoma tumor cells, which laid the foundation and provided experimental evidence for additional future in vivo studies of intervention targeting malignant melanoma tumor growth in nude mice. PMID:27313729

  17. siRNAs targeting PB2 and NP genes potentially inhibit replication of Highly Pathogenic H5N1 Avian Influenza Virus.

    Science.gov (United States)

    Behera, Padmanava; Nagarajan, Shanmugasundaram; Murugkar, Harshad V; Kalaiyarasu, Semmannan; Prakash, Anil; Gothalwal, Ragini; Dubey, Shiv Chandra; Kulkarni, Diwakar D; Tosh, Chakradhar

    2015-06-01

    Highly Pathogenic Avian Influenza (HPAI) H5N1 virus is a threat to animal and public health worldwide. Till date, the H5N1 virus has claimed 402 human lives, with a mortality rate of 58 percent and has caused the death or culling of millions of poultry since 2003. In this study, we have designed three siRNAs (PB2-2235, PB2-479 and NP-865) targeting PB2 and NP genes of avian influenza virus and evaluated their potential, measured by hemagglutination (HA), plaque reduction and Real time RT-PCR assay, in inhibiting H5N1 virus (A/chicken/Navapur/7972/2006) replication in MDCK cells. The siRNAs caused 8- to 16-fold reduction in virus HA titers at 24 h after challenged with 100TCID50 of virus. Among these siRNAs, PB2-2235 offered the highest inhibition of virus replication with 16-fold reduction in virus HA titer, 80 percent reduction in viral plaque counts and 94 percent inhibition in expression of specific RNA at 24 h. The other two siRNAs had 68-73 percent and 87-88 percent reduction in viral plaque counts and RNA copy number, respectively. The effect of siRNA on H5N1 virus replication continued till 48h (maximum observation period). These findings suggest that PB2-2235 could efficiently inhibit HPAI H5N1 virus replication.

  18. Silencing of the HER2/neu Gene by siRNA Inhibits Proliferation and Induces Apoptosis in HER2/neu-Overexpressing Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Timo Faltus

    2004-11-01

    Full Text Available In eukaryotes, double-stranded (ds RNA induces sequence-specific inhibition of gene expression referred to as RNA interference (RNAi. We exploited RNAi to define the role of HER2/neu in the neoplastic proliferation of human breast cancer cells. We transfected SK-BR-3, BT-474, MCF-7, and MDA-MB-468 breast cancer cells with short interfering RNA (siRNA targeted against human HER2/neu and analyzed the specific inhibition of HER2/neu expression by Northern and Western blots. Transfection with HER2/neu-specific siRNA resulted in a sequence-specific decrease in HER2/neu mRNA and protein levels. Moreover, transfection with HER2/neu siRNA caused cell cycle arrest at G0/G1 in the breast cancer cell lines SKBR-3 and BT-474, consistent with a powerful RNA silencing effect. siRNA treatment resulted in an antiproliferative and apoptotic response in cells overexpressing HER2/neu, but had no influence in cells with almost no expression of HER2/neu proteins like MDA-MB-468 cells. These data indicate that HER2/neu function is essential for the proliferation of HER2/neuoverexpressing breast cancer cells. Our observations suggest that siRNA targeted against human HER2/neu may be valuable tools as anti proliferative agents that display activity against neoplastic cells at very low doses.

  19. siRNAs targeting PB2 and NP genes potentially inhibit replication of Highly Pathogenic H5N1 Avian Influenza Virus

    Indian Academy of Sciences (India)

    Padmanava Behera; Shanmugasundaram Nagarajan; Harshad V Murugkar; Semmannan Kalaiyarasu; Anil Prakash; Ragini Gothalwal; Shiv Chandra Dubey; Diwakar D Kulkarni; Chakradhar Tosh

    2015-06-01

    Highly Pathogenic Avian Influenza (HPAI) H5N1 virus is a threat to animal and public health worldwide. Till date, the H5N1 virus has claimed 402 human lives, with a mortality rate of 58% and has caused the death or culling of millions of poultry since 2003. In this study, we have designed three siRNAs (PB2-2235, PB2-479 and NP-865) targeting PB2 and NP genes of avian influenza virus and evaluated their potential, measured by hemagglutination (HA), plaque reduction and Real time RT-PCR assay, in inhibiting H5N1 virus (A/chicken/Navapur/7972/2006) replication in MDCK cells. The siRNAs caused 8- to 16-fold reduction in virus HA titers at 24 h after challenged with 100TCID50 of virus. Among these siRNAs, PB2-2235 offered the highest inhibition of virus replication with 16-fold reduction in virus HA titer, 80% reduction in viral plaque counts and 94% inhibition in expression of specific RNA at 24 h. The other two siRNAs had 68–73% and 87–88% reduction in viral plaque counts and RNA copy number, respectively. The effect of siRNA on H5N1 virus replication continued till 48h (maximum observation period). These findings suggest that PB2-2235 could efficiently inhibit HPAI H5N1 virus replication.

  20. Strategies and advances in nanomedicine for targeted siRNA delivery.

    Science.gov (United States)

    Nimesh, Surendra; Gupta, Nidhi; Chandra, Ramesh

    2011-06-01

    siRNA are a rapidly emerging class of new therapeutic molecules for the treatment of inherited and acquired diseases. However, poor cellular uptake and instability in physiological conditions limits its therapeutic potential, hence a need to develop a delivery system that can protect and efficiently transport siRNA to the target cells has arisen. Nanoparticles have been proposed as suitable delivery vectors with reduced cytotoxicity and enhanced efficacy. These delivery vectors form condensed complexes with siRNA which, in turn, provides protection to siRNA against enzymatic degradation and further leads to tissue and cellular targeting. Nanoparticles derived from polymers, such as chitosan and polyethylenimine have found numerous applications owing to ease of manipulation, high stability, low cost and high gene carrying capability. This article focuses on various aspects of nanomedicine based siRNA delivery with emphasis on targeted delivery to tumors.

  1. [Screening efficient siRNAs in vitro as the candidate genes for chicken anti-avian influenza virus H5N1 breeding].

    Science.gov (United States)

    Zhang, P; Wang, J G; Wan, J G; Liu, W Q

    2010-01-01

    The frequent disease outbreaks caused by avian influenza virus not only affect the poultry industry but also pose a threat to human safety. To address the problem, RNA interference (RNAi) has recently been widely used as a potential antiviral approach. Transgenesis in combination with RNAi to specifically inhibit avian enza virus gene expression has been proposed to make chickens resistant to the infection. For the transgenic breeding, screening in vitro efficient siRNAs as the candidate genes is one of the most important tasks. Here, we combined an online search tool and a series of bioinformatics programs with a set of rules for designing siRNAs targeted towards different mRNA regions of H5N1 avian influenza virus. Five rational siRNAs were chosen by this method, five U6 promoter-driven shRNA expression plasmids containing the siRNA genes were constructed and used for producing stably transfected MDCK cells. The data obtained by virus titration, IFA, PI-stained flow cytometry, real-time quantitative RT-PCR, and DAS-ELISA analyses showed that all five stably transfected cell lines we re resistant to virusreplication when exposed to 100 CCID50 of avian influenza virus H5N1. Finally, most effective plasmids (pSi-604i and pSi-1597i) as the candidates for making the transgenic chickens were chosen. These findings provide baseline information on use of RNAi technique for breeding transgenic chickens resistant to avian influenza virus.

  2. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation

    OpenAIRE

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-01-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21–22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, di...

  3. Lentivirus vectors construction of SiRNA targeting interferenceGPC3 gene and its biological effects on liver cancer cell lines Huh-7

    Institute of Scientific and Technical Information of China (English)

    Chang-Jiang Lei; Chun Yao; Qing-Yun Pan; Hao-Cheng Long; Lei Li; Shu-Ping Zheng; Cheng Zeng; Jian-Bin Huang

    2014-01-01

    Objective:To buildGPC3 gene short hairpin interferenceRNA(shRNA) slow virus vector, observe expression ofHuh-7GPC3 gene in human liver cell line proliferation apoptosis and the effect ofGPC3 gene influencing on liver cancer cell growth, and provide theoretical basis for gene therapy of liver cancer.Methods:Hepatocellular carcinoma cell lineHuh-7 was transfected by aRNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitativePCR.TargetedGPC3 gene sequences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell lines of siRNA were screened and established with the help of liposomes(lipofectamineTM2000) as carrier transfection of human liver cell lines.In order to validate siRNA interference efficiency,GPC3 siRNA mRNA expression was detected after transfection by usingRT-PCR andWestern blot.The absorbance value of the cells of blank group, untransfection group and transfection group, the cell cycle and cell apoptosis were calculated, and effects ofGPC3 gene onHuh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell linesHuh-7,GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructed successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linesHuh-7 can obviously inhibitGPC3 mRNA expression level.Conclusions:The targetedGPC3 siRNA can effectively inhibit the expression ofGPC3.

  4. Systemic siRNA Delivery via Peptide-Tagged Polymeric Nanoparticles, Targeting PLK1 Gene in a Mouse Xenograft Model of Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Meenakshi Malhotra

    2013-01-01

    Full Text Available Polymeric nanoparticles were developed from a series of chemical reactions using chitosan, polyethylene glycol, and a cell-targeting peptide (CP15. The nanoparticles were complexed with PLK1-siRNA. The optimal siRNA loading was achieved at an N : P ratio of 129.2 yielding a nanoparticle size of >200 nm. These nanoparticles were delivered intraperitoneally and tested for efficient delivery, cytotoxicity, and biodistribution in a mouse xenograft model of colorectal cancer. Both unmodified and modified chitosan nanoparticles showed enhanced accumulation at the tumor site. However, the modified chitosan nanoparticles showed considerably, less distribution in other organs. The relative gene expression as evaluated showed efficient delivery of PLK1-siRNA (0.5 mg/kg with 50.7±19.5% knockdown (P=0.031 of PLK1 gene. The in vivo data reveals no systemic toxicity in the animals, when tested for systemic inflammation and liver toxicity. These results indicate a potential of using peptide-tagged nanoparticles for systemic delivery of siRNA at the targeted tumor site.

  5. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation.

    Science.gov (United States)

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-06-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21-22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, displayed pronounced reduction in MYMV DNA accumulation. Thus, silencing of the TrAP gene, a suppressor of gene silencing, emerged as an effective strategy to control MYMV.

  6. Preparation and evaluation of nanoparticles loading plasmid DNAs inserted with siRNA fragments targeting c-Myc gene.

    Science.gov (United States)

    Ma, Tao; Jiang, Jin-Ling; Liu, Ying; Ye, Zheng-Bao; Zhang, Jun

    2014-09-01

    c-Myc plays a key role in glioma cancer stem cell maintenance. A drug delivery system, nanoparticles loading plasmid DNAs inserted with siRNA fragments targeting c-Myc gene (NPs-c-Myc-siRNA-pDNAs), for the treatment of glioma, has not previously been reported. NPs-c-Myc-siRNA-pDNAs were prepared and evaluated in vitro. Three kinds of c-Myc-siRNA fragments were separately synthesized and linked with empty siRNA expression vectors in the mole ratio of 3:1 by T4 DNA ligase. The linked products were then separately transfected into Escherichia coli. DH5α followed by extraction with Endofree plasmid Mega kit (Qiagen, Hilden, Germany) obtained c-Myc-siRNA-pDNAs. Finally, the recombinant c-Myc-siRNA3-pDNAs, generating the highest transfection efficiency and the greatest apoptotic ability, were chosen for encapsulation into NPs by the double-emulsion solvent-evaporation procedure, followed by stability, transfection efficiency, as well as qualitative and quantitative apoptosis evaluation. NPs-c-Myc-siRNA3-pDNAs were obtained with spherical shape in uniform size below 150 nm, with the zeta potential about -18 mV, the encapsulation efficiency and loading capacity as 76.3 ± 5.4% and 1.91 ± 0.06%, respectively. The stability results showed that c-Myc-siRNA3-pDNAs remained structurally and functionally stable after encapsulated into NPs, and NPs could prevent the loaded c-Myc-siRNA3-pDNAs from DNase degradation. The transfection efficiency of NPs-c-Myc-siRNA3-pDNAs was proven to be positive. Furthermore, NPs-c-Myc-siRNA3-pDNAs produced significant apoptosis with the apoptotic rate at 24.77 ± 5.39% and early apoptosis cells observed. Methoxy-poly-(ethylene-glycol)-poly-(lactide-co-glycolide) nanoparticles (MPEG-PLGA-NPs) are potential delivery carriers for c-Myc-siRNA3-pDNAs.

  7. Construction of Ang2-siRNA chitosan magnetic nanoparticles and the effect on Ang2 gene expression in human malignant melanoma cells.

    Science.gov (United States)

    Liu, Zhao-Liang; You, Cai-Lian; Wang, Biao; Lin, Jian-Hong; Hu, Xue-Feng; Shan, Xiu-Ying; Wang, Mei-Shui; Zheng, Hou-Bing; Zhang, Yan-Ding

    2016-06-01

    The aim of the present study was to construct angiopoietin-2 (Ang2)-small interfering (si)RNA chitosan magnetic nanoparticles and to observe the interference effects of the nanoparticles on the expression of the Ang2 gene in human malignant melanoma cells. Ang2-siRNA chitosan magnetic nanoparticles were constructed and transfected into human malignant melanoma cells in vitro. Red fluorescent protein expression was observed, and the transfection efficiency was analyzed. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to assess the inhibition efficiency of Ang2 gene expression. Ang2-siRNA chitosan magnetic nanoparticles were successfully constructed, and at a mass ratio of plasmid to magnetic chitosan nanoparticles of 1:100, the transfection efficiency into human malignant melanoma cells was the highest of the ratios assessed, reaching 61.17%. RT-qPCR analysis showed that the magnetic chitosan nanoparticles effectively inhibited Ang2 gene expression in cells, and the inhibition efficiency reached 59.56% (Pconstructed. The in vitro studies showed that the nanoparticles inhibited Ang2 gene expression in human malignant melanoma tumor cells, which laid the foundation and provided experimental evidence for additional future in vivo studies of intervention targeting malignant melanoma tumor growth in nude mice.

  8. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.

    Science.gov (United States)

    Matsuda, Shigeo; Keiser, Kristofer; Nair, Jayaprakash K; Charisse, Klaus; Manoharan, Rajar M; Kretschmer, Philip; Peng, Chang G; V Kel'in, Alexander; Kandasamy, Pachamuthu; Willoughby, Jennifer L S; Liebow, Abigail; Querbes, William; Yucius, Kristina; Nguyen, Tuyen; Milstein, Stuart; Maier, Martin A; Rajeev, Kallanthottathil G; Manoharan, Muthiah

    2015-05-15

    Asialoglycoprotein receptor (ASGPR) mediated delivery of triantennary N-acetylgalactosamine (GalNAc) conjugated short interfering RNAs (siRNAs) to hepatocytes is a promising paradigm for RNAi therapeutics. Robust and durable gene silencing upon subcutaneous administration at therapeutically acceptable dose levels resulted in the advancement of GalNAc-conjugated oligonucleotide-based drugs into preclinical and clinical developments. To systematically evaluate the effect of display and positioning of the GalNAc moiety within the siRNA duplex on ASGPR binding and RNAi activity, nucleotides carrying monovalent GalNAc were designed. Evaluation of clustered and dispersed incorporation of GalNAc units to the sense (S) strand indicated that sugar proximity is critical for ASGPR recognition, and location of the clustered ligand impacts the intrinsic potency of the siRNA. An array of nucleosidic GalNAc monomers resembling a trivalent ligand at or near the 3' end of the S strand retained in vitro and in vivo siRNA activity, similar to the parent conjugate design. This work demonstrates the utility of simple, nucleotide-based, cost-effective siRNA-GalNAc conjugation strategies.

  9. High Density of Aligned Nanowire Treated with Polydopamine for Efficient Gene Silencing by siRNA According to Cell Membrane Perturbation.

    Science.gov (United States)

    Nair, Baiju G; Hagiwara, Kyoji; Ueda, Motoki; Yu, Hsiao-Hua; Tseng, Hsian-Rong; Ito, Yoshihiro

    2016-07-27

    High aspect ratio nanomaterials, such as vertically aligned silicon nanowire (SiNW) substrates, are three-dimensional topological features for cell manipulations. A high density of SiNWs significantly affects not only cell adhesion and proliferation but also the delivery of biomolecules to cells. Here, we used polydopamine (PD) that simply formed a thin coating on various material surfaces by the action of dopamine as a bioinspired approach. The PD coating not only enhanced cell adhesion, spreading, and growth but also anchored more siRNA by adsorption and provided more surface concentration for substrate-mediated delivery. By comparing plain and SiNW surfaces with the same amount of loaded siRNA, we quantitatively found that PD coating efficiently anchored siRNA on the surface, which knocked down the expression of a specific gene by RNA interference. It was also found that the interaction of SiNWs with the cell membrane perturbed the lateral diffusion of lipids in the membrane by fluorescence recovery after photobleaching. The perturbation was considered to induce the effective delivery of siRNA into cells and allow the cells to carry out their biological functions. These results suggest promising applications of PD-coated, high-density SiNWs as simple, fast, and versatile platforms for transmembrane delivery of biomolecules.

  10. A Comparison of Target Gene Silencing using Synthetically Modified siRNA and shRNA That Express Recombinant Lentiviral Vectors.

    Science.gov (United States)

    Spirin, P V; Baskaran, D; Rubtsov, P M; Zenkova, M A; Vlassov, V V; Chernolovskaya, E L; Prassolov, V S

    2009-07-01

    RNA-interference is an effective natural mechanism of post-transcriptional modulation of gene expression. RNA-interference mechanism exist as in high eukaryotes both animals and plants as well in lower eukaryotes and viruses. RNA-interference is now used as a powerful tool in study of functional gene activity and many essential for fundamental biology results was obtained with this approach. Also it's widely believed that RNA-interference could be used in working out of new therapeutic medicine against malignant, infectious and hereditary diseases. One of the main problems of these developments is search of effective methods of siRNA transfer into the target cells. At present time for these purpose different sorts of transfect ions or viral transduction are used. At present article the results of comparison of inhibition of expression of oncogene AML-ET O by synthetic siRNA and by recombinant lentiviruses coding for corresponding shRNA are presented.

  11. Silencing livin gene by siRNA leads to apoptosis induction, cell cycle arrest, and proliferation inhibition in malignant melanoma LiBr cells

    Institute of Scientific and Technical Information of China (English)

    Hao WANG; Sheng-shun TAN; Xin-yang WANG; Dong-hua LIU; Chun-shui YU; Zhuan-li BAI; Da-lin HE; Jun ZHAO

    2007-01-01

    Aim: The aim of the present study was to investigate the effects of silencing the livin gene by small interfering RNA (siRNA) on the expression of livin and the effects on apoptosis, cell cycle, and proliferation in human malignant melanoma LiBr cells. Methods: Three chemically-synthetic siRNA duplexes targeting livin were transiently transfected into the LiBr cells, and the effects on livin expression were detected both at the mRNA level by real-time RT-PCR and at the protein level by Western blotting. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, flow cytometric analysis, and the expression of procaspase-3 and activated caspase-3 analysis by Western blotting. Cell cycle was analyzed by flow cytometry. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Results: One of the 3 designed siRNA could effectively knock down the livin expression both at the mRNA and protein levels in dose- and time-dependent manners; 100 nmol/L with maximum downregulation on mRNA at 48 h, and on the protein at 72 h after transfection. Silencing livin could significantly induce apoptosis, arrest cell cycle at the GJG1 phase, and inhibit proliferation in LiBr cells. Meanwhile, caspase-3 was activated. Conclusion: The livin gene could serve as a potential molecular target for gene therapy by siRNA for malignant melanoma.

  12. Computational analysis of siRNA recognition by the Ago2 PAZ domain and identification of the determinants of RNA-induced gene silencing.

    Science.gov (United States)

    Kandeel, Mahmoud; Kitade, Yukio

    2013-01-01

    RNA interference (RNAi) is a highly specialized process of protein-siRNA interaction that results in the regulation of gene expression and cleavage of target mRNA. The PAZ domain of the Argonaute proteins binds to the 3' end of siRNA, and during RNAi the attaching end of the siRNA switches between binding and release from its binding pocket. This biphasic interaction of the 3' end of siRNA with the PAZ domain is essential for RNAi activity; however, it remains unclear whether stronger or weaker binding with PAZ domain will facilitate or hinder the overall RNAi process. Here we report the correlation between the binding of modified siRNA 3' overhang analogues and their in vivo RNAi efficacy. We found that higher RNAi efficacy was associated with the parameters of lower Ki value, lower total intermolecular energy, lower free energy, higher hydrogen bonding, smaller total surface of interaction and fewer van der Waals interactions. Electrostatic interaction was a minor contributor to compounds recognition, underscoring the presence of phosphate groups in the modified analogues. Thus, compounds with lower binding affinity are associated with better gene silencing. Lower binding strength along with the smaller interaction surface, higher hydrogen bonding and fewer van der Waals interactions were among the markers for favorable RNAi activity. Within the measured parameters, the interaction surface, van der Waals interactions and inhibition constant showed a statistically significant correlation with measured RNAi efficacy. The considerations provided in this report will be helpful in the design of new compounds with better gene silencing ability.

  13. STAT3基因 siRNA 表达载体的构建及鉴定%The Building and Identification of siRNA STAT3 Gene Expression Vector

    Institute of Scientific and Technical Information of China (English)

    刘洋; 靳秋月; 段美庆; 樊淑珍; 陈立军

    2015-01-01

    Objective This study used RNA interference technology,selected STAT3 gene as the tar-get,and then design and construction a recombinant vector and then identification the gene sequence analy-sis.Methods Design a DNA sequence with a small hairpin structure,PCR amplification a siRNA expression cassettes,cloning vector to construct recombinants in psiLentGeneTM into E.coli DH5αstrain,extract plas-mid after enzyme digestion sequencing.Results Recombinant plasmid by EcoR Ⅴ enzyme,electrophoresis and Sequencing analysis showed that the inserted sequence is correct,the recombinant plasmids were success-fully constructed.Conclusion The successful construction of STAT3 targeted RNA interference recombi-nant,provides some new methods for gene therapy of cancer research at the molecular level,cellular level and the overall level of.%目的:利用RNA干扰技术,以信号转导和转录激活因子3(STAT3)为靶基因,设计构建重组体,并进行序列分析鉴定。方法设计一条有小发夹结构的 DNA 序列,聚合酶链反应( PCR)扩增合成 siRNA 表达框架(SECs)表达框架,克隆至载体 psiLentGeneTM 中构建重组体,转化 E.coli DH5α菌株,提取质粒进行酶切鉴定后测序分析。结果重组质粒经 EcoRⅤ酶切、电泳、测序分析表明插入序列正确,重组质粒构建成功。结论成功构建了 STAT3靶向 RNA 干扰重组体,为肿瘤的基因治疗在分子水平、细胞水平和整体水平的研究提供一些新方法。

  14. Hepatocyte-specific delivery of siRNAs conjugated to novel non-nucleosidic trivalent N-acetylgalactosamine elicits robust gene silencing in vivo.

    Science.gov (United States)

    Rajeev, Kallanthottathil G; Nair, Jayaprakash K; Jayaraman, Muthusamy; Charisse, Klaus; Taneja, Nate; O'Shea, Jonathan; Willoughby, Jennifer L S; Yucius, Kristina; Nguyen, Tuyen; Shulga-Morskaya, Svetlana; Milstein, Stuart; Liebow, Abigail; Querbes, William; Borodovsky, Anna; Fitzgerald, Kevin; Maier, Martin A; Manoharan, Muthiah

    2015-04-13

    We recently demonstrated that siRNAs conjugated to triantennary N-acetylgalactosamine (GalNAc) induce robust RNAi-mediated gene silencing in the liver, owing to uptake mediated by the asialoglycoprotein receptor (ASGPR). Novel monovalent GalNAc units, based on a non-nucleosidic linker, were developed to yield simplified trivalent GalNAc-conjugated oligonucleotides under solid-phase synthesis conditions. Synthesis of oligonucleotide conjugates using monovalent GalNAc building blocks required fewer synthetic steps compared to the previously optimized triantennary GalNAc construct. The redesigned trivalent GalNAc ligand maintained optimal valency, spatial orientation, and distance between the sugar moieties for proper recognition by ASGPR. siRNA conjugates were synthesized by sequential covalent attachment of the trivalent GalNAc to the 3'-end of the sense strand and resulted in a conjugate with in vitro and in vivo potency similar to that of the parent trivalent GalNAc conjugate design.

  15. Gene therapy for colorectal cancer by adenovirus-mediated siRNA targeting CD147 based on loss of the IGF2 imprinting system.

    Science.gov (United States)

    Pan, Yuqin; He, Bangshun; Chen, Jie; Sun, Huiling; Deng, Qiwen; Wang, Feng; Ying, Houqun; Liu, Xian; Lin, Kang; Peng, Hongxin; Xie, Hongguang; Wang, Shukui

    2015-11-01

    Colorectal cancer (CRC) is one of the most common malignant tumors worldwide. Loss of imprinting (LOI) of the insulin-like growth factor 2 (IGF2) gene is an epigenetic abnormality phenomenon in CRC. Recently observed association of CRC with cluster of differentiation 147 (CD147) could provide a novel approach for gene therapy. In the present study, we investigated the feasibility of using adenovirus‑mediated siRNA targeting CD147 based on the IGF2 LOI system for targeted gene therapy of CRC. A novel adenovirus-mediated siRNA targeting CD147, rAd-H19-CD147mirsh, which was driven by the IGF2 imprinting system, was constructed. The results showed that the EGFP expression was detected only in the IGF2 LOI cell lines (HT-29 and HCT-8), but that no EGFP was produced in cell lines with maintenance of imprinting (MOI) (HCT116). Moreover, rAd-H19-CD147mirsh significantly inhibited the expression of CD147, decreased cell viability and invasive ability, and increased sensitivity to chemotherapeutic drugs only in the LOI cell lines in vitro. Furthermore, mice bearing HT-29 xenografted tumors, which received intratumoral administration of the rAd-H19-CD147mirsh, showed significantly reduced tumor growth and enhanced survival. We conclude that recombinant adenovirus-mediated siRNA targeting CD147 based on the IGF2 LOI system inhibited the growth of the LOI cells in vitro and in vivo, which would provide a novel approach for targeted CRC gene therapy.

  16. Protective mechanism of NALP3-siRNA on rat renal tubular epithelial cells from hypoxia/reoxygenation injury

    Institute of Scientific and Technical Information of China (English)

    冯娟

    2013-01-01

    Objective To explore the mechanism of protecting cells from hypoxia/reoxygenation(H/R) injury by constructing specific small interference RNA(siRNA) to inhibit NALP3 expression in rat renal tubular epithelial

  17. Construction and Expression of Eukaryotic Expression Vector and Plasmid Expressing siRNA of Human Protection of Telomeres 1

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    1 IntroductionThe POT1 (protection of telomeres 1) protein binds the single-stranded overhang at the ends of chromosomes in diverse eukaryocytes. It is essential for chromosome end-protection in the fission yeast Schizosaccharomyces pombe, and it is involved in regulation of telomere length in human cells. Human POT1 had been identified in 2001 year. Its amino terminal is highly conservative in eukaryocytes. Since Pot1 can bind internal loops and directly adjacent DNA-binding sites, it is likely to fully co...

  18. Human DMBT1-Derived Cell-Penetrating Peptides for Intracellular siRNA Delivery

    DEFF Research Database (Denmark)

    Tuttolomondo, Martina; Casella, Cinzia; Hansen, Pernille Lund

    2017-01-01

    Small interfering RNA (siRNA) is a promising molecule for gene therapy, but its therapeutic administration remains problematic. Among the recently proposed vectors, cell-penetrating peptides show great promise in in vivo trials for siRNA delivery. Human protein DMBT1 (deleted in malignant brain...... an electrophoretic mobility shift assay and UV spectra, we identified two DMBT1 peptides that could encapsulate the siRNA with a self- and co-assembly mechanism. The complexes were stable for at least 2 hr in the presence of either fetal bovine serum (FBS) or RNase A, with peptide-dependent time span protection. ζ...... line to exploit DMBT1-peptide nanocomplexes for therapeutic siRNA delivery....

  19. Solid-phase synthesis of siRNA oligonucleotides.

    Science.gov (United States)

    Beaucage, Serge L

    2008-03-01

    Since the discovery of RNA interference (RNAi) as a means to silence the expression of specific genes, small interfering RNA (siRNA) oligonucleotides have been recognized as powerful tools for targeting therapeutically important mRNAs and eliciting their destruction. This discovery has created a high demand for synthetic oligoribonucleotides as potential therapeutics and has spurred a renaissance in the development of rapid, efficient methods for solid-phase RNA synthesis. The design and implementation of 2'-hydroxyl protecting groups that provide ribonucleoside phosphoramidites with coupling kinetics and coupling efficiencies comparable to those of deoxyribonucleoside phosphoramidites are key to the production of RNA oligonucleotides in sufficient quantity and purity for pharmaceutical applications. In this context, various siRNAs were chemically modified to identify the biophysical and biochemical parameters necessary for effective and stable RNAi-mediated gene-silencing activities.

  20. Effects of the Lipophilic Core of Polymer Nanoassemblies on Intracellular Delivery and Transfection of siRNA

    Directory of Open Access Journals (Sweden)

    Steven Rheiner

    2015-07-01

    Full Text Available Despite effective gene silencing in vitro, in vivo delivery and transfection of siRNA remain challenging due to the lack of carriers that protect siRNA stably in the body. This study is focused to elucidate the correlation between complex stability and transfection efficiency of siRNA carriers. The carriers were prepared by using polymer nanoassemblies made of a cationic branched polymer [poly(ethylene imine: bPEI] to which hydrophilic poly(ethylene glycol polymers were tethered covalently. These polymer tethered nanoassemblies (TNAs were further modified with lipophilic chains (palmitate: PAL in the core to stabilize siRNA TNAs complexes through ionic and hydrophobic interactions in combination. The effects of PAL in the core of TNAs were investigated with respect to in vitro transfection, intracellular gene delivery, and toxicity of the complexes, using a human colon cancer HT29 cell line stably expressing a luciferase reporter gene. A commercial transfection agent (RNAiMax was used as a control. TNAs entrapping siRNA showed the greatest complex stability in the absence of PAL although TNAs with a greater PAL content induced effective intracellular siRNA delivery, while luciferase expression decreased as the amount of PAL increased in the core of TNAs. These results demonstrate that lipophilic components in carriers affect not only complex stability but also intracellular distribution and transfection of siRNA in cancer cells.

  1. Construction of a PLGA based, targeted siRNA delivery system for treatment of osteoporosis.

    Science.gov (United States)

    Sezlev Bilecen, Deniz; Rodriguez-Cabello, Jose Carlos; Uludag, Hasan; Hasirci, Vasif

    2017-11-01

    Osteoporosis, a systemic skeletal disorder, occurs when bone turnover balance is disrupted. With the identification of the genes involved in the pathogenesis of the disease, studies on development of new treatments has intensified. Short interfering RNA (siRNA) is used to knockdown disease related gene expressions. Targeting siRNA in vivo is challenging. The maintenance of therapeutic plasma level is hampered by clearance of siRNA from the body. Targeted systems are useful in increasing the drug concentration at the target site and decreasing side effects. Aim of the present study was to develop an injectable siRNA delivery system to protect siRNA during systemic distribution and target the siRNA to bone tissue using a thermoresponsive, genetically engineered, elastin-like recombinamer (ELR), designed to interact with the mineral component of bone. The delivery system consisted of DNAoligo as a siRNA substitute complexed with the cationic polymer, polyethyleneimine (PEI), at N/P ratio of 20. The complex was encapsulated in poly(lactic acid-co-glycolic acid) (PLGA) nanocapsules. PLGA capsules were characterized by SEM, TEM and XPS. FTIR was used to show the preferential attachment of ELR to HAp. Encapsulation efficiency of the complex in PLGA nanocapsules was 48%. The release kinetics of the complex fits the Higuchi release kinetics.

  2. [The study of siRNA interference after laryngeal cancer Hep-2 cells to cisplatin sensitivity of β-catenin gene expression].

    Science.gov (United States)

    Yu, Feng; Huang, Xin; Ai, Maomao; Lin, Ying

    2015-07-01

    To investigate the changes of laryngeal cancer Hep-2 cells to cisplatin chemosensitivity after the interference of siRNA of β-catenin gene expression. Using a small interference RNA (siRNA) technology interfere β-catenin gene of Hep-2 cells . The mRNA and protein levels of β-catenin in the Hep-2 cells of different groups were detected by qPCR and Western blot. It was divided into siRNA-β-catenin-Hep-2 siRNA group, β-catenin-Neg negative control group and blank control group. Cell proliferation inhibition rate of different concentrations of cisplatin on three groups was detected by MTT assay. Calculate the 50% inhibitory effective concentration IC50 value. Check the change of three groups of cells' apoptosis rate by flow cytometry after the same concentrations of cisplatin stimulation. β-catenin-siRNA interference fragment can specifically reduce the expression levels of β-catenin mRNA and protein. qPCR illustrated the expression of mRNA in β-catenin-siR-NA-Hep-2 interference group decreased 70% (P < 0.05) compared with the control group, Western blot results showed that the β-catenin protein expression of interference group (0. 545 ± 0.111) decreased significantly compared with blank control group (1.507 ± 0.139) and negative control group (1.429 ± 0.089), P < 0.05. The IC50 calculation software showed that IC50 of cisplatin on β-catenin-siRNA IC50 interference group is (5.81 ± 0.46)μg/ml, the blank control group is (10.10 ± 1.01) μg/ml, the difference between the two groups has statistical signifi- cance (P < 0.01). Cell apoptosis rate of β-catenin-siRNA interference group was (26.15 ± 0.60)%, significantly higher than the control group (14.16 ± 0.05)%, P < 0.05. To interfere the expression of β-catenin can effectively enhance the sensitivity of laryngeal cancer cells to chemotherapeutic drugs cisplatin. It provides a theoretical support for the reduction of laryngeal cancer chemotherapy drug cisplatin dosage.

  3. Single-walled carbon nanotubes noncovalently functionalized with lipid modified polyethylenimine for siRNA delivery in vitro and in vivo.

    Science.gov (United States)

    Siu, King S; Zheng, Xiufen; Liu, Yanling; Zhang, Yujuan; Zhang, Xusheng; Chen, Di; Yuan, Ken; Gillies, Elizabeth R; Koropatnick, James; Min, Wei-Ping

    2014-10-15

    siRNA can downregulate the expression of specific genes. However, delivery to specific cells and tissues in vivo presents significant challenges. Modified carbon nanotubes (CNTs) have been shown to protect siRNA and facilitate its entry into cells. However, simple and efficient methods to functionalize CNTs are needed. Here, noncovalent functionalization of CNTs is performed and shown to effectively deliver siRNA to target cells. Specifically, single-walled CNTs were functionalized by noncovalent association with a lipopolymer. The lipopolymer (DSPE-PEG) was composed of a phospholipid 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and poly(ethylene glycol) (PEG). Three different ratios of polyethylenimine (PEI) to DSPE-PEG were synthesized and characterized and the products were used to disperse CNTs. The resulting materials were used for siRNA delivery in vitro and in vivo. The structural, biophysical, and biological properties of DGI/C and their complexes formed with siRNA were investigated. Cytotoxicity of the materials was low, and effective gene silencing in B16-F10 cells was demonstrated in vitro. In addition, significant uptake of siRNA as well as gene silencing in the liver was found following intravenous injection. This approach provides a new strategy for siRNA delivery and could provide insight for the development of noncovalently functionalized CNTs for siRNA therapy.

  4. Investigation of a miRNA-Induced Gene Silencing Technique in Petunia Reveals Alterations in miR173 Precursor Processing and the Accumulation of Secondary siRNAs from Endogenous Genes.

    Directory of Open Access Journals (Sweden)

    Yao Han

    Full Text Available MIGS (miRNA-induced gene silencing is a straightforward and efficient gene silencing technique in Arabidopsis. It works by exploiting miR173 to trigger the production of phasiRNAs (phased small interfering RNAs. MIGS can be used in plant species other than Arabidopsis by co-expression of miR173 and target gene fragments fused to an upstream miR173 target site. However, the efficiency and technical mechanisms have not been thoroughly investigated in other plants. In this work, two vectors, pMIGS-chs and pMIGS-pds, were constructed and transformed into petunia plants. The transgenic plants showed CHS (chalcone synthase and PDS (phytoene desaturase gene-silencing phenotypes respectively, indicating that MIGS functions in petunia. MIGS-chs plants were used to investigate the mechanisms of this technique in petunia. Results of 5'- RACE showed that the miR173 target site was cleaved at the expected position and that endogenous CHS genes were cut at multiple positions. Small RNA deep sequencing analysis showed that the processing of Arabidopsis miR173 precursors in MIGS-chs transgenic petunia plants did not occur in exactly the same way as in Arabidopsis, suggesting differences in the machinery of miRNA processing between plant species. Small RNAs in-phase with the miR173 cleavage register were produced immediately downstream from the cleavage site and out-of-phase small RNAs were accumulated at relatively high levels from processing cycle 5 onwards. Secondary siRNAs were generated from multiple sites of endogenous CHS-A and CHS-J genes, indicating that miR173 cleavage induced siRNAs have the same ability to initiate siRNA transitivity as the siRNAs functioning in co-suppression and hpRNA silencing. On account of the simplicity of vector construction and the transitive amplification of signals from endogenous transcripts, MIGS is a good alternative gene silencing method for plants, especially for silencing a cluster of homologous genes with redundant

  5. Ultrasound-targeted microbubble destruction to deliver siRNA cancer therapy.

    Science.gov (United States)

    Carson, Andrew R; McTiernan, Charles F; Lavery, Linda; Grata, Michelle; Leng, Xiaoping; Wang, Jianjun; Chen, Xucai; Villanueva, Flordeliza S

    2012-12-01

    Microbubble contrast agents can specifically deliver nucleic acids to target tissues when exposed to ultrasound treatment parameters that mediate microbubble destruction. In this study, we evaluated whether microbubbles and ultrasound-targeted microbubble destruction (UTMD) could be used to enhance delivery of EGF receptor (EGFR)-directed siRNA to murine squamous cell carcinomas. Custom-designed microbubbles efficiently bound siRNA and mediated RNAse protection. UTMD-mediated delivery of microbubbles loaded with EGFR-directed siRNA to murine squamous carcinoma cells in vitro reduced EGFR expression and EGF-dependent growth, relative to delivery of control siRNA. Similarly, serial UTMD-mediated delivery of EGFR siRNA to squamous cell carcinoma in vivo decreased EGFR expression and increased tumor doubling time, relative to controls receiving EGFR siRNA-loaded microbubbles but not ultrasound or control siRNA-loaded microbubbles and UTMD. Taken together, our results offer a preclinical proof-of-concept for customized microbubbles and UTMD to deliver gene-targeted siRNA for cancer therapy.

  6. Human DMBT1-Derived Cell-Penetrating Peptides for Intracellular siRNA Delivery

    Directory of Open Access Journals (Sweden)

    Martina Tuttolomondo

    2017-09-01

    Full Text Available Small interfering RNA (siRNA is a promising molecule for gene therapy, but its therapeutic administration remains problematic. Among the recently proposed vectors, cell-penetrating peptides show great promise in in vivo trials for siRNA delivery. Human protein DMBT1 (deleted in malignant brain tumor 1 is a pattern recognition molecule that interacts with polyanions and recognizes and aggregates bacteria. Taking advantage of these properties, we investigated whether specific synthetic DMBT1-derived peptides could be used to formulate nanoparticles for siRNA administration. Using an electrophoretic mobility shift assay and UV spectra, we identified two DMBT1 peptides that could encapsulate the siRNA with a self- and co-assembly mechanism. The complexes were stable for at least 2 hr in the presence of either fetal bovine serum (FBS or RNase A, with peptide-dependent time span protection. ζ-potential, circular dichroism, dynamic light scattering, and transmission electron microscopy revealed negatively charged nanoparticles with an average diameter of 10–800 nm, depending on the reaction conditions, and a spherical or rice-shaped morphology, depending on the peptide and β-helix conformation. We successfully transfected human MCF7 cells with fluorescein isothiocyanate (FITC-DMBT1-peptide-Cy3-siRNA complexes. Finally, DMBT1 peptides encapsulating an siRNA targeting a fluorescent reporter gene showed efficient gene silencing in MCF7-recombinant cells. These results lay the foundation for a new research line to exploit DMBT1-peptide nanocomplexes for therapeutic siRNA delivery.

  7. Analysis of antisense expression by whole genome tiling microarrays and siRNAs suggests mis-annotation of Arabidopsis orphan protein-coding genes.

    Directory of Open Access Journals (Sweden)

    Casey R Richardson

    Full Text Available BACKGROUND: MicroRNAs (miRNAs and trans-acting small-interfering RNAs (tasi-RNAs are small (20-22 nt long RNAs (smRNAs generated from hairpin secondary structures or antisense transcripts, respectively, that regulate gene expression by Watson-Crick pairing to a target mRNA and altering expression by mechanisms related to RNA interference. The high sequence homology of plant miRNAs to their targets has been the mainstay of miRNA prediction algorithms, which are limited in their predictive power for other kingdoms because miRNA complementarity is less conserved yet transitive processes (production of antisense smRNAs are active in eukaryotes. We hypothesize that antisense transcription and associated smRNAs are biomarkers which can be computationally modeled for gene discovery. PRINCIPAL FINDINGS: We explored rice (Oryza sativa sense and antisense gene expression in publicly available whole genome tiling array transcriptome data and sequenced smRNA libraries (as well as C. elegans and found evidence of transitivity of MIRNA genes similar to that found in Arabidopsis. Statistical analysis of antisense transcript abundances, presence of antisense ESTs, and association with smRNAs suggests several hundred Arabidopsis 'orphan' hypothetical genes are non-coding RNAs. Consistent with this hypothesis, we found novel Arabidopsis homologues of some MIRNA genes on the antisense strand of previously annotated protein-coding genes. A Support Vector Machine (SVM was applied using thermodynamic energy of binding plus novel expression features of sense/antisense transcription topology and siRNA abundances to build a prediction model of miRNA targets. The SVM when trained on targets could predict the "ancient" (deeply conserved class of validated Arabidopsis MIRNA genes with an accuracy of 84%, and 76% for "new" rapidly-evolving MIRNA genes. CONCLUSIONS: Antisense and smRNA expression features and computational methods may identify novel MIRNA genes and other non

  8. Retrovirus-delivered siRNA

    Directory of Open Access Journals (Sweden)

    Devroe Eric

    2002-08-01

    Full Text Available Abstract Background The ability of transfected synthetic small interfering (si RNAs to suppress the expression of specific transcripts has proved a useful technique to probe gene function in mammalian cells. However, high production costs limit this technology's utility for many laboratories and experimental situations. Recently, several DNA-based plasmid vectors have been developed that direct transcription of small hairpin RNAs, which are processed into functional siRNAs by cellular enzymes. Although these vectors provide certain advantages over chemically synthesized siRNAs, numerous disadvantages remain including merely transient siRNA expression and low and variable transfection efficiency. Results To overcome several limitations of plasmid-based siRNA, a retroviral siRNA delivery system was developed based on commerically available vectors. As a pilot study, a vector was designed to target the human Nuclear Dbf2-Related (NDR kinase. Cells infected with the anti-NDR siRNA virus dramatically downregulate NDR expression, whereas control viruses have no effect on total NDR levels. To confirm and extend these findings, an additional virus was constructed to target a second gene, transcriptional coactivator p75. Conclusion The experiments presented here demonstrate that retroviruses are efficient vectors for delivery of siRNA into mammalian cells. Retrovirus-delivered siRNA provides significant advancement over previously available methods by providing efficient, uniform delivery and immediate selection of stable "knock-down" cells. This development should provide a method to rapidly assess gene function in established cell lines, primary cells, or animals.

  9. Predicting siRNA efficacy based on multiple selective siRNA representations and their combination at score level

    Science.gov (United States)

    He, Fei; Han, Ye; Gong, Jianting; Song, Jiazhi; Wang, Han; Li, Yanwen

    2017-03-01

    Small interfering RNAs (siRNAs) may induce to targeted gene knockdown, and the gene silencing effectiveness relies on the efficacy of the siRNA. Therefore, the task of this paper is to construct an effective siRNA prediction method. In our work, we try to describe siRNA from both quantitative and qualitative aspects. For quantitative analyses, we form four groups of effective features, including nucleotide frequencies, thermodynamic stability profile, thermodynamic of siRNA-mRNA interaction, and mRNA related features, as a new mixed representation, in which thermodynamic of siRNA-mRNA interaction is introduced to siRNA efficacy prediction for the first time to our best knowledge. And then an F-score based feature selection is employed to investigate the contribution of each feature and remove the weak relevant features. Meanwhile, we encode the siRNA sequence and existed empirical design rules as a qualitative siRNA representation. These two kinds of siRNA representations are combined to predict siRNA efficacy by supported Vector Regression (SVR) at score level. The experimental results indicate that our method may select the features with powerful discriminative ability and make the two kinds of siRNA representations work at full capacity. The prediction results also demonstrate that our method can outperform other popular siRNA efficacy prediction algorithms.

  10. Non-Covalently Functionalized of Single-Walled Carbon Nanotubes by DSPE-PEG-PEI for SiRNA Delivery.

    Science.gov (United States)

    Siu, King Sun; Zhang, Yujuan; Zheng, Xiufen; Koropatnick, James; Min, Wei-Ping

    2016-01-01

    The expression of a gene can be specifically downregulated by small interfering RNA (SiRNA). Modified carbon nanotubes (CNT) can be used to protect SiRNA and facilitate its entry into cells. Regardless of that, simple and efficient functionalization of CNT is lacking. Effective SiRNA delivery can be carried out using non-covalently functionalized CNT, where non-covalent (versus covalent) functionalization is simpler and more expeditious. Non-covalently functionalized single walled carbon nanotubes (SWCNT) that include a lipopolymer are described here. Polyethylenimine (PEI) conjugated to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) was generated and the products used to disperse CNT to form DSPE-PEG-PEI/CNT (DGI/C), an agent capable of facilitating SiRNA delivery to cells in vitro and organs and cells in vivo.

  11. Chapter 17 - Engineering cationic liposome siRNA complexes for in vitro and in vivo delivery.

    Science.gov (United States)

    Podesta, Jennifer E; Kostarelos, Kostas

    2009-01-01

    RNA interference, the sequence-specific silencing of gene expression by introduction of short interfering RNA (siRNA) is a powerful tool that that the potential to act as a therapeutic agent and the advantage of decreasing toxic effects on normal tissue sometimes seen with conventional treatments i.e. small molecule inhibitors. Naked, unmodified siRNA is poorly taken up by cells and is subject to degradation when exposed to blood proteins during systemic administration. It has also been shown to produce non-specific immune response as well as having the potential to generate 'off-target' effects. Therefore there is a requirement for a delivery system to not only protect the siRNA and facilitate its uptake, but additionally to offer the potential for targeted delivery with an aim of exploiting the high specificity afforded by RNA interference. Cationic liposomes are the most studied, non-viral delivery system used for nucleic acid delivery. As such, the use of cationic liposomes is promising for siRNA for delivery. Furthermore, polyethylene glycol (PEG) can be incorporated into the liposome formulation to create sterically stabilized or 'stealth' liposomes. Addition of PEG can reduce recognition by the reticuloendothelial system (RES) thereby prolonging circulation time. Here we describe a methodology for the complexation of siRNA with cationic liposomes and PEGylated liposomes using two protocols: mixing and encapsulation. Moreover, the different formulations are compared head to head to demonstrate their efficacy for gene silencing.

  12. Dual-functionalized graphene oxide for enhanced siRNA delivery to breast cancer cells.

    Science.gov (United States)

    Imani, Rana; Shao, Wei; Taherkhani, Samira; Emami, Shahriar Hojjati; Prakash, Satya; Faghihi, Shahab

    2016-11-01

    The aim of this study is to improve hydrocolloid stability and siRNA transfection ability of a reduced graphene oxide (rGO) based nano-carrier using a phospholipid-based amphiphilic polymer (PL-PEG) and cell penetrating peptide (CPPs). The dual functionalized nano-carrier is comprehensively characterized for its chemical structure, size, surface charge and morphology as well as thermal stability. The nano-carrier cytocompatibility, siRNA condensation ability both in the presence and absence of enzyme, endosomal buffering capacity, cellular uptake and intracellular localization are also assessed. The siRNA loaded nano-carrier is used for internalization to MCF-7 cells and its gene silencing ability is compared with AllStars Hs Cell Death siRNA as a model gene. The nano-carrier remains stable in biological solution, exhibits excellent cytocompatibility, retards the siRNA migration and protects it against enzyme degradation. The buffering capacity analysis shows that incorporation of the peptide in nano-carrier structure would increase the resistance to endo/lysosomal like acidic condition (pH 6-4) The functionalized nano-carrier which is loaded with siRNA in an optimal N:P ratio presents superior internalization efficiency (82±5.1% compared to HiPerFect(®)), endosomal escape quality and capable of inducing cell death in MCF-7 cancer cells (51±3.1% compared to non-treated cells). The success of siRNA-based therapy is largely dependent on the safe and efficient delivery system, therefore; the dual functionalized rGO introduced here could have a great potential to be used as a carrier for siRNA delivery with relevancy in therapeutics and clinical applications.

  13. Function and anatomy of plant siRNA pools derived from hairpin transgenes

    Directory of Open Access Journals (Sweden)

    Lee Kevin AW

    2007-11-01

    Full Text Available Abstract Background RNA interference results in specific gene silencing by small-interfering RNAs (siRNAs. Synthetic siRNAs provide a powerful tool for manipulating gene expression but high cost suggests that novel siRNA production methods are desirable. Strong evolutionary conservation of siRNA structure suggested that siRNAs will retain cross-species function and that transgenic plants expressing heterologous siRNAs might serve as useful siRNA bioreactors. Here we report a detailed evaluation of the above proposition and present evidence regarding structural features of siRNAs extracted from plants. Results Testing the gene silencing capacity of plant-derived siRNAs in mammalian cells proved to be very challenging and required partial siRNA purification and design of a highly sensitive assay. Using the above assay we found that plant-derived siRNAs are ineffective for gene silencing in mammalian cells. Plant-derived siRNAs are almost exclusively double-stranded and most likely comprise a mixture of bona fide siRNAs and aberrant partially complementary duplexes. We also provide indirect evidence that plant-derived siRNAs may contain a hitherto undetected physiological modification, distinct from 3' terminal 2-O-methylation. Conclusion siRNAs produced from plant hairpin transgenes and extracted from plants are ineffective for gene silencing in mammalian cells. Thus our findings establish that a previous claim that transgenic plants offer a cost-effective, scalable and sustainable source of siRNAs is unwarranted. Our results also indicate that the presence of aberrant siRNA duplexes and possibly a plant-specific siRNA modification, compromises the gene silencing capacity of plant-derived siRNAs in mammalian cells.

  14. Efficacious cellular codelivery of doxorubicin and EGFP siRNA mediated by the composition of PLGA and PEI protected gold nanoparticles.

    Science.gov (United States)

    Kumar, Krishan; Vulugundam, Gururaja; Jaiswal, Pradeep Kumar; Shyamlal, Bharti Rajesh Kumar; Chaudhary, Sandeep

    2017-09-15

    This study reports the simultaneous delivery of EGFP siRNA and the chemotherapeutic drug, doxorubicin by means of the composition that results from the electrostatic interaction between positively charged siRNA-complexes of gold nanoparticles (AuNPs) capped with PEI, 25kDa (P25-AuNPs) and negatively charged carboxymethyl cellulose formulated PLGA nanoparticles loaded with doxorubicin. The nanoparticles and their facile interaction were studied by means of dynamic light scattering (DLS), zeta potential, transmission electron microscopic (TEM) measurements. The flow cytometric and confocal microscopic analysis evidenced the simultaneous internalization of both labelled siRNA and doxorubin into around 55% of the HeLa cancer cell population. Fluorescence microscopic studies enabled the visual analysis of EGFP expressing HeLa cells which suggested that the composition mediated codelivery resulted in a substantial downregulation of EGFP expression and intracellular accumulation of doxorubicin. Interestingly, codelivery treatment resulted in an increased cellular delivery of doxorubicin when compared to PLGA-DOX alone treatment. On the other hand, the activity of siRNA complexes of PEI-AuNPs was completely retained even when they were part of composition. The results suggest that this formulation can serve as promising tool for delivery applications in combinatorial anticancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Inhibition of Androgen-Independent Growth of Prostate Cancer by siRNA- Mediated Androgen Receptor Gene Silencing

    Science.gov (United States)

    2008-02-01

    Center, Kansas City, KS 66160, USA Introduction Prostate cancer is the most common cancer diagnosed after skin cancers and the... cancer cell growth: androgen regulation of CDK2, CDK4 , and CKI p16 genes. Cancer Res. 1997; 57:4511-4516. 109. Gregory CW, Hamil KG, Kim D, Hall SH...the most diagnosed non- skin cancer and the second leading cause of cancer -related death [2]. Currently, prostate-specific antigen (PSA) is the most

  16. An siRNA screen of membrane trafficking genes highlights pathways common to HIV-1 and M-PMV virus assembly and release.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Wen

    Full Text Available The assembly and release of retroviruses from the host cells requires a coordinated series of interactions between viral structural proteins and cellular trafficking pathways. Although a number of cellular factors involved in retrovirus assembly have been identified, it is likely that retroviruses utilize additional trafficking factors to expedite their assembly and budding that have not yet been defined. We performed a screen using an siRNA library targeting host membrane trafficking genes in order to identify new host factors that contribute to retrovirus assembly or release. We utilized two retroviruses that follow very distinct assembly pathways, HIV-1 and Mason-Pfizer monkey virus (M-PMV in order to identify host pathways that are generally applicable in retrovirus assembly versus those that are unique to HIV or M-PMV. Here we report the identification of 24 host proteins identified in the screen and subsequently validated in follow-up experiments as contributors to the assembly or release of both viruses. In addition to identifying a number of previously unsuspected individual trafficking factors, we noted multiple hits among proteins involved in modulation of the actin cytoskeleton, clathrin-mediated transport pathways, and phosphoinositide metabolism. Our study shows that distant genera of retroviruses share a number of common interaction strategies with host cell trafficking machinery, and identifies new cellular factors involved in the late stages of retroviral replication.

  17. Polyethylene glycol–poly(ε-benzyloxycarbonyl-l-lysine)-conjugated VEGF siRNA for antiangiogenic gene therapy in hepatocellular carcinoma

    Science.gov (United States)

    Wang, Gangmin; Gao, XiaoLong; Gu, GuoJun; Shao, ZhiHong; Li, MingHua; Wang, PeiJun; Yang, JianRong; Cai, XiaoJun; Li, YongYong

    2017-01-01

    A polyethylene glycol–poly(ε-benzyloxycarbonyl-l-lysine) (PEG-SS-PLL) block copolymer based on a disulfide-linked, novel biodegradable catiomer bearing a PEG-sheddable shell was developed to avoid “PEG dilemma” in nanoparticle intracellular tracking of PEG-PLL where PEG was nondegradable. However, PEG-SS-PLL catiomers have not been used to deliver small interfering VEGF RNA (siVEGF) in antiangiogenesis gene therapy. In this study, we aimed to investigate whether this novel biodegradable catiomer can deliver siVEGF into cancer cells and at the same time have an antitumor effect in a xenograft mouse model. It was found that PEG-SS-PLL efficiently delivered siVEGF with negligible cytotoxicity, and significantly decreased the expression of VEGF at both the messenger-RNA and protein levels both in vitro and in vivo, and thus tumor growth was inhibited. Our findings demonstrated that PEG-SS-PLL/siVEGF could potentially be applied to antiangiogenesis gene therapy for hepatocellular carcinoma. PMID:28533682

  18. Construction of a siRNA vector targeting Smad3 gene and gene-silencing effect%Smad3基因siRNA表达载体的构建及沉默效应

    Institute of Scientific and Technical Information of China (English)

    王爱丽; 吴志宏; 徐顺; 顾耀辉; 黄静; 陈波; 贾卿

    2011-01-01

    BACKGROUND: Previous studies demonstrated that inhibition of Smad3 can suppress fibroplasias or collagen over expression.OBJECTIVE: To construct an expression vector of siRNA targeting human Smad3 gene and observe its gene-silencing effect in hypertrophic scar (HS) fibroblasts METHODS: The siRNA sequences targeting Smad3 gene were designed and synthesized with two complementary oligonucleotide strands. The oligonucleotide strands were annealed and recombined into pRNAT-U6 vector, which was identified by sequencing following transformation and amplification. The siRNA expression vector pRNAT -U6- Smad3 was transfected into human HS fibroblasts. Smad3 expressions in mRNA and protein levels were examined by real-time-PCR and Western blotting.RESULTS AND CONCLUSION: The result of recombinant sequence was the same as aim sequence. Real-time RCR and Western blot showed that, compared to blank, the mRNA and protein levels of Smad3 were significantly decreased. Thereinto,the most effective siRNA sequence was AGA CAG ACT GTG ACC AGT A (1156). The gene-silent efficiency was 45%.Smad3-siRNA recombinant was constructed successfully and can be transfected into HS fibroblasts to inhibit the expression of Smad3 gene.%背景:研究提示抑制Smad3可望抑制纤维增生和胶原的大量产生.目的:构建针对Smad3基因的siRNA表达载体,观察RNA干扰对增生性瘢痕成纤维细胞Smad3基因表达的抑制作用.方法:根据siRNA设计原则,设计3条针对Smad3基因的siRNA靶序列,分别合成两条互补的寡核苷酸链,退火后与载体pRNAT-U6连接,然后进行酶切鉴定和DNA序列测定.用脂质体包裹转染增生性瘢痕成纤维细胞,荧光定量PCR和Western blot方法检测Smad3基因的表达情况.结果与结论:测序分析结果显示:克隆入pRNAT-U6载体的针对Smad3基因的siRNA的双链寡核苷酸片段插入正确;荧光定量PCR和Western blot检测显示:转染的增生性瘢痕成纤维细胞Smad3基因的表

  19. HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector

    Directory of Open Access Journals (Sweden)

    Akkina Ramesh

    2005-01-01

    Full Text Available Abstract Background RNA interference (RNAi mediated by small interfering RNAs (siRNAs has proved to be a highly effective gene silencing mechanism with great potential for HIV/AIDS gene therapy. Previous work with siRNAs against cellular coreceptors CXCR4 and CCR5 had shown that down regulation of these surface molecules could prevent HIV-1 entry and confer viral resistance. Since monospecific siRNAs targeting individual coreceptors are inadequate in protecting against both T cell tropic (X4 and monocyte tropic (R5 viral strains simultaneously, bispecific constructs with dual specificity are required. For effective long range therapy, the bispecific constructs need to be stably transduced into HIV-1 target cells via integrating viral vectors. Results To achieve this goal, lentiviral vectors incorporating both CXCR4 and CCR5 siRNAs of short hairpin design were constructed. The CXCR4 siRNA was driven by a U6 promoter whereas the CCR5 siRNA was driven by an H1 promoter. A CMV promoter driven EGFP reporter gene is also incorporated in the bispecific construct. High efficiency transduction into coreceptor expressing Magi and Ghost cell lines with a concomitant down regulation of respective coreceptors was achieved with lentiviral vectors. When the siRNA expressing transduced cells were challenged with X4 and R5 tropic HIV-1, they demonstrated marked viral resistance. HIV-1 resistance was also observed in bispecific lentiviral vector transduced primary PBMCs. Conclusions Both CXCR4 and CCR5 coreceptors could be simultaneously targeted for down regulation by a single combinatorial lentiviral vector incorporating respective anti-coreceptor siRNAs. Stable down regulation of both the coreceptors protects cells against infection by both X4 and R5 tropic HIV-1. Stable down regulation of cellular molecules that aid in HIV-1 infection will be an effective strategy for long range HIV gene therapy.

  20. Effective siRNA targets screening for human telomerase reverse transcriptase

    Institute of Scientific and Technical Information of China (English)

    Yun Xia; Ru-Xian Lin; Su-Jun Zheng; Ying Yang; Xiao-Chen Bo; Dao-Yin Zhu; Sheng-Qi Wang

    2005-01-01

    AIM: To study the inhibitory effects of siRNAs targeting different hTERT sequences and to screen the effective siRNA sequence.METHODS: Five double-stranded siRNAs targeting coding and non-coding regions of hTERT gene were designed and synthesized by T7 transcription system in vitro. siRNA4sequence was screened by full length gene targeting technique and the rest of the siRNA sequences were selected randomly. After being purified by ethanol precipitation, the siRNAs were transfected to the human hepatocellular carcinoma cell (HepG2) by Lipofectamine 2000TM. At 48-72 h after siRNAs transfection, MTT assay,RT-PCR and Western-blot were applied to evaluate the effects of siRNAs on cell growth, mRNA and protein expression level of hTERT gene, respectively.RESULTS: Compared to the control cells, the cells treated with the five double-stranded siRNAs exhibited different degrees of inhibition of cell proliferation in a dose-dependent manner. siRNA2 and siRNA4, exhibited obvious effects of inhibiting hTERT mRNA and protein expression in HepG2cells.CONCLUSION: siRNAs targeting different hTERT sequences have significantly various inhibitory effects on hTERT gene expression. The siRNA sequence screened by full length gene targeting technique has comparable inhibitory effect with the rest siRNA sequences screened by random selection, suggesting that siRNAs and antisense oligonucleic acids may have the same effective target sites. Compared with chemical synthesis method,synthesizing double-stranded siRNA by T7 transcription system in vitro is a rapid, simple, and inexpensive method suitable for screening high-effect siRNA targeting site for specific gene.

  1. Efficacy of TRAIL treatment against HPV16 infected cervical cancer cells undergoing senescence following siRNA knockdown of E6/E7 genes.

    Science.gov (United States)

    Eaton, Seron; Wiktor, Peter; Thirstrup, Derek; Lake, Douglas; Nagaraj, Vinay Janthakahalli

    2011-02-04

    In this study we investigated E6 and E7 oncogenes from the Human Papilloma Virus as targets for siRNA knockdown in order to boost the efficacy of the anti-cancer drug 'tumor necrosis factor-related apoptosis inducing ligand' (TRAIL). SiHa cells were treated with TRAIL following transfection with E6/E7 siRNA and the expression of death receptors DR4 and DR5, cell viability, apoptosis, senescence and cell cycle analysis were undertaken using flow cytometry, MTT viability assay and cellular β-galactosidase activity assays. E6/E7 siRNA resulted in significant upregulation of death receptors DR4 and DR5 but did not result in an enhanced sensitivity to TRAIL. Our results indicate that E6/E7-siRNA induces senescence rather than apoptosis in SiHa cells. The occurrence of senescence in drug resistant cervical cancer cells such as the SiHa cell line by E6/E7 siRNA, among other factors, may prevent TRAIL induced activation of extrinsic and intrinsic pathways that lead to apoptotic cell death. Our findings are significant for combinatorial strategies for cancer therapy since the induction of senescence can preclude apoptosis rendering cells to be recalcitrant to TRAIL treatment. Copyright © 2010 Elsevier Inc. All rights reserved.

  2. Silencing of Inducible Immunoproteasome Subunit Expression by Chemically Modified siRNA and shRNA.

    Science.gov (United States)

    Gvozdeva, Olga V; Prassolov, Vladimir S; Zenkova, Marina A; Vlassov, Valentin V; Chernolovskaya, Elena L

    2016-08-01

    Overexpression of inducible subunits of immunoproteasome is related to pathogenesis of some chronic diseases. Specific inhibition of the immunosubunits may be used for the treatment of these diseases and RNA interference is one of the potent methods used in this area. We designed 2'-O-methyl modified siRNAs with selectively protected nuclease-sensitive sites, which efficiently silence LMP2, LMP7, and MECL-1 genes expression. To provide stable long-lasting inhibition of target genes, short-hairpin RNAs (shRNA) expressed by lentiviral vectors were constructed. Our results demonstrated that chemically modified siRNAs inhibited the expression of target genes with similar efficiency or with efficiency exceeding that of corresponding shRNAs and provide silencing effect for 5 days.

  3. The Scope of Gene Patent Protection and the TRIPS Agreement

    DEFF Research Database (Denmark)

    Sommer, Tine

    2007-01-01

    The Scope of Gene Patent Protection and the TRIPS Agreement - An Exclusively Nondiscriminatory Approach?   Gene patenting in Europe has provoked much debate both before and since the adoption of Directive 98/44/EC on the legal protection of biotechnological inventions. Some of the major points...... of discussion have been focused on the scope of protection (e.g. purpose-bound protection) and gene patents being subject to a specific DNA regime on patent rights. The Directive can be interpreted as favouring such a solution, but so far the European Commission has decided neither to support nor reject...

  4. Co-delivery of Se nanoparticles and pooled SiRNAs for overcoming drug resistance mediated by P-glycoprotein and class III β-tubulin in drug-resistant breast cancers.

    Science.gov (United States)

    Zheng, Wenjing; Yin, Tiantian; Chen, Qingchang; Qin, Xiuying; Huang, Xiaoquan; Zhao, Shuang; Xu, Taoyuan; Chen, Lanmei; Liu, Jie

    2016-02-01

    Drug resistance mediated by P-glycoprotein (P-gp) and class III β-tubulin (β-tubulin III) is a major barrier in microtubule-targeting cancer chemotherapy. In this study, layered double hydroxide nanoparticles (LDHs) were employed to simultaneously deliver selenium (Se) and pooled small interfering RNAs (siRNAs) to achieve therapeutic efficacy. LDH-supported Se nanoparticles (Se@LDH) were compacted with siRNAs (anti-P-gp and anti-β-tubulin III) via electrostatic interactions, which could protect siRNA from degradation. Se@LDH showed excellent abilities to deliver siRNA into cells, including enhancing siRNA internalization, and promoting siRNA escape from endosomes. siRNA transfection experiments further confirmed a higher gene silencing efficiency of Se@LDH than LDH. Interestingly, we found Se@LDH may be a microtubule (MT) stabilizing agent which could inhibit cell proliferation by blocking cell cycle at G2/M phase, disrupting normal mitotic spindle formation and inducing cell apoptosis. When complexed with different specific siRNAs, Se@LDH/siRNA nanoparticles, especially the Se@LDH-pooled siRNAs, exhibit an efficient gene-silencing effect that significantly downregulate the expression of P-gp and β-tubulin III. Moreover, Se@LDH-pooled siRNAs could induce cell apoptosis, change cell morphology and increase cellular ROS levels through change the expression of Bcl-2/Bax, activation of caspase-3, PI3K/AKT/mTOR and MAPK/ERK pathways. These results suggested that co-delivery of Se and pooled siRNAs may be a promising strategy for overcoming the drug resistance mediated by P-gp and β-tubulin III in drug-resistant breast cancers. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  5. Utilizing Selected Di- and Trinucleotides of siRNA to Predict RNAi Activity

    Directory of Open Access Journals (Sweden)

    Ye Han

    2017-01-01

    Full Text Available Small interfering RNAs (siRNAs induce posttranscriptional gene silencing in various organisms. siRNAs targeted to different positions of the same gene show different effectiveness; hence, predicting siRNA activity is a crucial step. In this paper, we developed and evaluated a powerful tool named “siRNApred” with a new mixed feature set to predict siRNA activity. To improve the prediction accuracy, we proposed 2-3NTs as our new features. A Random Forest siRNA activity prediction model was constructed using the feature set selected by our proposed Binary Search Feature Selection (BSFS algorithm. Experimental data demonstrated that the binding site of the Argonaute protein correlates with siRNA activity. “siRNApred” is effective for selecting active siRNAs, and the prediction results demonstrate that our method can outperform other current siRNA activity prediction methods in terms of prediction accuracy.

  6. Efficient delivery of Notch1 siRNA to SKOV3 cells by cationic cholesterol derivative-based liposome

    Science.gov (United States)

    Zhao, Yun-Chun; Zhang, Li; Feng, Shi-Sen; Hong, Lu; Zheng, Hai-Li; Chen, Li-Li; Zheng, Xiao-Ling; Ye, Yi-Qing; Zhao, Meng-Dan; Wang, Wen-Xi; Zheng, Cai-Hong

    2016-01-01

    A novel cationic cholesterol derivative-based small interfering RNA (siRNA) interference strategy was suggested to inhibit Notch1 activation in SKOV3 cells for the gene therapy of ovarian cancer. The cationic cholesterol derivative, N-(cholesterylhemisuccinoyl-amino-3-propyl)-N, N-dimethylamine (DMAPA-chems) liposome, was incubated with siRNA at different nitrogen-to-phosphate ratios to form stabilized, near-spherical siRNA/DMAPA-chems nanoparticles with sizes of 100–200 nm and zeta potentials of 40–50 mV. The siRNA/DMAPA-chems nanoparticles protected siRNA from nuclease degradation in 25% fetal bovine serum. The nanoparticles exhibited high cell uptake and Notch1 gene knockdown efficiency in SKOV3 cells at an nitrogen-to-phosphate ratio of 100 and an siRNA concentration of 50 nM. They also inhibited the growth and promoted the apoptosis of SKOV3 cells. These results may provide the potential for using cationic cholesterol derivatives as efficient nonviral siRNA carriers for the suppression of Notch1 activation in ovarian cancer cells.

  7. Targeted Sterically Stabilized Phospholipid siRNA Nanomedicine for Hepatic and Renal Fibrosis

    Directory of Open Access Journals (Sweden)

    Fatima Khaja

    2016-01-01

    Full Text Available Since its discovery, small interfering RNA (siRNA has been considered a potent tool for modulating gene expression. It has the ability to specifically target proteins via selective degradation of messenger RNA (mRNA not easily accessed by conventional drugs. Hence, RNA interference (RNAi therapeutics have great potential in the treatment of many diseases caused by faulty protein expression such as fibrosis and cancer. However, for clinical application siRNA faces a number of obstacles, such as poor in vivo stability, and off-target effects. Here we developed a unique targeted nanomedicine to tackle current siRNA delivery issues by formulating a biocompatible, biodegradable and relatively inexpensive nanocarrier of sterically stabilized phospholipid nanoparticles (SSLNPs. This nanocarrier is capable of incorporating siRNA in its core through self-association with a novel cationic lipid composed of naturally occuring phospholipids and amino acids. This overall assembly protects and delivers sufficient amounts of siRNA to knockdown over-expressed protein in target cells. The siRNA used in this study, targets connective tissue growth factor (CTGF, an important regulator of fibrosis in both hepatic and renal cells. Furthermore, asialoglycoprotein receptors are targeted by attaching the galactosamine ligand to the nanocarries which enhances the uptake of nanoparticles by hepatocytes and renal tubular epithelial cells, the major producers of CTGF in fibrosis. On animals this innovative nanoconstruct, small interfering RNA in sterically stabilized phospholipid nanoparticles (siRNA-SSLNP, showed favorable pharmacokinetic properties and accumulated mostly in hepatic and renal tissues making siRNA-SSLNP a suitable system for targeting liver and kidney fibrotic diseases.

  8. The Influence of RNA Secondary Structure on the Efficiency of siRNA Silencing

    Institute of Scientific and Technical Information of China (English)

    SUN Ying; GUI Jian-bin; CHEN Zhao-xue

    2015-01-01

    In the application of RNAi technology, it is an essential step to design siRNA applicable to target gene. At present, there are many researches and conclusions on siRNA design. This paper aims to the influences of mRNA secondary structure or siRNA antisense-strand secondary structure on siRNA silence efficiency. The paper also discusses the problems and sets out further insights in the research.

  9. Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

    Science.gov (United States)

    Warburton, Philip J; Amodeo, Nina; Roberts, Adam P

    2016-12-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.

  10. Alkane-modified short polyethyleneimine for siRNA delivery.

    Science.gov (United States)

    Schroeder, Avi; Dahlman, James E; Sahay, Gaurav; Love, Kevin T; Jiang, Shan; Eltoukhy, Ahmed A; Levins, Christopher G; Wang, Yingxia; Anderson, Daniel G

    2012-06-10

    RNA interference (RNAi) is a highly specific gene-silencing mechanism triggered by small interfering RNA (siRNA). Effective intracellular delivery requires the development of potent siRNA carriers. Here, we describe the synthesis and screening of a series of siRNA delivery materials. Short polyethyleneimine (PEI, Mw 600) was selected as a cationic backbone to which lipid tails were conjugated at various levels of saturation. In solution these polymer-lipid hybrids self-assemble to form nanoparticles capable of complexing siRNA. The complexes silence genes specifically and with low cytotoxicity. The efficiency of gene knockdown increased as the number of lipid tails conjugated to the PEI backbone increased. This is explained by reducing the binding affinity between the siRNA strands to the complex, thereby enabling siRNA release after cellular internalization. These results highlight the importance of complexation strength when designing siRNA delivery materials. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Silencing GADD153/CHOP gene expression protects against Alzheimer's disease-like pathology induced by 27-hydroxycholesterol in rabbit hippocampus.

    Directory of Open Access Journals (Sweden)

    Jaya R P Prasanthi

    Full Text Available Endoplasmic reticulum (ER stress is suggested to play a key role in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD. Sustained ER stress leads to activation of the growth arrest and leucine zipper transcription factor, DNA damage inducible gene 153 (gadd153; also called CHOP. Activated gadd153 can generate oxidative damage and reactive oxygen species (ROS, increase β-amyloid (Aβ levels, disturb iron homeostasis and induce inflammation as well as cell death, which are all pathological hallmarks of AD. Epidemiological and laboratory studies suggest that cholesterol dyshomeostasis contributes to the pathogenesis of AD. We have previously shown that the cholesterol oxidized metabolite 27-hydroxycholesterol (27-OHC triggers AD-like pathology in organotypic slices. However, the extent to which gadd153 mediates 27-OHC effects has not been determined. We silenced gadd153 gene with siRNA and determined the effects of 27-OHC on AD hallmarks in organotypic slices from adult rabbit hippocampus. siRNA to gadd153 reduced 27-OHC-induced Aβ production by mechanisms involving reduction in levels of β-amyloid precursor protein (APP and β-secretase (BACE1, the enzyme that initiates cleavage of APP to yield Aβ peptides. Additionally, 27-OHC-induced tau phosphorylation, ROS generation, TNF-α activation, and iron and apoptosis-regulatory protein levels alteration were also markedly reduced by siRNA to gadd153. These data suggest that ER stress-mediated gadd153 activation plays a central role in the triggering of AD pathological hallmarks that result from incubation of hippocampal slices with 27-OHC. Our results add important insights into cellular mechanisms that underlie the potential contribution of cholesterol metabolism in AD pathology, and suggest that preventing gadd153 activation protects against AD related to cholesterol oxidized products.

  12. Chemosensitization of cancer cells by siRNA using targeted nanogel delivery

    Directory of Open Access Journals (Sweden)

    Blackburn William H

    2010-01-01

    Full Text Available Abstract Background Chemoresistance is a major obstacle in cancer treatment. Targeted therapies that enhance cancer cell sensitivity to chemotherapeutic agents have the potential to increase drug efficacy while reducing toxic effects on untargeted cells. Targeted cancer therapy by RNA interference (RNAi is a relatively new approach that can be used to reversibly silence genes in vivo by selectively targeting genes such as the epidermal growth factor receptor (EGFR, which has been shown to increase the sensitivity of cancer cells to taxane chemotherapy. However, delivery represents the main hurdle for the broad development of RNAi therapeutics. Methods We report here the use of core/shell hydrogel nanoparticles (nanogels functionalized with peptides that specially target the EphA2 receptor to deliver small interfering RNAs (siRNAs targeting EGFR. Expression of EGFR was determined by immunoblotting, and the effect of decreased EGFR expression on chemosensitization of ovarian cancer cells after siRNA delivery was investigated. Results Treatment of EphA2 positive Hey cells with siRNA-loaded, peptide-targeted nanogels decreased EGFR expression levels and significantly increased the sensitivity of this cell line to docetaxel (P 0.05. Conclusion This study suggests that targeted delivery of siRNAs by nanogels may be a promising strategy to increase the efficacy of chemotherapy drugs for the treatment of ovarian cancer. In addition, EphA2 is a viable target for therapeutic delivery, and the siRNAs are effectively protected by the nanogel carrier, overcoming the poor stability and uptake that has hindered clinical advancement of therapeutic siRNAs.

  13. An inside job for siRNAs.

    Science.gov (United States)

    Golden, Daniel E; Gerbasi, Vincent R; Sontheimer, Erik J

    2008-08-08

    Among the three main categories of small silencing RNAs in insects and mammals-siRNAs, miRNAs, and piRNAs-siRNAs were thought to arise primarily from exogenous sources, whereas miRNAs and piRNAs arise from endogenous loci. Recent work in flies and mice reveals several classes of endogenous siRNAs (endo-siRNAs) that contribute to functions previously reserved for miRNAs and piRNAs, including gene regulation and transposon suppression.

  14. Generation of siRNA Nanosheets for Efficient RNA Interference

    Science.gov (United States)

    Kim, Hyejin; Lee, Jae Sung; Lee, Jong Bum

    2016-04-01

    After the discovery of small interference RNA (siRNA), nanostructured siRNA delivery systems have been introduced to achieve an efficient regulation of the target gene expression. Here we report a new siRNA-generating two dimensional nanostructure in a formation of nanosized sheet. Inspired by tunable mechanical and functional properties of the previously reported RNA membrane, siRNA nanosized sheets (siRNA-NS) with multiple Dicer cleavage sites were prepared. The siRNA-NS has two dimensional structure, providing a large surface area for Dicer to cleave the siRNA-NS for the generation of functional siRNAs. Furthermore, downregulation of the cellular target gene expression was achieved by delivery of siRNA-NS without chemical modification of RNA strands or conjugation to other substances.

  15. A surface-mediated siRNA delivery system developed with chitosan/hyaluronic acid-siRNA multilayer films through layer-by-layer self-assembly

    Science.gov (United States)

    Wu, Lijuan; Wu, Changlin; Liu, Guangwan; Liao, Nannan; Zhao, Fang; Yang, Xuxia; Qu, Hongyuan; Peng, Bo; Chen, Li; Yang, Guang

    2016-12-01

    siRNA delivery remains highly challenging because of its hydrophilic and anionic nature and its sensitivity to nuclease degradation. Effective siRNA loading and improved transfection efficiency into cells represents a key problem. In our study, we prepared Chitosan/Hyaluronic acid-siRNA multilayer films through layer-by-layer self-assembly, in which siRNAs can be effectively loaded and protected. The construction process was characterized by FTIR, 13C NMR (CP/MAS), UV-vis spectroscopy, and atomic force microscopy (AFM). We presented the controlled-release performance of the films during incubation in 1 M NaCl solution for several days through UV-vis spectroscopy and polyacrylamide gel electrophoresis (PAGE). Additionally, we verified the stability and integrity of the siRNA loaded on multilayer films. Finally, the biological efficacy of the siRNA delivery system was evaluated via cells adhesion and gene silencing analyses in eGFP-HEK 293T cells. This new type of surface-mediated non-viral multilayer films may have considerable potential in the localized and controlled-release delivery of siRNA in mucosal tissues, and tissue engineering application.

  16. Hybrid PET/CT for noninvasive pharmacokinetic evaluation of dynamic PolyConjugates, a synthetic siRNA delivery system.

    Science.gov (United States)

    Mudd, Sarah R; Trubetskoy, Vladimir S; Blokhin, Andrei V; Weichert, Jamey P; Wolff, Jon A

    2010-07-21

    Positron emission tomography/computed tomography (PET/CT) hybrid imaging can be used to gain insights into a synthetic siRNA delivery system targeted to the liver. Either siRNA or the delivery vehicle was labeled with (64)Cu via 1, 4, 7, 10- tetraazacyclododecane- 1, 4, 7, 10- tetraacetic acid (DOTA) chelation. This study confirmed that the siRNA delivery system was successfully targeted to the liver. Incorporation of the siRNA into the delivery system protected the siRNA from renal filtration long enough so that the siRNA could be delivered to the liver. PET/CT imaging was important for confirming biodistribution and for determining differences in the distribution of labeled siRNA, siRNA incorporated into the delivery system, and the delivery system without siRNA.

  17. DNMT1、DNMT3b siRNA重组质粒的构建及鉴定%Construction and identification of siRNA recombinant plasmid of DNMT1 and DNMT3b genes

    Institute of Scientific and Technical Information of China (English)

    刘毅; 张旋; 魏涧; 李虎宜; 梁志恒; 张栩亮; 张有顺

    2012-01-01

    Objective: To construct and identificate the siRNA reccmbinant plasmid of DNM1 and DNMT3 b genes.Methods: Four types of sENA reccmbinant plasmid of DNMT1 or DNMT3 b gene were constructed, and Hentificated. Results: After digested with Bam H Ⅰ andPst Ⅰ enzymes, all plasmids showed positive for reccmbinant vector plasmids. Two clones of each type ofvectorplasimid were selected for, sequencing identification. The results of sequencing identification were consistent with the inserted oligonucleotides Conclusions: The siRNA reccmbinant plasmds of DNMT1 and DNMT3 b genes were constructed corectly and could be used for further study.%目的:构建DNA甲基转移酶(DNMT1)、DNMT3b 微干扰RNA(siRNA)重组质粒,并鉴定其正确性.方法:构建DNMT1 siRNA重组质粒和DNMT3b siRNA重组质粒各4种,分别用BamH Ⅰ和Pst Ⅰ酶切、测序、鉴定.结果:用BamH Ⅰ和Pst Ⅰ进行双酶切后所有质粒均为阳性重组载体,每一种载体选择两个克隆进行测序鉴定,测序结果与插入的寡核苷酸的序列完全相符.结论:DNMT1 siRNA重组质粒和DNMT3b siRNA重组质粒正确,可用于后续实验.

  18. The study of gene expression of siRNA inhibited magT1 in rat cadiocyte%siRNA抑制大鼠心肌细胞MagT1基因表达的研究

    Institute of Scientific and Technical Information of China (English)

    滕飞翔; 吴隽松

    2013-01-01

    Objective Mg2+is one of the most abundant cations in living cells which is cofactor of a variety of enzymes and maintained the conformation of the macromolecules activity ,regulated lipids and phosphoinositide second messenger.Mg2+plays im-portant role in mammal′s life activity which is necessary to the regulation of many kinds of ion channels and transporters .Clinical ob-servation showed that arrhythmia phenomenon appeared when the concentration of Mg 2+which in myocardial cells is low.In order to study the function of MagT 1 gene in myocardial cells deeply ,this study tried to determined the MagT 1 siRNA which with good silence effect,and to lay the foundation for follow -up study.Methods Isolated rat myocardial cells in vitro and checked the purity of myo-cardial cells ,entrusted Shanghai Jima Biotech Corp to designed four pairs of siRNA and mixed then dyed into rat cadiocyte ,tested the effect of siRNA silenced MagT 1 gene by the method of RT -PCR.Results Only one pairs of siRNA silence effect were found best though the test of RT -PCR,the sequence were 5′-GAUUGGACUUGUUGUGUUATT-3′;5′-UAACACAACAAGUCCAAUCTT-3′.Conclusion The siRNA that have better silence effect was found which laid the foundation for study the function of MagT 1 deep-ly by the method of RNA interference.%目的: Mg2+是活细胞中含量最丰富的二价阳离子之一,是多种酶的辅助因子,还是维持大分子有活性的构象,脂类和磷酸肌醇类第二信使的调控,多种离子通道和转运体的调控所必需的在哺乳动物细胞生命活动中起着重要作用。临床观察表明,心肌细胞内游离Mg2+浓度过低时会导致心律失常等现象。为了深入研究MagT1基因在心肌细胞中的功能,本研究试图确定沉默效果好的MagT1 siRNA,为后续实验奠定基础。方法体外分离大鼠心肌细胞,并检验心肌细胞的纯度;委托上海吉玛生物技术公司设计4对siRNA,与转染试剂混合并转

  19. Effect of Silencing CD105 Gene with siRNA on Proliferation of HUVECs and Expression of CD105%RNA干扰对体外培养HUVECs增殖和CD105表达的影响

    Institute of Scientific and Technical Information of China (English)

    丁怡; 张明昌

    2011-01-01

    Objective To investigate the effect of silencing CD105 gene expression with siRNA on the proliferation of human umbilical vein endothelial cells(HUVECs)and CD105 mRNA and protein expression. Methods HUVECs were cultured in vilro and transfected with siRNA of CD105 by lipofectamine 2000. The expression levels of CD105 mRNA and protein before and after transfection were detected by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR)and Western blot respectively. Apoptosis of HUVECs induced by CD105 siRNA was assayed by MTT. Results The expression level of CD105 mRNA in HUVECs was inhibited by the special siRNA. The expression of CD105 protein was down-regulated in HUVECs after interfered with siRNA. The cell proliferation in RNAi group was significantly lower than that in the negative control and control groups. Conclusion RNA interference can down-regulate the level of CD105 mRNA and protein expression, inducing the apoptosis of HUVECs. CD105 can accelerate the growth of HUVECs. CD105 may be used as a gene therapy target on corneal neovascularization.%目的 应用RNA干扰技术(RNAi)研究针对CD105的小片段RNA(siRNA)抑制CD105在体外培养的人脐静脉内皮细胞(HUVECs)中表达的情况,观察CD105沉默后对细胞生长的影响.方法 根据人的CD105 mRNA序列选择3个不同的靶位点,分别合成3条CD105 siRNA,并同时合成阴性对照和阳性对照siRNA.采用脂质体介导的基因法转染至体外培养的HUVECs中,采用RT-PCR检测各组细胞CD105表达水平,Western blot检测干扰前后CD105蛋白表达水平,并用MTT法检测人HUVECs的细胞活力.结果 RT-PCR和Western blot检测表明,3号CD105 siRNA能最有效沉默CD105的表达,转染后的HUVECs中CD105 mRNA水平和蛋白表达水平明显下降.MTT结果表明CD105的下调导致了HUVECs活力的下降.结论 特异性的siRNA可以下调HUVECs中CD105 mRNA及蛋白表达水平,减低细胞活力.也提示CD105可能对体外培

  20. Biodegradable Multiamine Polymeric Vector for siRNA Delivery.

    Science.gov (United States)

    Yuan, Yuanyuan; Gong, Faming; Cao, Yang; Chen, Weicai; Cheng, Du; Shuai, Xintao

    2015-04-01

    The gene silencing activity of small interfering RNA (siRNA) has led to their use as tools for target validation and as potential therapeutics for a variety of diseases. A major challenge is the development of vectors with high delivery efficiency and low toxicity. Although poly(ethylenimine) (PEI) has been regarded as the most promising polymeric vector for nucleic acid delivery, the nonbiodegradable structure greatly hinders its clinical application. In the present study, a diblock copolymer, PEG-PAsp(DIP-DETA), of poly(ethylene glycol) (PEG) and poly(L-aspartic acid) (PAsp) randomly grafted with pH-sensitive 2-(diisopropylamino)ethylamine (DIP) and diethylenetriamine (DETA) groups was synthesized via ring-opening polymerization and aminolysis reaction. Similar to polyethylenimine (PEI), the copolymer possesses a multiamine structure that not only allows effective siRNA complexation at neutral pH but also facilitates lysosomal release of siRNA via a proton buffering effect. Moreover, the poly(L-aspartic acid) backbone renders the vector biodegradability, which is not achievable with PEI. This novel polymeric vector can mediate effective intracellular siRNA delivery in various cancer cells. Consequently, the delivery of BCL-2 siRNA resulted in target gene silencing, inducing apoptosis and inhibiting the growth of cancer cells. These results show the potential of this non-PEI based polymeric vector with proton buffering capacity and biodegradability for siRNA delivery in cancer therapy.

  1. Enhancing endosomal escape for nanoparticle mediated siRNA delivery

    Science.gov (United States)

    Ma, Da

    2014-05-01

    Gene therapy with siRNA is a promising biotechnology to treat cancer and other diseases. To realize siRNA-based gene therapy, a safe and efficient delivery method is essential. Nanoparticle mediated siRNA delivery is of great importance to overcome biological barriers for systemic delivery in vivo. Based on recent discoveries, endosomal escape is a critical biological barrier to be overcome for siRNA delivery. This feature article focuses on endosomal escape strategies used for nanoparticle mediated siRNA delivery, including cationic polymers, pH sensitive polymers, calcium phosphate, and cell penetrating peptides. Work has been done to develop different endosomal escape strategies based on nanoparticle types, administration routes, and target organ/cell types. Also, enhancement of endosomal escape has been considered along with other aspects of siRNA delivery to ensure target specific accumulation, high cell uptake, and low toxicity. By enhancing endosomal escape and overcoming other biological barriers, great progress has been achieved in nanoparticle mediated siRNA delivery.

  2. Cloning of the Protective Antigen Gene of Bacillus anthracis

    Science.gov (United States)

    1983-09-01

    of the complicated precedents of duplicate toxin genes in chro- muumm mosomall and plasmid DNA of B. thuringiensis (Schnepf and Whitely, 1981; Klier...OiL V4. 34. S-W7. SW 1v 99 CwI 0193 by MT 0 009-7483/06O-002.00/0 mU"- - 1*;)-0Cloning of the Protective Antigen Gene OCT 19 MI L Sof Bacillus ...Sumnler uncertain, it is probably caused by other Bacillus antigens, 4 t which may include LF and EF. PA produced from recom- A The - "w t of a

  3. Albumin-mediated delivery of siRNA

    DEFF Research Database (Denmark)

    Bienk, Konrad

    2015-01-01

    . The human body, however, possesses several natural transport mechanisms for active transport of molecules. Amongst these is albumin, which is the most abundant plasma protein and has a circulatory half-life of ~21 days, partially due to engagement and recycling by the neonatal Fc receptor (FcRn). Albumin......The recent development of protein, peptide and nucleotide therapeutics is an important step towards the successful treatment of many severe diseases caused by altered gene expression. Small interfering RNA (siRNA) can be used for regulation of cellular gene expression to revert pathologic states...... vehicle. This proof of concept silencing showed that siRNA can be used for therapeutic purposes without the use of non-biocompatible polymer or lipid materials. This work, therefore, provides a novel technology platform for the safe delivery of siRNA therapeutics....

  4. Thymosin beta 4 protects cardiomyocytes from oxidative stress by targeting anti-oxidative enzymes and anti-apoptotic genes.

    Directory of Open Access Journals (Sweden)

    Chuanyu Wei

    Full Text Available BACKGROUND: Thymosin beta-4 (Tβ4 is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. The mechanism by which Tβ4 modulates cardiac protection under oxidative stress is not known. The purpose of this study is to dissect the cardioprotective mechanism of Tβ4 on H(2O(2 induced cardiac damage. METHODS: Rat neonatal cardiomyocytes with or without Tβ4 pretreatment were exposed to H(2O(2 and expression of antioxidant, apoptotic, and anti-inflammatory genes was evaluated by quantitative real-time PCR and western blotting. ROS levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant, anti-inflammatory and antiapoptotic genes were silenced by siRNA transfections in neonatal cardiomyocytes and effect of Tβ4 on H(2O(2-induced cardiac damage was evaluated. RESULTS: Pre-treatment of Tβ4 resulted in reduction of the intracellular ROS levels induced by H(2O(2 in cardiomyocytes. Tβ4 pretreatment also resulted in an increase in the expression of antiapoptotic proteins and reduction of Bax/BCl(2 ratio in the cardiomyocytes. Pretreatment with Tβ4 resulted in stimulating the expression of antioxidant enzymes copper/zinc SOD and catalase in cardiomyocytes at both transcription and translation levels. Tβ4 treatment resulted in the increased expression of anti-apoptotic and anti-inflammatory genes. Silencing of Cu/Zn SOD and catalase gene resulted in apoptotic cell death in the cardiomyocytes which was prevented by treatment with Tβ4. CONCLUSION: This is the first report that demonstrates the effect of Tβ4 on cardiomyocytes and its capability to selectively upregulate anti-oxidative enzymes, anti-inflammatory genes, and antiapoptotic enzymes in the neonatal cardiomyocytes thus preventing cell death thereby protecting the myocardium. Tβ4 treatment resulted in decreased oxidative stress and inflammation in the

  5. Effects of CD147 Gene Silencing by siRNA on Apoptosis and Proliferation of Hepatoma Cells%siRNA沉默CD147基因对肝癌细胞的影响

    Institute of Scientific and Technical Information of China (English)

    赵先胜; 任志群

    2011-01-01

    Objective To evaluate small interfering RNAs (siRNAs) for downregulation of the CD147 expression in HepG2. Methods According to the gene sequence of CD147 in Genbank, one DNA sequence containing a small hairpin structure were designed and synthesized. The complement form was cloned into vector pSilencerTM 4. 1- CMV nco, and the recombinant plasmid was transfected into HepG2. The inhibitory effects were detected by Western blotting, the cell proliferation was detected by MTT, and the cell apoptosis was measured by flow cytometry. Results The recombinant plasmid was successful cloned and inhibited CD147 protein expression specifically. The cell growth in siRNA group was significantly inhibited after 48 h in cell growth medium. Conclusion Successful cloning of the recombinant plasmid may help to discover new gene therapies for hepatoma.%目的 观察小分子干扰RNA(siRNA)沉默CD147基因在肝癌细胞HepG2中的表达,探讨CD147基因对肝癌细胞生长、凋亡的影响.方法 根据CD147 cDNA序列设计具有短发夹结构的两条DNA序列,与载体pSileneerTM4.1-CMV neo构建重组表达载体,鉴定后转染至HepG2细胞,Western blotting法检测抑制效果,MTT法检测细胞增殖情况,流式细胞术检测肿瘤细胞凋亡情况.结果 成功构建了针对CD147基因表达的干扰质粒,有效抑制了HepG2细胞增殖,促进了HepG2细胞凋亡.结论 CD147靶向RNA干扰重组表达栽体为肝癌的基因治疗提供了可能.

  6. MysiRNA-designer: a workflow for efficient siRNA design.

    Directory of Open Access Journals (Sweden)

    Mohamed Mysara

    Full Text Available The design of small interfering RNA (siRNA is a multi factorial problem that has gained the attention of many researchers in the area of therapeutic and functional genomics. MysiRNA score was previously introduced that improves the correlation of siRNA activity prediction considering state of the art algorithms. In this paper, a new program, MysiRNA-Designer, is described which integrates several factors in an automated work-flow considering mRNA transcripts variations, siRNA and mRNA target accessibility, and both near-perfect and partial off-target matches. It also features the MysiRNA score, a highly ranked correlated siRNA efficacy prediction score for ranking the designed siRNAs, in addition to top scoring models Biopredsi, DISR, Thermocomposition21 and i-Score, and integrates them in a unique siRNA score-filtration technique. This multi-score filtration layer filters siRNA that passes the 90% thresholds calculated from experimental dataset features. MysiRNA-Designer takes an accession, finds conserved regions among its transcript space, finds accessible regions within the mRNA, designs all possible siRNAs for these regions, filters them based on multi-scores thresholds, and then performs SNP and off-target filtration. These strict selection criteria were tested against human genes in which at least one active siRNA was designed from 95.7% of total genes. In addition, when tested against an experimental dataset, MysiRNA-Designer was found capable of rejecting 98% of the false positive siRNAs, showing superiority over three state of the art siRNA design programs. MysiRNA is a freely accessible (Microsoft Windows based desktop application that can be used to design siRNA with a high accuracy and specificity. We believe that MysiRNA-Designer has the potential to play an important role in this area.

  7. Carbohydrate-interactive pDNA and siRNA gene vectors based on boronic acid functionalized poly(amido amine)s

    NARCIS (Netherlands)

    Piest, Martin; Ankone, Martinus J.K.; Engbersen, Johannes F.J.

    2013-01-01

    In order to evaluate the influence of incorporation of boronic acid groups on the properties of poly(amido amine)s as gene vectors, a novel poly(amido amine) copolymer p(CBA-ABOL/2AMPBA) containing ortho-aminomethylphenylboronic acid (2AMPBA) moieties was prepared by Michael-type polyaddition of a m

  8. HIVsirDB: a database of HIV inhibiting siRNAs.

    Directory of Open Access Journals (Sweden)

    Atul Tyagi

    Full Text Available BACKGROUND: Human immunodeficiency virus (HIV is responsible for millions of deaths every year. The current treatment involves the use of multiple antiretroviral agents that may harm patients due to their toxic nature. RNA interference (RNAi is a potent candidate for the future treatment of HIV, uses short interfering RNA (siRNA/shRNA for silencing HIV genes. In this study, attempts have been made to create a database HIVsirDB of siRNAs responsible for silencing HIV genes. DESCRIPTIONS: HIVsirDB is a manually curated database of HIV inhibiting siRNAs that provides comprehensive information about each siRNA or shRNA. Information was collected and compiled from literature and public resources. This database contains around 750 siRNAs that includes 75 partially complementary siRNAs differing by one or more bases with the target sites and over 100 escape mutant sequences. HIVsirDB structure contains sixteen fields including siRNA sequence, HIV strain, targeted genome region, efficacy and conservation of target sequences. In order to facilitate user, many tools have been integrated in this database that includes; i siRNAmap for mapping siRNAs on target sequence, ii HIVsirblast for BLAST search against database, iii siRNAalign for aligning siRNAs. CONCLUSION: HIVsirDB is a freely accessible database of siRNAs which can silence or degrade HIV genes. It covers 26 types of HIV strains and 28 cell types. This database will be very useful for developing models for predicting efficacy of HIV inhibiting siRNAs. In summary this is a useful resource for researchers working in the field of siRNA based HIV therapy. HIVsirDB database is accessible at http://crdd.osdd.net/raghava/hivsir/.

  9. A Nonpolycationic Fully Proteinaceous Multiagent System for Potent Targeted Delivery of siRNA

    Science.gov (United States)

    Liu, David V; Yang, Nicole J; Wittrup, K Dane

    2014-01-01

    Protein-based methods of targeted short-interfering RNA (siRNA) delivery have the potential to solve some of the problems faced by nanoparticle-based methods, such as poor pharmacokinetics and biodistribution, low tumor penetration, and polydispersity. However, protein-based targeted delivery has been limited to fusion proteins with polycationic peptides as siRNA carriers, whose high charge density in some cases results in undesirable biophysical and in vivo properties. Here, we present a fully proteinaceous, multiagent approach for targeted siRNA delivery to epidermal growth factor receptor (EGFR), using a nonpolycationic carrier for siRNA. Each agent contributes a fundamentally different mechanism of action that work together for potent targeted RNA interference. The first agent is an EGFR-targeted fusion protein that uses a double-stranded RNA-binding domain as a nonpolycationic siRNA carrier. This double-stranded RNA-binding domain fusion protein can deliver siRNA to the endosomes of an EGFR-expressing cell line. A second agent delivers the cholesterol-dependent cytolysin, perfringolysin O, in a targeted manner, which enhances the endosomal escape of siRNA and induces gene silencing. A third agent that clusters EGFR increases gene-silencing potency and decreases cytolysin toxicity. Altogether, this system is potent, with only 16 nmol/l siRNA required for gene silencing and a therapeutic window that spans two orders of magnitude of targeted cytolysin concentrations. PMID:24825362

  10. Prediction of siRNA potency using sparse logistic regression.

    Science.gov (United States)

    Hu, Wei; Hu, John

    2014-06-01

    RNA interference (RNAi) can modulate gene expression at post-transcriptional as well as transcriptional levels. Short interfering RNA (siRNA) serves as a trigger for the RNAi gene inhibition mechanism, and therefore is a crucial intermediate step in RNAi. There have been extensive studies to identify the sequence characteristics of potent siRNAs. One such study built a linear model using LASSO (Least Absolute Shrinkage and Selection Operator) to measure the contribution of each siRNA sequence feature. This model is simple and interpretable, but it requires a large number of nonzero weights. We have introduced a novel technique, sparse logistic regression, to build a linear model using single-position specific nucleotide compositions which has the same prediction accuracy of the linear model based on LASSO. The weights in our new model share the same general trend as those in the previous model, but have only 25 nonzero weights out of a total 84 weights, a 54% reduction compared to the previous model. Contrary to the linear model based on LASSO, our model suggests that only a few positions are influential on the efficacy of the siRNA, which are the 5' and 3' ends and the seed region of siRNA sequences. We also employed sparse logistic regression to build a linear model using dual-position specific nucleotide compositions, a task LASSO is not able to accomplish well due to its high dimensional nature. Our results demonstrate the superiority of sparse logistic regression as a technique for both feature selection and regression over LASSO in the context of siRNA design.

  11. Nanoparticles containing siRNA to silence CD4 and CCR5 reduce expression of these receptors and inhibit HIV-1 infection in human female reproductive tract tissue explants

    Directory of Open Access Journals (Sweden)

    Susan K. Eszterhas

    2011-09-01

    Full Text Available Human Immunodeficiency Virus-type 1 (HIV- 1 binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV- 1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA sequences. Explants were then exposed two days later to HIV-1, and HIV-1 reverse transcripts were measured five days post-infection. Explants treated with nanoparticles containing CD4- and CCR5-specific siRNA showed reduced levels of CD4 and CCR5 transcripts, and significantly lower levels of HIV-1 reverse transcripts compared to those treated with an irrelevant siRNA. In female reproductive tract explants and in peripheral blood cell cultures, siRNA transfection induced the secretion of IFN-alpha (IFN-α, a potent antiviral cytokine. In female mice, murine-specific Cd4-siRNA nanoparticles instilled within the uterus significantly reduced murine Cd4 transcripts by day 3. Our findings demonstrate that siRNA nanoparticles reduce expression of HIV-1 infectivity receptors in human female reproductive tract tissues and also inhibit HIV-1 infection. Murine studies demonstrate that nanoparticles can penetrate the reproductive tract tissues in vivo and silence gene expression. The induction of IFN-α after siRNA transfection can potentially contribute to the antiviral effect. These findings support the therapeutic development of nanoparticles to deliver siRNA molecules to silence host cell receptors in the female reproductive tract as a novel microbicide to inhibit mucosal HIV-1 transmission.

  12. Menin missense mutants encoded by the MEN1 gene that are targeted to the proteasome: restoration of expression and activity by CHIP siRNA.

    Science.gov (United States)

    Canaff, Lucie; Vanbellinghen, Jean-François; Kanazawa, Ippei; Kwak, Hayeon; Garfield, Natasha; Vautour, Line; Hendy, Geoffrey N

    2012-02-01

    In multiple endocrine neoplasia type 1 (MEN1) characterized by tumors of parathyroid, enteropancreas, and anterior pituitary, missense mutations in the MEN1 gene product, menin, occur in a subset of cases. The mutant proteins are degraded by the proteasome. However, whether their expression and activity can be restored is not known. Our objective was to functionally characterize a panel of 16 menin missense mutants, including W423R and S443Y identified in new MEN1 families, with respect to protein stability, targeting to the proteasome and restoration of expression by proteasome inhibitors and expression and function by small interfering RNA technology. Flag-tagged wild-type (WT) and missense menin mutant expression vectors were transiently transfected in human embryonic kidney (HEK293) and/or rat insulinoma (Rin-5F) cells. The majority of mutants were short-lived, whereas WT menin was stable. Proteasome inhibitors MG132 and PS-341 and inhibition of the chaperone, heat-shock protein 70 (Hsp70), or the ubiquitin ligase, COOH terminus of Hsp70-interacting protein (CHIP), by specific small interfering RNA, restored the levels of the mutants, whereas that of WT menin was largely unaffected. Inhibition of CHIP restored the ability of mutants to mediate normal functions of menin: TGF-β up-regulation of the promoters of its target genes, the cyclin-dependent kinase inhibitors p15 and p21 as well as TGF-β inhibition of cell numbers. When the levels of missense menin mutants that are targeted to the proteasome are normalized they may function similarly to WT menin. Potentially, targeting specific components of the proteasome chaperone pathway could be beneficial in treating a subset of MEN1 cases.

  13. Combination of nanoparticle-delivered siRNA for Astrocyte elevated gene-1 (AEG-1) and all-trans retinoic acid (ATRA): an effective therapeutic strategy for hepatocellular carcinoma (HCC)

    Science.gov (United States)

    Rajasekaran, Devaraja; Srivastava, Jyoti; Ebeid, Kareem; Gredler, Rachel; Akiel, Maaged; Jariwala, Nidhi; Robertson, Chadia L.; Shen, Xue-Ning; Siddiq, Ayesha; Fisher, Paul B.; Salem, Aliasger K.; Sarkar, Devanand

    2016-01-01

    Hepatocellular carcinoma (HCC) is a fatal cancer with no effective therapy. Astrocyte elevated gene-1 (AEG-1) plays a pivotal role in hepatocarcinogenesis and inhibits retinoic acid-induced gene expression and cell death. Combination of a lentivirus expressing AEG-1 shRNA and all-trans retinoic acid (ATRA) profoundly and synergistically inhibited subcutaneous human HCC xenografts in nude mice. We now have developed liver-targeted nanoplexes by conjugating poly(amidoamine) (PAMAM) dendrimers with polyethylene glycol (PEG) and lactobionic acid (Gal) (PAMAM-PEG-Gal) which were complexed with AEG-1 siRNA (PAMAM-AEG-1si). The polymer conjugate was characterized by 1H-NMR, MALDI and mass spectrometry, and optimal nanoplex formulations were characterized for surface charge, size and morphology. Orthotopic xenografts of human HCC cell QGY-7703 expressing luciferase (QGY-luc) were established in the livers of athymic nude mice and tumor development was monitored by bioluminescence imaging (BLI). Tumor-bearing mice were treated with PAMAM-siCon, PAMAM-siCon+ATRA, PAMAM-AEG-1si and PAMAM-AEG-1si+ATRA. In the control group the tumor developed aggressively. ATRA showed little effect due to high AEG-1 levels in QGY-luc cells. PAMAM-AEG-1si showed significant reduction in tumor growth and the combination of PAMAM-AEG-1si+ATRA showed profound and synergistic inhibition so that the tumors were almost undetectable by BLI. A marked decrease in AEG-1 level was observed in tumor samples treated with PAMAM-AEG-1si. The group treated with PAMAM-AEG-1si+ATRA nanoplexes showed increased necrosis, inhibition of proliferation and increased apoptosis when compared to other groups. Liver is an ideal organ for RNAi therapy and ATRA is an approved anti-cancer agent. Our exciting observations suggest that the combinatorial approach might be an effective way to combat HCC. PMID:26079152

  14. The direct effect of Focal Adhesion Kinase (FAK, dominant-negative FAK, FAK-CD and FAK siRNA on gene expression and human MCF-7 breast cancer cell tumorigenesis

    Directory of Open Access Journals (Sweden)

    Zhang Li

    2009-08-01

    Full Text Available Abstract Background Focal adhesion kinase (FAK is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis. Methods To study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD, and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation in vitro, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD in vivo, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors. Results Induction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability in vitro and inhibited tumorigenesis in vivo. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p Conclusion Thus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis in vivo and reveal

  15. Whole-genome thermodynamic analysis reduces siRNA off-target effects.

    Directory of Open Access Journals (Sweden)

    Xi Chen

    Full Text Available Small interfering RNAs (siRNAs are important tools for knocking down targeted genes, and have been widely applied to biological and biomedical research. To design siRNAs, two important aspects must be considered: the potency in knocking down target genes and the off-target effect on any nontarget genes. Although many studies have produced useful tools to design potent siRNAs, off-target prevention has mostly been delegated to sequence-level alignment tools such as BLAST. We hypothesize that whole-genome thermodynamic analysis can identify potential off-targets with higher precision and help us avoid siRNAs that may have strong off-target effects. To validate this hypothesis, two siRNA sets were designed to target three human genes IDH1, ITPR2 and TRIM28. They were selected from the output of two popular siRNA design tools, siDirect and siDesign. Both siRNA design tools have incorporated sequence-level screening to avoid off-targets, thus their output is believed to be optimal. However, one of the sets we tested has off-target genes predicted by Picky, a whole-genome thermodynamic analysis tool. Picky can identify off-target genes that may hybridize to a siRNA within a user-specified melting temperature range. Our experiments validated that some off-target genes predicted by Picky can indeed be inhibited by siRNAs. Similar experiments were performed using commercially available siRNAs and a few off-target genes were also found to be inhibited as predicted by Picky. In summary, we demonstrate that whole-genome thermodynamic analysis can identify off-target genes that are missed in sequence-level screening. Because Picky prediction is deterministic according to thermodynamics, if a siRNA candidate has no Picky predicted off-targets, it is unlikely to cause off-target effects. Therefore, we recommend including Picky as an additional screening step in siRNA design.

  16. Development and characterization of siRNA lipoplexes: Effect of different lipids, in vitro evaluation in cancerous cell lines and in vivo toxicity study.

    Science.gov (United States)

    Khatri, Nirav; Baradia, Dipesh; Vhora, Imran; Rathi, Mohan; Misra, Ambikanandan

    2014-12-01

    Cationic liposomes have long been used as non-viral vectors for small interfering RNA (siRNA) delivery but are associated with high toxicity, less transfection efficiency, and in vivo instability. In this investigation, we have developed siRNA targeted to RRM1 that is responsible for development of resistance to gemcitabine in cancer cells. Effect of different lipid compositions has been evaluated on formation of stable and less toxic lipoplexes. Optimized cationic lipoplex (D2CH) system was comprised of dioleoyl-trimethylammoniumpropane (DOTAP), dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), hydrogenated soya phosphocholine (HSPC), cholesterol, and methoxy(polyethyleneglycol)2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG2000-DSPE). D2CH lipoplexes have shown particle size (147.5 ± 2.89 nm) and zeta potential (12.26 ± 0.54 mV) characteristics essential for their in vivo use. In vitro cytotoxicity study has shown low toxicity of developed lipoplexes as compared with lipofectamine-2000 up to N/P ratio as high as 7.5. Cell uptake studies and gene expression studies have confirmed intracellular availability of siRNA. In addition, developed lipoplexes also showed ~3 times less hemolytic potential as compared with DOTAP/DOPE lipoplexes at lipid concentration of 5 mg/mL. Lipoplexes also maintained particle size less than 200 nm on exposure to high electrolyte concentration and showed >70% siRNA retention in presence of serum showing siRNA protection conferred by lipoplexes. Furthermore, in vivo acute toxicity studies in mice showed that formulation was non-toxic up to a dosage of 0.75 mg of siRNA/kg as lipoplexes and 300 mg lipid/kg as blank liposomes indicating tolerability of lipoplexes at a dose much higher than required for therapeutic use. Promising results of this study warrant further investigation of developed siRNA lipoplexes for cancer treatment.

  17. VRP09 Reduction of Corneal Scanning Following Blast and Burn Injuries to Cornea Using siRNAs Targeting TGFb and CTGF

    Science.gov (United States)

    2011-10-01

    the three target gene mRNAs using cultures of rabbit corneal fibroblasts (RCF). The optimal siRNA for each gene was then formulated into double and...each of the three target genes, TGFb, TGFbRII and CTGF, using cultures of rabbit corneal fibroblasts (RCF). We will then test the optimal siRNA for... corneal fibroblasts for use in assessing effects of siRNAs on expression of the three target genes. We successfully established cultures of rabbit

  18. siRNA machinery in whitefly (Bemisia tabaci).

    Science.gov (United States)

    Upadhyay, Santosh Kumar; Dixit, Sameer; Sharma, Shailesh; Singh, Harpal; Kumar, Jitesh; Verma, Praveen C; Chandrashekar, K

    2013-01-01

    RNA interference has been emerged as an utmost tool for the control of sap sucking insect pests. Systemic response is necessary to control them in field condition. Whitefly is observed to be more prone to siRNA in recent studies, however the siRNA machinery and mechanism is not well established. To identify the core siRNA machinery, we curated transcriptome data of whitefly from NCBI database. Partial mRNA sequences encoding Dicer2, R2D2, Argonaute2 and Sid1 were identified by tblastn search of homologous sequences from Aphis glycines and Tribolium castaneum. Complete encoding sequences were obtained by RACE, protein sequences derived by Expasy translate tool and confirmed by blastp analysis. Conserved domain search and Prosite-Scan showed similar domain architecture as reported in homologs from related insects. We found helicase, PAZ, RNaseIIIa, RNaseIIIb and double-stranded RNA-binding fold (DSRBF) in Dicer2; DsRBD in R2D2; and PAZ and PIWI domains in Argonaute2. Eleven transmembrane domains were detected in Sid1. Sequence homology and phylogenetic analysis revealed that RNAi machinery of whitefly is close to Aphids. Real-time PCR analysis showed similar expression of these genes in different developmental stages as reported in A. glycines and T. castaneum. Further, the expression level of above genes was quite similar to the housekeeping gene actin. Availability of core siRNA machinery including the Sid1 and their universal expression in reasonable quantity indicated significant response of whitefly towards siRNA. Present report opens the way for controlling whitefly, one of the most destructive crop insect pest.

  19. Synthesis of polyethylenimine grafted with copolymers of polyethylene glycol and polycaprolactone and its potential for siRNA delivery

    Institute of Scientific and Technical Information of China (English)

    Wei Huang; Ming Lv; Zhong Gao Gao

    2011-01-01

    Copolymers mPEG-PCL were prepared and grafted onto polyethylenimine (PEI) to synthesize copolymers mPEG-PCL-g-PEI with low cytotoxicity. The mPEG-PCL-g-PEI could condense siRNA to form nanoparticles with positive zeta potential. These nanoparticles could delivery siRNA into cells to effectively inhibit the expression of target gene, which suggested that mPEG-PCL-g-PEl could serve as a highly efficient vector for siRNA delivery.

  20. Inhibiting the expression of CD28 costimulatory molecule on human lymphocytes by special siRNA

    Institute of Scientific and Technical Information of China (English)

    XU Kai-lin; ZHANG Ying; PAN Xiu-ying; LU Qun-xian

    2005-01-01

    Background The B7/CD28 pathway provides critical costimulatory signals for complete T cell activation, and members of this pathway have served as useful targets for immunotherapeutic strategies. In this study, we investigated the RNA interference (RNAi) effect induced by small interfering RNA (siRNA) targeting CD28 mRNA on human lymphocytes and its specificity.Methods According to CD28 gene sequence, we designed and synthysized three different siRNAs (siRNA-1, siRNA-2, siRNA-3) containing 21 bases using SilencerTM siRNA construction kit. These siRNAs were transfected into freshly isolated human lymphocytes with Lipofectamine 2000 reagent. At 24-hour, 48-hour and 72-hour post transfection, these cells were collected and analyzed. The changes of surface expression of CD28 gene were detected by flow cytometry, and the changes of CD28 mRNA levels were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The cell viability of transfected lymphocytes was determined by methyl thiazolyl tetrazolium (MTT) assay and trypan blue dye exclusion assay. Results Three siRNAs (siRNA-1, siRNA-2, siRNA-3) specifically targeting CD28 mRNA were successfully designed and constructed. Flow cytometry analysis showed that a decrease in CD28 expression was detectable at 24-hour post transfection. Different siRNA showed different inhibition effects on CD28 expression. At 48-hour post transfection, the degrees of reduction with siRNA-1, siRNA-2 and siRNA-3 were 22.10%±1.63%, 73.50%±1.02% and 42.90%±0.89% respectively compared with the control (P0.05). Moreover, lymphocytes treated with siRNA-co showed no marked reduction in CD28 expression (5.07%±0.96%) (P>0.05). The results of semi-quantitative RT-PCR assay indicated CD28 mRNA level was inhibited after transfection of specific siRNAs. At least 4-fold of reduction in siRNA-2 group occurred at 48-hour post transfection compared with the control (P0.05). Conclusions Three different siRNAs were

  1. Suppression of tumor growth in lung cancer xenograft model mice by poly(sorbitol-co-PEI)-mediated delivery of osteopontin siRNA.

    Science.gov (United States)

    Cho, Won-Young; Hong, Seong-Ho; Singh, Bijay; Islam, Mohammad Ariful; Lee, Somin; Lee, Ah Young; Gankhuyag, Nomundelger; Kim, Ji-Eun; Yu, Kyeong-Nam; Kim, Kwang-Ho; Park, Young-Chan; Cho, Chong-Su; Cho, Myung-Haing

    2015-08-01

    Small interfering RNA (siRNA)-mediated gene silencing represents a promising strategy for treating diseases such as cancer; however, specific gene silencing requires an effective delivery system to overcome the instability and low transfection efficiency of siRNAs. To address this issue, a polysorbitol-based transporter (PSOT) was prepared by low molecular weight branched polyethylenimine (bPEI) crosslinked with sorbitol diacrylate (SDA). Osteopontin (OPN) gene, which is highly associated with non-small cell lung cancer (NSCLC) was targeted by siRNA therapy using siRNA targeting OPN (siOPN). Characterization study confirmed that PSOT formed compact complexes with siOPN and protected siOPN against degradation by RNase. PSOT/siOPN complexes demonstrated low cytotoxicity and enhanced transfection efficiency in vitro, suggesting that this carrier may be suitable for gene silencing. In the A549 and H460 lung cancer cell lines, PSOT/siOPN complexes demonstrated significant silencing efficiency at both RNA and protein levels. To study in vivo tumor growth suppression, two lung cancer cell-xenograft mouse models were prepared and PSOT/siOPN complexes were delivered into the mice through intravenous injection. The siOPN-treated groups demonstrated significantly reduced OPN expression at both the RNA and protein levels as well as suppression of tumor volume and weight. Taken together, siOPN delivery using PSOT may present an effective and novel therapeutic system for lung cancer treatment.

  2. RNA干扰下调HE4基因表达对人卵巢癌SK-OV-3细胞增殖和侵袭的影响%Effect of HE4 gene silenced by siRNA on the proliferation and invasiveness of ovarian cancer cell SK-OV-3

    Institute of Scientific and Technical Information of China (English)

    房青; 王在; 游嘉; 詹雪梅; 杨爱莲; 魏继红; 房昭

    2014-01-01

    目的应用 RNA 干扰技术下调人附睾蛋白 HE4基因表达水平,观察 HE4对人卵巢癌 SK-OV-3细胞增殖和侵袭能力的影响。  方法化学合成3对 HE4特异性小分子干扰 RNA ( siRNA ),脂质体法转染人卵巢癌细胞系 SK-OV-3( HE4-siRNA 组),同时以非特异序列 siRNA 转染的SK-OV-3细胞(阴性对照组)和正常培养的 SK-OV-3细胞(正常对照组)为对照,于转染后48 h,采用实时荧光定量 PCR 技术(RT-qPCR)和 Western blot 方法分别检测HE4 mRNA 及蛋白表达水平。采用 CCK8试剂盒检测卵巢癌细胞增殖活性的变化,穿膜小室模型测定 HE4对细胞侵袭能力的影响。  结果与正常对照组相比,HE4-siRNA 转染卵巢癌SK-OV-3细胞48 h 后,HE4 mRNA 的表达水平显著下降,仅为正常对照组的12.7%(P0.05)。HE4-siRNA 下调卵巢癌 SK-OV-3细胞 HE4表达后,细胞增殖受到明显抑制,细胞增殖活性仅为正常对照组的60%,阴性对照组细胞增殖未见明显变化。体外侵袭实验显示,HE4-siRNA 组穿膜细胞数为每视野(21.8±2.86)个,显著低于正常对照组(187.4±11.17)个(P 0.05)。  结论 HE4特异性 siRNA 能成功下调 SK-OV-3细胞中HE4基因的表达,显著降低卵巢癌细胞增殖和侵袭能力, HE4有可能成为人卵巢癌侵袭转移防治的重要靶点。%Objective To investigate the effect of HE4 gene knocked down by small interfering RNA (siRNA) on proliferation and invasiveness of ovarian cancer cell SK-OV-3. Methods 3 pairs of synthetic siRNAs targeted HE4 gene were transiently transfected into SK-OV-3 cells by liposomal method (HE4-siRNA group), siRNA of scrambled sequence were transfected at the same time as a negative control, and normal cultured SK-OV-3 without any transfection were used as normal control. HE4 expression level in SK-OV-3 cells was determined using real-time quantitative PCR method and Western blot. CCK8 assay was

  3. Translational siRNA therapeutics using liposomal carriers: prospects & challenges.

    Science.gov (United States)

    Bhavsar, Dhiraj; Subramanian, Krishnakumar; Sethuraman, Swaminathan; Krishnan, Uma Maheswari

    2012-08-01

    Gene silencing has emerged as a promising strategy for molecular therapy of various malignant, viral, hereditary and inflammatory disorders. However, its translation from lab to clinic is yet to gain momentum due to the numerous problems that plague its development. A multi-functional siRNA delivery system with desired properties such as enhanced immune compatibility, target specificity, high cell uptake and excellent silencing efficiency is required to understand the challenges involved in the selection and modification of small interfering RNA (siRNA), factors influencing the complexation process and the response of the biological system to the formulation. Liposomes have been used as delivery systems due to its versatility in handling different types of drugs, tunable size, charge and surface functionalities that improve its effectiveness in vivo. This review highlights the challenges involved in gene silencing and describes the progression of liposomal systems used in gene silencing. The rationale in introducing chemical modifications in siRNA, synthesizing designer cationic lipids and evolution of hybrid liposomal systems has been elaborated, emphasizing their merits and short-comings. Finally, a description of the current state of clinical trials involving liposomal formulations has been included to provide an unbiased perspective of the future of liposomal systems and gene silencing tools as therapeutic tools.

  4. Ultrasound assisted siRNA delivery using PEG-siPlex loaded microbubbles.

    Science.gov (United States)

    Vandenbroucke, Roosmarijn E; Lentacker, Ine; Demeester, Joseph; De Smedt, Stefaan C; Sanders, Niek N

    2008-03-20

    Short interfering RNA (siRNA) attracts much attention for the treatment of various diseases. However, its delivery, especially via systemic routes, remains a challenge. Indeed, naked siRNAs are rapidly degraded, while complexed siRNAs massively aggregate in the blood or are captured by macrophages. Although this can be circumvented by PEGylation, we found that PEGylation had a strong negative effect on the gene silencing efficiency of siRNA-liposome complexes (siPlexes). Recently, ultrasound combined with microbubbles has been used to deliver naked siRNA but the gene silencing efficiency is rather low and very high amounts of siRNA are required. To overcome the negative effects of PEGylation and to enhance the efficiency of ultrasound assisted siRNA delivery, we coupled PEGylated siPlexes (PEG-siPlexes) to microbubbles. Ultrasound radiation of these microbubbles resulted in massive release of unaltered PEG-siPlexes. Interestingly, PEG-siPlexes loaded on microbubbles were able to enter cells after exposure to ultrasound, in contrast to free PEG-siPlexes, which were not able to enter cells rapidly. Furthermore, these PEG-siPlex loaded microbubbles induced, in the presence of ultrasound, much higher gene silencing than free PEG-siPlexes. Additionally, the PEG-siPlex loaded microbubbles only silenced the expression of genes in the presence of ultrasound, which allows space and time controlled gene silencing.

  5. Delivery of siRNA using ternary complexes containing branched cationic peptides: the role of peptide sequence, branching and targeting.

    Science.gov (United States)

    Kudsiova, Laila; Welser, Katharina; Campbell, Frederick; Mohammadi, Atefeh; Dawson, Natalie; Cui, Lili; Hailes, Helen C; Lawrence, M Jayne; Tabor, Alethea B

    2016-03-01

    Ternary nanocomplexes, composed of bifunctional cationic peptides, lipids and siRNA, as delivery vehicles for siRNA have been investigated. The study is the first to determine the optimal sequence and architecture of the bifunctional cationic peptide used for siRNA packaging and delivery using lipopolyplexes. Specifically three series of cationic peptides of differing sequence, degrees of branching and cell-targeting sequences were co-formulated with siRNA and vesicles prepared from a 1 : 1 molar ratio of the cationic lipid DOTMA and the helper lipid, DOPE. The level of siRNA knockdown achieved in the human alveolar cell line, A549-luc cells, in both reduced serum and in serum supplemented media was evaluated, and the results correlated to the nanocomplex structure (established using a range of physico-chemical tools, namely small angle neutron scattering, transmission electron microscopy, dynamic light scattering and zeta potential measurement); the conformational properties of each component (circular dichroism); the degree of protection of the siRNA in the lipopolyplex (using gel shift assays) and to the cellular uptake, localisation and toxicity of the nanocomplexes (confocal microscopy). Although the size, charge, structure and stability of the various lipopolyplexes were broadly similar, it was clear that lipopolyplexes formulated from branched peptides containing His-Lys sequences perform best as siRNA delivery agents in serum, with protection of the siRNA in serum balanced against efficient release of the siRNA into the cytoplasm of the cell.

  6. Antineoplastic Effects of siRNA against TMPRSS2-ERG Junction Oncogene in Prostate Cancer.

    Directory of Open Access Journals (Sweden)

    Giorgia Urbinati

    Full Text Available TMPRSS2-ERG junction oncogene is present in more than 50% of patients with prostate cancer and its expression is frequently associated with poor prognosis. Our aim is to achieve gene knockdown by siRNA TMPRSS2-ERG and then to assess the biological consequences of this inhibition. First, we designed siRNAs against the two TMPRSS2-ERG fusion variants (III and IV, most frequently identified in patients' biopsies. Two of the five siRNAs tested were found to efficiently inhibit mRNA of both TMPRSS2-ERG variants and to decrease ERG protein expression. Microarray analysis further confirmed ERG inhibition by both siRNAs TMPRSS2-ERG and revealed one common down-regulated gene, ADRA2A, involved in cell proliferation and migration. The siRNA against TMPRSS2-ERG fusion variant IV showed the highest anti-proliferative effects: Significantly decreased cell viability, increased cleaved caspase-3 and inhibited a cluster of anti-apoptotic proteins. To propose a concrete therapeutic approach, siRNA TMPRSS2-ERG IV was conjugated to squalene, which can self-organize as nanoparticles in water. The nanoparticles of siRNA TMPRSS2-ERG-squalene injected intravenously in SCID mice reduced growth of VCaP xenografted tumours, inhibited oncoprotein expression and partially restored differentiation (decrease in Ki67. In conclusion, this study offers a new prospect of treatment for prostate cancer based on siRNA-squalene nanoparticles targeting TMPRSS2-ERG junction oncogene.

  7. Construction of a vector generating both siRNA and a fluorescent reporter: a siRNA study in cultured neurons.

    Science.gov (United States)

    Yoon, Seung Yong; Choi, Jung Eun; Hwang, Onyou; Hong, Hea Nam; Lee, Heuiran; Kim, Yoo Kyum; Cho, Sung-Woo; Kim, Hyun; Kim, DongHou

    2004-08-31

    RNA interference is an important tool for gene silencing. However, its application to primary cultured cells has been limited by low transfection efficiencies. In this work we developed a vector which encodes both siRNA and red fluorescent protein. Using this vector we could markedly suppress green fluorescent protein (GFP) and bim an endogenous gene. Primary cultured cortical neurons transfected with siRNA against doublecortin showed that doublecortin expression was significantly inhibited in nearly all the transfected neurons. This vector identifies the transfected cells and should be useful for loss-of-gene function studies in neurons.

  8. Structural Dynamics of Human Argonaute2 and Its Interaction with siRNAs Designed to Target Mutant tdp43

    Directory of Open Access Journals (Sweden)

    Vishwambhar Bhandare

    2016-01-01

    Full Text Available The human Argonaute2 protein (Ago2 is a key player in RNA interference pathway and small RNA recognition by Ago2 is the crucial step in siRNA mediated gene silencing mechanism. The present study highlights the structural and functional dynamics of human Ago2 and the interaction mechanism of Ago2 with a set of seven siRNAs for the first time. The human Ago2 protein adopts two conformations such as “open” and “close” during the simulation of 25 ns. One of the domains named as PAZ, which is responsible for anchoring the 3′-end of siRNA guide strand, is observed as a highly flexible region. The interaction between Ago2 and siRNA, analyzed using a set of siRNAs (targeting at positions 128, 251, 341, 383, 537, 1113, and 1115 of mRNA designed to target tdp43 mutants causing Amyotrophic Lateral Sclerosis (ALS disease, revealed the stable and strong recognition of siRNA by the Ago2 protein during dynamics. Among the studied siRNAs, the siRNA341 is identified as a potent siRNA to recognize Ago2 and hence could be used further as a possible siRNA candidate to target the mutant tdp43 protein for the treatment of ALS patients.

  9. Structural Dynamics of Human Argonaute2 and Its Interaction with siRNAs Designed to Target Mutant tdp43.

    Science.gov (United States)

    Bhandare, Vishwambhar; Ramaswamy, Amutha

    2016-01-01

    The human Argonaute2 protein (Ago2) is a key player in RNA interference pathway and small RNA recognition by Ago2 is the crucial step in siRNA mediated gene silencing mechanism. The present study highlights the structural and functional dynamics of human Ago2 and the interaction mechanism of Ago2 with a set of seven siRNAs for the first time. The human Ago2 protein adopts two conformations such as "open" and "close" during the simulation of 25 ns. One of the domains named as PAZ, which is responsible for anchoring the 3'-end of siRNA guide strand, is observed as a highly flexible region. The interaction between Ago2 and siRNA, analyzed using a set of siRNAs (targeting at positions 128, 251, 341, 383, 537, 1113, and 1115 of mRNA) designed to target tdp43 mutants causing Amyotrophic Lateral Sclerosis (ALS) disease, revealed the stable and strong recognition of siRNA by the Ago2 protein during dynamics. Among the studied siRNAs, the siRNA341 is identified as a potent siRNA to recognize Ago2 and hence could be used further as a possible siRNA candidate to target the mutant tdp43 protein for the treatment of ALS patients.

  10. Multifunctional selenium nanoparticles as carriers of HSP70 siRNA to induce apoptosis of HepG2 cells

    Directory of Open Access Journals (Sweden)

    Li Y

    2016-07-01

    Full Text Available Yinghua Li,1 Zhengfang Lin,1 Mingqi Zhao,1 Tiantian Xu,1 Changbing Wang,1 Huimin Xia,1,* Hanzhong Wang,2,* Bing Zhu1,* 1Guangzhou Women and Children’s Medical Center, Guangzhou, Guangdong, 2State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People’s Republic of China *These authors contributed equally to this work Abstract: Small interfering RNA (siRNA as a new therapeutic modality holds promise for cancer treatment, but it is unable to cross cell membrane. To overcome this limitation, nanotechnology has been proposed for mediation of siRNA transfection. Selenium (Se is a vital dietary trace element for mammalian life and plays an essential role in the growth and functioning of humans. As a novel Se species, Se nanoparticles have attracted more and more attention for their higher anticancer efficacy. In the present study, siRNAs with polyethylenimine (PEI-modified Se nanoparticles (Se@PEI@siRNA have been demonstrated to enhance the apoptosis of HepG2 cells. Heat shock protein (HSP-70 is overexpressed in many types of human cancer and plays a significant role in several biological processes including the regulation of apoptosis. The objective of this study was to silence inducible HSP70 and promote the apoptosis of Se-induced HepG2 cells. Se@PEI@siRNA were successfully prepared and characterized by various microscopic methods. Se@PEI@siRNA showed satisfactory size distribution, high stability, and selectivity between cancer and normal cells. The cytotoxicity of Se@PEI@siRNA was lower for normal cells than tumor cells, indicating that these compounds may have fewer side effects. The gene-silencing efficiency of Se@PEI@siRNA was significantly much higher than Lipofectamine 2000@siRNA and resulted in a significantly reduced HSP70 mRNA and protein expression in cancer cells. When the expression of HSP70 was diminished, the function of cell protection was also removed and cancer cells became more

  11. Peptide- and Amine-Modified Glucan Particles for the Delivery of Therapeutic siRNA

    DEFF Research Database (Denmark)

    Cohen, Jessica L.; Shen, Yuefei; Aouadi, Myriam

    2016-01-01

    manufacturing and future commercialization. Here we report the synthesis and evaluation of a simplified glucan-based particle (GP) capable of delivering siRNA in vivo to selectively silence macrophage genes. Covalent attachment of small-molecule amines and short peptides containing weak bases to GPs facilitated...... to efficiently deliver siRNA to peritoneal macrophages in lean, healthy mice. In an animal model of obesity-induced inflammation, i.p. administration of one of the peptide-modified GPs (GP-EP14) bound to siRNA selectively reduced the expression of target inflammatory cytokines in the visceral adipose tissue...

  12. Soft computing model for optimized siRNA design by identifying off target possibilities using artificial neural network model.

    Science.gov (United States)

    Murali, Reena; John, Philips George; Peter S, David

    2015-05-15

    The ability of small interfering RNA (siRNA) to do posttranscriptional gene regulation by knocking down targeted genes is an important research topic in functional genomics, biomedical research and in cancer therapeutics. Many tools had been developed to design exogenous siRNA with high experimental inhibition. Even though considerable amount of work has been done in designing exogenous siRNA, design of effective siRNA sequences is still a challenging work because the target mRNAs must be selected such that their corresponding siRNAs are likely to be efficient against that target and unlikely to accidentally silence other transcripts due to sequence similarity. In some cases, siRNAs may tolerate mismatches with the target mRNA, but knockdown of genes other than the intended target could make serious consequences. Hence to design siRNAs, two important concepts must be considered: the ability in knocking down target genes and the off target possibility on any nontarget genes. So before doing gene silencing by siRNAs, it is essential to analyze their off target effects in addition to their inhibition efficacy against a particular target. Only a few methods have been developed by considering both efficacy and off target possibility of siRNA against a gene. In this paper we present a new design of neural network model with whole stacking energy (ΔG) that enables to identify the efficacy and off target effect of siRNAs against target genes. The tool lists all siRNAs against a particular target with their inhibition efficacy and number of matches or sequence similarity with other genes in the database. We could achieve an excellent performance of Pearson Correlation Coefficient (R=0. 74) and Area Under Curve (AUC=0.906) when the threshold of whole stacking energy is ≥-34.6 kcal/mol. To the best of the author's knowledge, this is one of the best score while considering the "combined efficacy and off target possibility" of siRNA for silencing a gene. The proposed model

  13. siRNA in milk exosomes is resistant to digestion and cross intestinal barrier in vitro.

    Science.gov (United States)

    Shandilya, Shruti; Rani, Payal; Onteru, Suneel Kumar; Singh, Dheer

    2017-10-02

    Milk is not only a composite of nutrients but emerged as a source of exosomes acting as a promising drug delivery vehicle for siRNA. siRNA is known for its immense therapeutic potential but has various physiological limitations including stable delivery. To investigate the suitability of siRNA for physiological stability and oral delivery, we encapsulated scrambled AF-488 siRNA in milk whey exosomes using lipofection and evaluated stability against the digestive processes along with their uptake and trans-epithelial transport by intestinal epithelial cells. Milk exosomal siRNA were found resistant to different digestive juices including saliva, gastric, bile and pancreatic juice in vitro and were internalized by Caco-2 cells. The stable delivery of exosomal-AF488 siRNA along with their trans-epithelial transport was confirmed by fluorescence microscopy and fluorescence intensity measurements. In summary, the encapsulation of siRNA in milk exosomes resists harsh digestive processes, improved intestinal permeability and payload protection.

  14. Synthesis and characterization of amino acid-functionalized calcium phosphate nanoparticles for siRNA delivery.

    Science.gov (United States)

    Bakan, Feray; Kara, Goknur; Cokol Cakmak, Melike; Cokol, Murat; Denkbas, Emir Baki

    2017-06-27

    Small interfering RNAs (siRNA) are short nucleic acid fragments of about 20-27 nucleotides, which can inhibit the expression of specific genes. siRNA based RNAi technology has emerged as a promising method for the treatment of a variety of diseases. However, a major limitation in the therapeutic use of siRNA is its rapid degradation in plasma and cellular cytoplasm, resulting in short half-life. In addition, as siRNA molecules cannot penetrate into the cell efficiently, it is required to use a carrier system for its delivery. In this work, chemically and morphologically different calcium phosphate (CaP) nanoparticles, including spherical-like hydroxyapatite (HA-s), needle-like hydroxyapatite (HA-n) and calcium deficient hydroxyapatite (CDHA) nanoparticles were synthesized by the sol-gel technique and the effects of particle characteristics on the binding capacity of siRNA were investigated. In order to enhance the gene loading efficiency, the nanoparticles were functionalized with arginine and the morphological and their structural characteristics were analyzed. The addition of arginine did not significantly change the particle sizes; however, it provided a significantly increased binding of siRNA for all types of CaP nanoparticles, as revealed by spectrophotometric measurements analysis. Arginine functionalized HA-n nanoparticles showed the best binding behavior with siRNA among the other nanoparticles due to its high, positive zeta potential (+18.8mV) and high surface area of Ca(++) rich "c" plane. MTT cytotoxicity assays demonstrated that all the nanoparticles tested herein were biocompatible. Our results suggest that high siRNA entrapment in each of the three modified non-toxic CaP nanoparticles make them promising candidates as a non-viral vector for delivering therapeutic siRNA molecules to treat cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Lipid-Modified Aminoglycoside Derivatives for In Vivo siRNA Delivery

    OpenAIRE

    2013-01-01

    Rationally designed siRNA delivery materials that are enabled by lipid-modified aminoglycosides are demonstrated. Leading materials identified are able to self-assemble with siRNA into well-defined nanoparticles and induce efficient gene knockdown both in vitro and in vivo. Histology studies and liver function tests reveal that no apparent toxicity is caused by these nanoparticles at doses over two orders of magnitude.

  16. Lipid-modified aminoglycoside derivatives for in vivo siRNA delivery.

    Science.gov (United States)

    Zhang, Yunlong; Pelet, Jeisa M; Heller, Daniel A; Dong, Yizhou; Chen, Delai; Gu, Zhen; Joseph, Brian J; Wallas, Jasmine; Anderson, Daniel G

    2013-09-06

    Rationally designed siRNA delivery materials that are enabled by lipid-modified aminoglycosides are demonstrated. Leading materials identified are able to self-assemble with siRNA into well-defined nanoparticles and induce efficient gene knockdown both in vitro and in vivo. Histology studies and liver function tests reveal that no apparent toxicity is caused by these nanoparticles at doses over two orders of magnitude.

  17. SiRNA and epigenetic aberrations in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Hamed Mirzaei

    2016-01-01

    Full Text Available Ovarian cancer has the most noteworthy lethal rate around gynecologic malignancies, and it is also considered as the fourth most frequent cancer in the woman in world. Two most critical barriers to treatment of ovarian malignancy are absence of early diagnostic markers and advancement of drug resistance after therapy, especially in advanced stages. Various epigenetic changes have been recognized in ovarian cancer. Recent progresses in our understanding of molecular pathogenesis of ovarian malignancy have dramatically provided potential new targets for molecularly targeted therapies. In very recent years, small interfering RNA (siRNA-mediated gene silencing has been emerging as a novel treatment modality in preclinical studies in the light of its strong gene-specific silencing. Gene suppression mediated by RNA interference (RNAi significantly suppressed gene expression at the messenger RNA (mRNA and protein levels. SiRNAs have therapeutic potential for ovarian cancer through various mechanisms. In this review, we not only provide an overview of siRNA designing for epigenetic silencing of genes aberrantly expressed in ovarian cancer but also we will highlight that the epigenetically silenced genes offer new targets for therapeutic approaches based on re-expression of tumor suppressor genes via demethylating and deacetylating drugs.

  18. Increasing the efficiency of homologous recombination vec-tor-mediated end joining repair by inhibition of Lig4 gene using siRNA in sheep embryo fibroblasts%siRNA 干扰绵羊胚胎成纤维细胞 Lig4基因增加同源重组载体重连修复效率

    Institute of Scientific and Technical Information of China (English)

    王伟; 王玉霜; 黄兰兰; 简子健; 王新华; 刘守仁; 皮文辉

    2016-01-01

    In animal cells, inhibition of non-homologous end joining (NHEJ) pathway improves the efficiency of homologous recombination (HR)-mediated double-strand brakes (DSBs) repair. To improve the efficiency of HR in sheep embryo fibroblasts, the NHEJ key molecule DNA ligase 4 (Lig4) was suppressed by siRNA interference. Four pairs of siRNA targeting Lig4 were designed and chemically synthesized. These siRNA were electro-transferred into sheep embryo fibroblasts respectively. Compared with the control groups, two pairs of siRNA were identified to ef-fectively inhibit the expression of sheep Lig4 gene by qRT-PCR and Western blotting. The plasmid rejoining assay was adopted for examining the efficiency of HR-mediated DSB repair. I-SceⅠ endonuclease linearized vector and siRNA were co-transfected into sheep embryo fibroblasts. Flow cytometry analysis of cells after transfection for 72 h showed that suppression of Lig4 using siRNAs increased the rejoining efficiency of HR vector by 3-4 times compared with the control groups. Therefore, enhanced HR vector rejoining frequency by instant inhabition of Lig4 gene pro-vides theoretical basis for improving gene targeting efficiency of sheep embryo fibroblasts.%在动物细胞中,抑制非同源末端连接(Non-homologous end joining, NHEJ)修复途径,可以提高同源重组(Homologous recombination, HR)修复基因组双链断裂(Double-strand brakes, DSBs)的发生概率。为了提高绵羊胚胎成纤维细胞的 HR 效率,针对 NHEJ 修复途径中的关键因子 Lig4(DNA ligase 4)基因,本文设计合成4个具有靶向性的 siRNA(Small interfering RNA)。绵羊胚胎成纤维细胞经电转染导入 siRNA,通过实时荧光定量PCR(qRT-PCR)和 Western blotting 检测,筛选出有效抑制 Lig4基因表达的2个 siRNA。应用质粒重连法检测HR 修复效率,将 I-SceⅠ酶线性化的 HR 质粒和 siRNA 共转染绵羊胚胎成纤维细胞,经72 h 培养及流式细胞仪检测,与对照

  19. 应用小干扰RNA技术阻断VEGF-C基因体内抑制乳癌细胞增殖%BLOCKING VASCULAR ENDOTHELIAL GROWTH FACTOR-C GENE TO INHIBIT BREAST CANCER CELL PROLIFERATION IN VIVO BY USING siRNA TECHNIQUE

    Institute of Scientific and Technical Information of China (English)

    李鲲; 葛银林; 张金玉

    2009-01-01

    目的 应用化学修饰的小干扰RNA(siRNA)阻断裸鼠乳癌移植瘤中血管内皮生长因子C(VEGF-C)基因, 探讨其对人乳癌MCF-7裸鼠移植瘤生长的影响.方法 于雌裸鼠皮下种植MCF-7细胞,将成瘤阳性的裸鼠随机分为VEGF-C siRNA处理组、脂质体组和对照组,每组5只.VEGF-C siRNA处理组肿瘤局部注射VEGF-C siRNA 1 mg/kg和PEITM,脂质体组肿瘤局部注射PEITM和PBS,对照组仅注射PBS,每3 d注射1次,连续注射8次.22 d后拉颈处死全部动物, 取肿瘤,采用半定量 RT-PCR 分析VEGF-C mRNA水平,Western blotting检测VEGF-C蛋白表达水平.结果 VEGF-C siRNA处理组瘤组织的增长受到明显抑制,VEGF-C基因的mRNA和蛋白表达水平显著降低(F=73.64~197.15,q=8.74~25.56,P<0.05).对照组各指标无显著变化.结论 化学修饰的siRNA介导的RNAi在体内能成功下调人乳癌裸鼠移植瘤中VEGF-C基因的表达, 抑制肿瘤的生长,是潜在的肿瘤基因治疗新方法.%Objective Using chemically-modified siRNA technique, vascular endothelial growth factor-C (VEGF-C) gene in transplanted breast cancer in nude mice was blocked and its effects on the growth of transplanted tumor investigated. MethodsHuman breast cancer cells MCF-7 were subcutaneously transplanted into the female mice, those with tumor-positive were rando-mized to three groups as VEGF-C siRNA, liposome and control, with five mice in each group. For VEGF-C siRNA group: VEGF-C siRNA 1 mg/kg and in vivo jetPEITM were injected locally; for liposome group: intra-tumor injection of in vivo jetPEITM and PBS was done; for control group: PBS alone was given. The medication was given every three days for eight times. The experimental animals were sacrificed 22 days later and tumors taken. Semiquantitative RT-PCR was applied for VEGF-C Mrna detection, and Western blotting for VEGF-C. Results The growth of tumor in VEGF-C siRNA group was greatly inhibited, the expressions of VEGF-C gene and protein

  20. Coupling of double-stranded RNA synthesis and siRNA generation in fission yeast RNAi.

    Science.gov (United States)

    Colmenares, Serafin U; Buker, Shane M; Buhler, Marc; Dlakić, Mensur; Moazed, Danesh

    2007-08-03

    The fission yeast centromeric repeats are transcribed and ultimately processed into small interfering RNAs (siRNAs) that are required for heterochromatin formation. siRNA generation requires dsRNA synthesis by the RNA-directed RNA polymerase complex (RDRC) and processing by the Dicer ribonuclease. Here we show that Dcr1, the fission yeast Dicer, is physically associated with RDRC. Dcr1 generates siRNAs in an ATP-dependent manner that requires its conserved N-terminal helicase domain. Furthermore, C-terminal truncations of Dcr1 that abolish its interaction with RDRC, but can generate siRNA in vitro, abolish siRNA generation and heterochromatic gene silencing in vivo. Finally, reconstitution experiments show that the association of Dcr1 with RDRC strongly stimulates the dsRNA synthesis activity of RDRC. Our results suggest that heterochromatic dsRNA synthesis and siRNA generation are physically coupled processes. This coupling has implications for cis-restriction of siRNA-mediated heterochromatin assembly and for mechanisms that give rise to siRNA strand polarity.

  1. Bolaamphiphiles as carriers for siRNA delivery: From chemical syntheses to practical applications.

    Science.gov (United States)

    Gupta, Kshitij; Afonin, Kirill A; Viard, Mathias; Herrero, Virginia; Kasprzak, Wojciech; Kagiampakis, Ioannis; Kim, Taejin; Koyfman, Alexey Y; Puri, Anu; Stepler, Marissa; Sappe, Alison; KewalRamani, Vineet N; Grinberg, Sarina; Linder, Charles; Heldman, Eliahu; Blumenthal, Robert; Shapiro, Bruce A

    2015-09-10

    In this study we have investigated a new class of cationic lipids--"bolaamphiphiles" or "bolas"--for their ability to efficiently deliver small interfering RNAs (siRNAs) to cancer cells. The bolas of this study consist of a hydrophobic chain with one or more positively charged head groups at each end. Recently, we reported that micelles of the bolas GLH-19 and GLH-20 (derived from vernonia oil) efficiently deliver siRNAs, while having relatively low toxicities in vitro and in vivo. Our previous studies validated that; bolaamphiphiles can be designed to vary the magnitude of siRNA shielding, its delivery, and its subsequent release. To further understand the structural features of bolas critical for siRNAs delivery, new structurally related bolas (GLH-58 and GLH-60) were designed and synthesized from jojoba oil. Both bolas have similar hydrophobic domains and contain either one, in GLH-58, or two, in GLH-60 positively charged head groups at each end of the hydrophobic core. We have computationally predicted and experimentally validated that GLH-58 formed more stable nano sized micelles than GLH-60 and performed significantly better in comparison to GLH-60 for siRNA delivery. GLH-58/siRNA complexes demonstrated better efficiency in silencing the expression of the GFP gene in human breast cancer cells at concentrations of 5μg/mL, well below the toxic dose. Moreover, delivery of multiple different siRNAs targeting the HIV genome demonstrated further inhibition of virus production.

  2. SiRNA sequence model: redesign algorithm based on available genome-wide libraries.

    Science.gov (United States)

    Kozak, Karol

    2013-12-01

    The evolution of RNA interference (RNAi) and the development of technologies exploiting its biology have enabled scientists to rapidly examine the consequences of depleting a particular gene product in cells. Design tools have been developed based on experimental data to increase the knockdown efficiency of siRNAs. Not all siRNAs that are developed to a given target mRNA are equally effective. Currently available design algorithms take an accession, identify conserved regions among their transcript space, find accessible regions within the mRNA, design all possible siRNAs for these regions, filter them based on multi-scores thresholds, and then perform off-target filtration. These different criteria are used by commercial suppliers to produce siRNA genome-wide libraries for different organisms. In this article, we analyze existing siRNA design algorithms and evaluate weight of design parameters for libraries produced in the last decade. We proved that not all essential parameters are currently applied by siRNA vendors. Based on our evaluation results, we were able to suggest an siRNA sequence pattern. The findings in our study can be useful for commercial vendors improving the design of RNAi constructs, by addressing both the issue of potency and the issue of specificity.

  3. Mussel-Inspired Modification of Nanofibers for REST siRNA Delivery: Understanding the Effects of Gene-Silencing and Substrate Topography on Human Mesenchymal Stem Cell Neuronal Commitment.

    Science.gov (United States)

    Low, Wei Ching; Rujitanaroj, Pim-On; Lee, Dong-Keun; Kuang, Jinghao; Messersmith, Phillip B; Chan, Jerry Kok Yen; Chew, Sing Yian

    2015-10-01

    In this study, we promote neuronal differentiation of human mesenchymal stem cells (MSCs) through scaffold-mediated sustained release of siRNA targeting RE-1 silencing transcription factor (REST). Poly (ϵ-caprolactone) nanofibers were surface modified with mussel inspired DOPA-melanin (DM) coating for adsorption of REST siRNA. DM modification increased siRNA-loading efficiency and reduced the initial burst release. Fiber alignment and DM modification enhanced REST knockdown efficiencies. Under non-specific differentiation condition, REST silencing and fiber topography enhanced MSC neuronal markers expressions and reduced glial cell commitment. Such scaffolds may find useful applications in enhancing MSCs neuronal differentiation under non-specific conditions such as an in vivo environment. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Targeted Delivery of siRNA to Transferrin Receptor Overexpressing Tumor Cells via Peptide Modified Polyethylenimine

    Directory of Open Access Journals (Sweden)

    Yuran Xie

    2016-10-01

    Full Text Available The use of small interference RNA (siRNA to target oncogenes is a promising treatment approach for cancer. However, siRNA cancer therapies are hindered by poor delivery of siRNA to cancer cells. Transferrin receptor (TfR is overexpressed in many types of tumor cells and therefore is a potential target for the selective delivery of siRNA to cancer cells. Here, we used the TfR binding peptide HAIYPRH (HAI peptide conjugated to cationic polymer branched polyethylenimine (bPEI, optimized the coupling strategy, and the TfR selective delivery of siRNA was evaluated in cells with high (H1299 and low TfR expression (A549 and H460. The HAI-bPEI conjugate exhibited chemico-physical properties in terms of size, zeta-potential, and siRNA condensation efficiency similar to unmodified bPEI. Confocal microscopy and flow cytometry results revealed that HAI-bPEI selectively delivered siRNA to H1299 cells compared with A549 or H460 cells. Moreover, HAI-bPEI achieved more efficient glyceraldehyde 3-phosphate dehydrogenase (GAPDH gene knockdown in H1299 cells compared with bPEI alone. However, despite optimization of the targeting peptide and coupling strategy, HAI-bPEI can only silence reporter gene enhanced green fluorescent protein (eGFP at the protein level when chloroquine is present, indicating that further optimization of the conjugate is required. In conclusion, the HAI peptide may be useful to target TfR overexpressing tumors in targeted gene and siRNA delivery approaches.

  5. Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA

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    Nathan M Belliveau

    2012-01-01

    Full Text Available Lipid nanoparticles (LNP are the leading systems for in vivo delivery of small interfering RNA (siRNA for therapeutic applications. Formulation of LNP siRNA systems requires rapid mixing of solutions containing cationic lipid with solutions containing siRNA. Current formulation procedures employ macroscopic mixing processes to produce systems 70-nm diameter or larger that have variable siRNA encapsulation efficiency, homogeneity, and reproducibility. Here, we show that microfluidic mixing techniques, which permit millisecond mixing at the nanoliter scale, can reproducibly generate limit size LNP siRNA systems 20 nm and larger with essentially complete encapsulation of siRNA over a wide range of conditions with polydispersity indexes as low as 0.02. Optimized LNP siRNA systems produced by microfluidic mixing achieved 50% target gene silencing in hepatocytes at a dose level of 10 µg/kg siRNA in mice. We anticipate that microfluidic mixing, a precisely controlled and readily scalable technique, will become the preferred method for formulation of LNP siRNA delivery systems.

  6. An inhalable β₂-adrenoceptor ligand-directed guanidinylated chitosan carrier for targeted delivery of siRNA to lung.

    Science.gov (United States)

    Luo, Yongfeng; Zhai, Xinyun; Ma, Chaonan; Sun, Peng; Fu, Zhiping; Liu, Wenguang; Xu, Jun

    2012-08-20

    SiRNA-based strategies appear to be an exciting new approach for the treatment of respiratory diseases. To extrapolate siRNA-mediated interventions from bench to bedside in this area, several aspects have to be jointly considered, including a safe and efficient gene carrier with pulmonary deposition efficiency, as well as in vivo method for siRNA/nanoparticles delivery. Accordingly, in this work, (i) a non-viral DNA vector, guanidinylated chitosan (GCS) that has been developed in our previous study [X.Y. Zhai, P. Sun, Y.F. Luo, C.N. Ma, J. Xu, W.G. Liu, 2011], was tested for siRNA delivery. We demonstrated that GCS was able to completely condense siRNA at weight ratio 40:1, forming nanosize particles of diameter ~100 nm, 15 mV in surface potential. Guanidinylation of chitosan not only decreased the cytotoxicity but also facilitated cellular internalization of siRNA nanoparticles, leading to an enhanced gene-silencing efficiency compared to the pristine chitosan (CS). (ii) We chemically coupled salbutamol, a β(2)-adrenoceptor agonist, to GCS (SGCS), which successfully improved targeting specificity of the green fluorescent protein (GFP)-siRNA carrier to lung cells harbored with β(2)-adrenergic receptor, and remarkably enhanced the efficacy of gene silence in vitro and in the lung of enhanced green fluorescent protein (EGFP)-transgenic mice in vivo. (iii) It was proved that this chitosan-based polymer was able to provide both the pDNA and siRNA with the protection against destructive shear forces generated by the mesh-based nebulizers. Aerosol treatment improved the nanoparticle size distribution, which should be in favor of enhancing the transfection efficiency. We suggest a potential application of the chitosan-derived nanodelivery vehicle (SGCS) in RNA interference therapy for lung diseases via aerosol inhalation.

  7. Cationic cell-penetrating peptides as vehicles for siRNA delivery.

    Science.gov (United States)

    Beloor, Jagadish; Zeller, Skye; Choi, Chang Seon; Lee, Sang-Kyung; Kumar, Priti

    2015-01-01

    RNA interference mediated gene silencing has tremendous applicability in fields ranging from basic biological research to clinical therapy. However, delivery of siRNA across the cell membrane into the cytoplasm, where the RNA silencing machinery is located, is a significant hurdle in most primary cells. Cell-penetrating peptides (CPPs), peptides that possess an intrinsic ability to translocate across cell membranes, have been explored as a means to achieve cellular delivery of siRNA. Approaches using CPPs by themselves or through incorporation into other siRNA delivery platforms have been investigated with the intent of improving cytoplasmic delivery. Here, we review the utilization of CPPs for siRNA delivery with a focus on strategies developed to enhance cellular uptake, endosomal escape and cytoplasmic localization of CPP/siRNA complexes.

  8. Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA

    Directory of Open Access Journals (Sweden)

    Zang X

    2016-08-01

    Full Text Available Xinlong Zang,1 Huaiwei Ding,2 Xiufeng Zhao,3 Xiaowei Li,1 Zhouqi Du,1 Haiyang Hu,1 Mingxi Qiao,1 Dawei Chen,1 Yuihui Deng,1 Xiuli Zhao1 1Department of Pharmaceutics, School of Pharmacy, 2Department of Pharmaceutical Chemistry, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China; 3Hongqi Hospital affiliated to Mudanjiang Medical University, Mudanjiang, People’s Republic of ChinaAbstract: Therapeutic delivery of small interfering RNA (siRNA is a major challenge that limits its potential clinical application. Here, a pH-sensitive cholesterol–Schiff base–polyethylene glycol (Chol–SIB–PEG-modified cationic liposome–siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph, was developed as an efficient nonviral siRNA delivery system. Chol–SIB–PEG was successfully synthesized and confirmed with FTIR and 1H-NMR. An Eph–PEG–SIB–Chol-modified liposome–siRNA complex (EPSLR was prepared and characterized by size, zeta potential, gel retardation, and encapsulation efficiency. Electrophoresis results showed that EPSLR was resistant to heparin replacement and protected siRNA from fetal bovine serum digestion. EPSLR exhibited only minor cytotoxicity in MCF-7/ADR cells. The results of flow cytometry and confocal laser scanning microscopy suggested that EPSLR enhanced siRNA transfection in MCF-7/ADR cells. Intracellular distribution experiment revealed that EPSLR could escape from the endo-lysosomal organelle and release siRNA into cytoplasm at 4 hours posttransfection. Western blot experiment demonstrated that EPSLR was able to significantly reduce the levels of MDR1 protein in MCF-7/ADR cells. The in vivo study of DIR-labeled complexes in mice bearing MCF-7/ADR tumor indicated that EPSLR could reach the tumor site rather than other organs more effectively. All these results demonstrate that EPSLR has much potential for effective siRNA delivery and may

  9. Strategies for ocular siRNA delivery: Potential and limitations of non-viral nanocarriers

    Directory of Open Access Journals (Sweden)

    Thakur Ajit

    2012-06-01

    Full Text Available Abstract Controlling gene expression via small interfering RNA (siRNA has opened the doors to a plethora of therapeutic possibilities, with many currently in the pipelines of drug development for various ocular diseases. Despite the potential of siRNA technologies, barriers to intracellular delivery significantly limit their clinical efficacy. However, recent progress in the field of drug delivery strongly suggests that targeted manipulation of gene expression via siRNA delivered through nanocarriers can have an enormous impact on improving therapeutic outcomes for ophthalmic applications. Particularly, synthetic nanocarriers have demonstrated their suitability as a customizable multifunctional platform for the targeted intracellular delivery of siRNA and other hydrophilic and hydrophobic drugs in ocular applications. We predict that synthetic nanocarriers will simultaneously increase drug bioavailability, while reducing side effects and the need for repeated intraocular injections. This review will discuss the recent advances in ocular siRNA delivery via non-viral nanocarriers and the potential and limitations of various strategies for the development of a ‘universal’ siRNA delivery system for clinical applications.

  10. Strategies for ocular siRNA delivery: Potential and limitations of non-viral nanocarriers.

    Science.gov (United States)

    Thakur, Ajit; Fitzpatrick, Scott; Zaman, Abeyat; Kugathasan, Kapilan; Muirhead, Ben; Hortelano, Gonzalo; Sheardown, Heather

    2012-06-11

    Controlling gene expression via small interfering RNA (siRNA) has opened the doors to a plethora of therapeutic possibilities, with many currently in the pipelines of drug development for various ocular diseases. Despite the potential of siRNA technologies, barriers to intracellular delivery significantly limit their clinical efficacy. However, recent progress in the field of drug delivery strongly suggests that targeted manipulation of gene expression via siRNA delivered through nanocarriers can have an enormous impact on improving therapeutic outcomes for ophthalmic applications. Particularly, synthetic nanocarriers have demonstrated their suitability as a customizable multifunctional platform for the targeted intracellular delivery of siRNA and other hydrophilic and hydrophobic drugs in ocular applications. We predict that synthetic nanocarriers will simultaneously increase drug bioavailability, while reducing side effects and the need for repeated intraocular injections. This review will discuss the recent advances in ocular siRNA delivery via non-viral nanocarriers and the potential and limitations of various strategies for the development of a 'universal' siRNA delivery system for clinical applications.

  11. Drug delivery of siRNA therapeutics: potentials and limits of nanosystems.

    Science.gov (United States)

    Reischl, Daniela; Zimmer, Andreas

    2009-03-01

    Gene therapy is a promising tool for the treatment of human diseases that cannot be cured by rational therapies. The major limitation for the use of small interfering RNA (siRNA), both in vitro and in vivo, is the inability of naked siRNA to passively diffuse through cellular membranes due to the strong anionic charge of the phosphate backbone and consequent electrostatic repulsion from the anionic cell membrane surface. Therefore, the primary success of siRNA applications depends on suitable vectors to deliver therapeutic genes. Cellular entrance is further limited by the size of the applied siRNA molecule. Multiple delivery pathways, both viral and nonviral, have been developed to bypass these problems and have been successfully used to gain access to the intracellular environment in vitro and in vivo, and to induce RNA interference (RNAi). This review focuses on different pathways for siRNA delivery and summarizes recent progress made in the use of vector-based siRNA technology.

  12. Pol IV-Dependent siRNA Production is Reduced in Brassica rapa

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    Rebecca A. Mosher

    2013-09-01

    Full Text Available Plants produce a diverse array of small RNA molecules capable of gene regulation, including Pol IV-dependent short interfering (p4-siRNAs that trigger transcriptional gene silencing. Small RNA transcriptomes are available for many plant species, but mutations affecting the synthesis of Pol IV-dependent siRNAs are characterized only in Arabidopsis and maize, leading to assumptions regarding nature of p4-siRNAs in all other species. We have identified a mutation in the largest subunit of Pol IV, NRPD1, that impacts Pol IV activity in Brassica rapa, an agriculturally important relative of the reference plant Arabidopsis. Using this mutation we characterized the Pol IV-dependent and Pol IV-independent small RNA populations in B. rapa. In addition, our analysis demonstrates reduced production of p4-siRNAs in B. rapa relative to Arabidopsis. B. rapa genomic regions are less likely to generate p4-siRNAs than Arabidopsis but more likely to generate Pol IV-independent siRNAs, including 24 nt RNAs mapping to transposable elements. These observations underscore the diversity of small RNAs produced by plants and highlight the importance of genetic studies during small RNA analysis.

  13. Trends in the Binding of Cell Penetrating Peptides to siRNA: A Molecular Docking Study

    Science.gov (United States)

    Gunathunge, B. G. C. M.; Wimalasiri, P. N.; Karunaratne, D. N.

    2017-01-01

    The use of gene therapeutics, including short interfering RNA (siRNA), is limited by the lack of efficient delivery systems. An appealing approach to deliver gene therapeutics involves noncovalent complexation with cell penetrating peptides (CPPs) which are able to penetrate the cell membranes of mammals. Although a number of CPPs have been discovered, our understanding of their complexation and translocation of siRNA is as yet insufficient. Here, we report on computational studies comparing the binding affinities of CPPs with siRNA, considering a variety of CPPs. Specifically, seventeen CPPs from three different categories, cationic, amphipathic, and hydrophobic CPPs, were studied. Molecular mechanics were used to minimize structures, while molecular docking calculations were used to predict the orientation and favorability of sequentially binding multiple peptides to siRNA. Binding scores from docking calculations were highest for amphipathic peptides over cationic and hydrophobic peptides. Results indicate that initial complexation of peptides will likely occur along the major groove of the siRNA, driven by electrostatic interactions. Subsequent binding of CPPs is likely to occur in the minor groove and later on bind randomly, to siRNA or previously bound CPPs, through hydrophobic interactions. However, hydrophobic CPPs do not show this binding pattern. Ultimately binding yields a positively charged nanoparticle capable of noninvasive cellular import of therapeutic molecules.

  14. Maternal control of Pol IV-dependent siRNAs in Arabidopsis endosperm.

    Science.gov (United States)

    Mosher, Rebecca A

    2010-04-01

    Small RNAs recently emerged as ubiquitous regulators of gene expression. However, the most abundant class of small RNAs in flowering plants is poorly understood. Known as Pol IV-dependent (p4-)siRNAs, these small RNAs are associated with transcriptional gene silencing, transposable elements and heterochromatin formation. Recent research demonstrates that they are initially expressed in the maternal gametophyte and uniparentally expressed from maternal chromosomes in developing endosperm. This unique expression pattern links p4-siRNAs to double fertilization, parental genome interactions and imprinted gene expression.

  15. Thermal Stability of siRNA Modulates Aptamer- conjugated siRNA Inhibition

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    Alexey Berezhnoy

    2012-01-01

    Full Text Available Oligonucleotide aptamer-mediated in vivo cell targeting of small interfering RNAs (siRNAs is emerging as a useful approach to enhance the efficacy and reduce the adverse effects resulting from siRNA-mediated genetic interference. A current main impediment in aptamer-mediated siRNA targeting is that the activity of the siRNA is often compromised when conjugated to an aptamer, often requiring labor intensive and time consuming design and testing of multiple configurations to identify a conjugate in which the siRNA activity has not been significantly reduced. Here, we show that the thermal stability of the siRNA is an important parameter of siRNA activity in its conjugated form, and that siRNAs with lower melting temperature (Tm are not or are minimally affected when conjugated to the 3′ end of 2′F-pyrimidine-modified aptamers. In addition, the configuration of the aptamer-siRNA conjugate retains activity comparable with the free siRNA duplex when the passenger strand is co-transcribed with the aptamer and 3′ overhangs on the passenger strand are removed. The approach described in this paper significantly reduces the time and effort necessary to screening siRNA sequences that retain biological activity upon aptamer conjugation, facilitating the process of identifying candidate aptamer-siRNA conjugates suitable for in vivo testing.

  16. Testing insecticidal activity of novel chemically synthesized siRNA against Plutella xylostella under laboratory and field conditions.

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    Liang Gong

    Full Text Available BACKGROUND: Over the last 60 years, synthetic chemical pesticides have served as a main tactic in the field of crop protection, but their availability is now declining as a result of the development of insect resistance. Therefore, alternative pest management agents are needed. However, the demonstration of RNAi gene silencing in insects and its successful usage in disrupting the expression of vital genes opened a door to the development of a variety of novel, environmentally sound approaches for insect pest management. METHODOLOGY/PRINCIPAL FINDINGS: Six small interfering RNAs (siRNAs were chemically synthesized and modified according to the cDNA sequence of P. xylostella acetylcholine esterase genes AChE1 and AChE2. All of them were formulated and used in insecticide activity screening against P. xylostella. Bioassay data suggested that Si-ace1_003 and Si-ace2_001 at a concentration of 3 µg cm(-2 displayed the best insecticidal activity with 73.7% and 89.0%, mortality, respectively. Additional bioassays were used to obtain the acute lethal concentrations of LC50 and LC90 for Si-ace2_001, which were 53.66 µg/ml and 759.71 µg/ml, respectively. Quantitative Real-time PCR was used to confirm silencing and detected that the transcript levels of P. xylostella AChE2 (PxAChE2 were reduced by 5.7-fold compared to the control group. Consequently, AChE activity was also reduced by 1.7-fold. Finally, effects of the siRNAs on treated plants of Brassica oleracea and Brassica alboglabra were investigated with different siRNA doses. Our results showed that Si-ace2_001 had no negative effects on plant morphology, color and growth of vein under our experimental conditions. CONCLUSIONS: The most important finding of this study is the discovery that chemically synthesized and modified siRNA corresponding to P. xylostella AChE genes cause significant mortality of the insect both under laboratory and field conditions, which provides a novel strategy to control P

  17. Peptide- and Amine-Modified Glucan Particles for the Delivery of Therapeutic siRNA.

    Science.gov (United States)

    Cohen, Jessica L; Shen, Yuefei; Aouadi, Myriam; Vangala, Pranitha; Tencerova, Michaela; Amano, Shinya U; Nicoloro, Sarah M; Yawe, Joseph C; Czech, Michael P

    2016-03-07

    Translation of siRNA technology into the clinic is limited by the need for improved delivery systems that target specific cell types. Macrophages are particularly attractive targets for RNAi therapy because they promote pathogenic inflammatory responses in a number of important human diseases. We previously demonstrated that a multicomponent formulation of β-1,3-d-glucan-encapsulated siRNA particles (GeRPs) can specifically and potently silence genes in mouse macrophages. A major advance would be to simplify the GeRP system by reducing the number of delivery components, thus enabling more facile manufacturing and future commercialization. Here we report the synthesis and evaluation of a simplified glucan-based particle (GP) capable of delivering siRNA in vivo to selectively silence macrophage genes. Covalent attachment of small-molecule amines and short peptides containing weak bases to GPs facilitated electrostatic interaction of the particles with siRNA and aided in the endosomal release of siRNA by the proton-sponge effect. Modified GPs were nontoxic and were efficiently internalized by macrophages in vitro. When injected intraperitoneally (i.p.), several of the new peptide-modified GPs were found to efficiently deliver siRNA to peritoneal macrophages in lean, healthy mice. In an animal model of obesity-induced inflammation, i.p. administration of one of the peptide-modified GPs (GP-EP14) bound to siRNA selectively reduced the expression of target inflammatory cytokines in the visceral adipose tissue macrophages. Decreasing adipose tissue inflammation resulted in an improvement of glucose metabolism in these metabolically challenged animals. Thus, modified GPs represent a promising new simplified system for the efficient delivery of therapeutic siRNAs specifically to phagocytic cells in vivo for modulation of inflammation responses.

  18. An atypical epigenetic mechanism affects uniparental expression of Pol IV-dependent siRNAs.

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    Rebecca A Mosher

    Full Text Available BACKGROUND: Small RNAs generated by RNA polymerase IV (Pol IV are the most abundant class of small RNAs in flowering plants. In Arabidopsis thaliana Pol IV-dependent short interfering (p4-siRNAs are imprinted and accumulate specifically from maternal chromosomes in the developing seeds. Imprinted expression of protein-coding genes is controlled by differential DNA or histone methylation placed in gametes. To identify epigenetic factors required for maternal-specific expression of p4-siRNAs we analyzed the effect of a series of candidate mutations, including those required for genomic imprinting of protein-coding genes, on uniparental expression of a representative p4-siRNA locus. RESULTS: Paternal alleles of imprinted genes are marked by DNA or histone methylation placed by DNA METHYLTRANSFERASE 1 or the Polycomb Repressive Complex 2. Here we demonstrate that repression of paternal p4-siRNA expression at locus 08002 is not controlled by either of these mechanisms. Similarly, loss of several chromatin modification enzymes, including a histone acetyltransferase, a histone methyltransferase, and two nucleosome remodeling proteins, does not affect maternal expression of locus 08002. Maternal alleles of imprinted genes are hypomethylated by DEMETER DNA glycosylase, yet expression of p4-siRNAs occurs irrespective of demethylation by DEMETER or related glycosylases. CONCLUSIONS: Differential DNA methylation and other chromatin modifications associated with epigenetic silencing are not required for maternal-specific expression of p4-siRNAs at locus 08002. These data indicate that there is an as yet unknown epigenetic mechanism causing maternal-specific p4-siRNA expression that is distinct from the well-characterized mechanisms associated with DNA methylation or the Polycomb Repressive Complex 2.

  19. Low molecular weight chitosan conjugated with folate for siRNA delivery in vitro: optimization studies

    Directory of Open Access Journals (Sweden)

    Shi Q

    2012-11-01

    Full Text Available Julio C Fernandes,1 Xingping Qiu,2 Francoise M Winnik,2 Mohamed Benderdour,1 Xiaoling Zhang,3 Kerong Dai,3 Qin Shi11Orthopaedics Research Laboratory, Research Centre, Sacré-Coeur Hospital, 2Department of Physical Chemistry and Polymer Science, Faculty of Pharmacy, University of Montreal, Montreal, Quebec, Canada; 3Orthopaedic Cellular and Molecular Biology Laboratories, Institute of Health Sciences, Chinese Academy of Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, ChinaAbstract: The low transfection efficiency of chitosan is one of its drawbacks as a gene delivery carrier. Low molecular weight chitosan may help to form small-sized polymer-DNA or small interfering RNA (siRNA complexes. Folate conjugation may improve gene transfection efficiency because of the promoted uptake of folate receptor-bearing cells. In the present study, chitosan was conjugated with folate and investigated for its efficacy as a delivery vector for siRNA in vitro. We demonstrate that the molecular weight of chitosan has a major influence on its biological and physicochemical properties, and very low molecular weight chitosan (below 10 kDa has difficulty in forming stable complexes with siRNA. In this study, chitosan 25 kDa and 50 kDa completely absorbed siRNA and formed nanoparticles (≤220 nm at a chitosan to siRNA weight ratio of 50:1. The introduction of a folate ligand onto chitosan decreased nanoparticle toxicity. Compared with chitosan-siRNA, folate-chitosan-siRNA nanoparticles improved gene silencing transfection efficiency. Therefore, folate-chitosan shows potential as a viable candidate vector for safe and efficient siRNA delivery.Keywords: nonviral vector, chitosan, gene delivery, folate-targeted, siRNA

  20. Self-assembled RNA interference microsponges for efficient siRNA delivery.

    Science.gov (United States)

    Lee, Jong Bum; Hong, Jinkee; Bonner, Daniel K; Poon, Zhiyong; Hammond, Paula T

    2012-04-01

    The encapsulation and delivery of short interfering RNA (siRNA) has been realized using lipid nanoparticles, cationic complexes, inorganic nanoparticles, RNA nanoparticles and dendrimers. Still, the instability of RNA and the relatively ineffectual encapsulation process of siRNA remain critical issues towards the clinical translation of RNA as a therapeutic. Here we report the synthesis of a delivery vehicle that combines carrier and cargo: RNA interference (RNAi) polymers that self-assemble into nanoscale pleated sheets of hairpin RNA, which in turn form sponge-like microspheres. The RNAi-microsponges consist entirely of cleavable RNA strands, and are processed by the cell's RNA machinery to convert the stable hairpin RNA to siRNA only after cellular uptake, thus inherently providing protection for siRNA during delivery and transport to the cytoplasm. More than half a million copies of siRNA can be delivered to a cell with the uptake of a single RNAi-microsponge. The approach could lead to novel therapeutic routes for siRNA delivery.

  1. Novel enabling technologies of gene isolation and plant transformation for improved crop protection

    Energy Technology Data Exchange (ETDEWEB)

    Torok, Tamas

    2013-02-04

    Meeting the needs of agricultural producers requires the continued development of improved transgenic crop protection products. The completed project focused on developing novel enabling technologies of gene discovery and plant transformation to facilitate the generation of such products.

  2. Suppression of diabetic retinopathy with GLUT1 siRNA.

    Science.gov (United States)

    You, Zhi-Peng; Zhang, Yu-Lan; Shi, Ke; Shi, Lu; Zhang, Yue-Zhi; Zhou, Yue; Wang, Chang-Yun

    2017-08-07

    To investigate the effect of glucose transporter-1 (GLUT1) inhibition on diabetic retinopathy, we divided forty-eight mice into scrambled siRNA, diabetic scrambled siRNA, and GLUT1 siRNA (intravitreally injected) groups. Twenty-one weeks after diabetes induction, we calculated retinal glucose concentrations, used electroretinography (ERG) and histochemical methods to assess photoreceptor degeneration, and conducted immunoblotting, leukostasis and vascular leakage assays to estimate microangiopathy. The diabetic scrambled siRNA and GLUT1 siRNA exhibited higher glucose concentrations than scrambled siRNA, but GLUT1 siRNA group concentrations were only 50.05% of diabetic scrambled siRNA due to downregulated GLUT1 expression. The diabetic scrambled siRNA and GLUT1 siRNA had lower ERG amplitudes and ONL thicknesses than scrambled siRNA. However, compared with diabetic scrambled siRNA, GLUT1 siRNA group amplitudes and thicknesses were higher. Diabetic scrambled siRNA cones were more loosely arranged and had shorter outer segments than GLUT1 siRNA cones. ICAM-1 and TNF-α expression levels, adherent leukocyte numbers, fluorescence leakage areas and extravasated Evans blue in diabetic scrambled siRNA were higher than those in scrambled siRNA. However, these parameters in the GLUT1 siRNA were lower than diabetic scrambled siRNA. Together, these results demonstrate that GLUT1 siRNA restricted glucose transport by inhibiting GLUT1 expression, which decreased retinal glucose concentrations and ameliorated diabetic retinopathy.

  3. Dose-ranging evaluation of intravitreal siRNA PF-04523655 for diabetic macular edema (the DEGAS study)

    DEFF Research Database (Denmark)

    Nguyen, Quan Dong; Schachar, Ronald A; Nduaka, Chudy I;

    2012-01-01

    To evaluate the safety and efficacy of three doses of PF-04523655, a 19-nucleotide methylated double stranded siRNA targeting the RTP801 gene, for the treatment of diabetic macular edema (DME) compared to focal/grid laser photocoagulation.......To evaluate the safety and efficacy of three doses of PF-04523655, a 19-nucleotide methylated double stranded siRNA targeting the RTP801 gene, for the treatment of diabetic macular edema (DME) compared to focal/grid laser photocoagulation....

  4. Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo

    DEFF Research Database (Denmark)

    Laursen, Maria B; Pakula, Malgorzata M; Gao, Shan;

    2010-01-01

    Small interfering RNAs (siRNAs) are now established as a favourite tool to reduce gene expression by RNA interference (RNAi) in mammalian cell culture. However, limitations in potency, duration, delivery and specificity of the gene knockdown (KD) are still major obstacles that need further addres...... in a xenograft model of human pancreas cancer. Hereby UNA constitutes an important type of chemical modification for future siRNA designs....

  5. Rational design of micro-RNA-like bifunctional siRNAs targeting HIV and the HIV coreceptor CCR5.

    Science.gov (United States)

    Ehsani, Ali; Saetrom, Pål; Zhang, Jane; Alluin, Jessica; Li, Haitang; Snøve, Ola; Aagaard, Lars; Rossi, John J

    2010-04-01

    Small-interfering RNAs (siRNAs) and micro-RNAs (miRNAs) are distinguished by their modes of action. SiRNAs serve as guides for sequence-specific cleavage of complementary mRNAs and the targets can be in coding or noncoding regions of the target transcripts. MiRNAs inhibit translation via partially complementary base-pairing to 3' untranslated regions (UTRs) and are generally ineffective when targeting coding regions of a transcript. In this study, we deliberately designed siRNAs that simultaneously direct cleavage and translational suppression of HIV RNAs, or cleavage of the mRNA encoding the HIV coreceptor CCR5 and suppression of translation of HIV. These bifunctional siRNAs trigger inhibition of HIV infection and replication in cell culture. The design principles have wide applications throughout the genome, as about 90% of genes harbor sites that make the design of bifunctional siRNAs possible.

  6. Self-assembled nanoscale coordination polymers carrying siRNAs and cisplatin for effective treatment of resistant ovarian cancer.

    Science.gov (United States)

    He, Chunbai; Liu, Demin; Lin, Wenbin

    2015-01-01

    Resistance to the chemotherapeutic agent cisplatin is a major limitation for the successful treatment of many cancers. Development of novel strategies to overcome intrinsic and acquired resistance to chemotherapy is of critical importance to effective treatment of ovarian cancer and other types of cancers. We have sought to re-sensitize resistant ovarian cancer cells to chemotherapy by co-delivering chemotherapeutics and pooled siRNAs targeting multi-drug resistance (MDR) genes using self-assembled nanoscale coordination polymers (NCPs). In this work, NCP-1 particles with trigger release properties were first constructed by linking cisplatin prodrug-based bisphosphonate bridging ligands with Zn(2+) metal-connecting points and then coated with a cationic lipid layer, followed by the adsorption of pooled siRNAs targeting three MDR genes including survivin, Bcl-2, and P-glycoprotein via electrostatic interactions. The resulting NCP-1/siRNA particles promoted cellular uptake of cisplatin and siRNA and enabled efficient endosomal escape in cisplatin-resistant ovarian cancer cells. By down-regulating the expression of MDR genes, NCP-1/siRNAs enhanced the chemotherapeutic efficacy as indicated by cell viability assay, DNA ladder, and flow cytometry. Local administration of NCP-1/siRNAs effectively reduced tumor sizes of cisplatin-resistant SKOV-3 subcutaneous xenografts. This work shows that the NCP-1/siRNA platform holds great promise in enhancing chemotherapeutic efficacy for the effective treatment of drug-resistant cancers.

  7. Does bilirubin protect against hemochromatosis gene (HFE) related mortality?

    NARCIS (Netherlands)

    Alizadeh, Behrooz Z.; Njajou, Omer T.; Houwing-Duistermaat, Jeanine J.; de Jong, Gerard; Vergeer, Jeannette M.; Hofman, Albert; Pols, Huibert A.P.; van Duijn, Cornelia M.

    2004-01-01

    Serum bilirubin is an important antioxidant that is found at increased levels in hereditary hemochromatosis patients. We hypothesized that increased levels of serum bilirubin may play a protective role against oxidative stress induced by iron overload in carriers of mutations in the hereditary hemoc

  8. 白细胞介素-12干扰RNA转染树突状细胞对淋巴细胞增殖的影响%The effects of IL-12 siRNA gene-modified dentritic cells on mixed lymphocyte reaction

    Institute of Scientific and Technical Information of China (English)

    罗建飞; 童仕伦; 陈必成; 陈忠华

    2009-01-01

    目的 观察白细胞介素(IL)-12两亚基(IL-12p35、IL-12p40)干扰RNA(IL-12p35siRNA、IL-12p40siRNA)重组腺病毒载体感染树突状细胞(DC)后对淋巴细胞增殖的影响.方法 用重组腺病毒载体(IL-12p35siRNA、IL-12p40siRNA、阴性对照HKsiRNA)感染DC,之后与同种小鼠T细胞行混合淋巴细胞培养,并测定上清液中IL-4和干扰素(IFN)-γ浓度以及淋巴细胞增殖反应.结果 IL-12p35siRNA、IL-12p40siRNA重组腺病毒载体分别感染树突状细胞后,只有IL-12p35siRNA重组腺病毒载体感染DC在混合淋巴细胞培养中引起的淋巴细胞增殖能力最低,并导致混合培养上清液中IL-4的上升和IFN-γ的下降.结论 IL-12p35亚基特异性siRNA抑制DC的共刺激活动,导致在体外TH2偏移.%Objective To explore the biological characteristics of IL-12p35 siRNA and IL-12p40 siRNA gene-modified dendritic cells (DC) in vitro. Methods The recombinant adenovirus expression plasmids(Ad-IL-12p35 siRNA,Ad-IL-12p40 siRNA, Ad-HKsiRNA) were transfected into bone marrow-derived DC (BMDC) respectively. Murine T cell proliferation stimulated by gene-modified DC was detec-ted by MTT assay. The levels of TH1/TH2 cytokines such as IFN-γ and IL-4 in the mixed lymphocyte re-action (MLR) supernatant were examined by ELISA. Results DC modified by IL-12p35 siRNA exhibited weak activity in stimulating the proliferation of allogene T cells. T cells in MLR expressed higher levels of TH2 cytokiues such as IL-4,but lower levels of TH1 cytokines such as IFN-γ in group of IL-12p35siRNA gene-modified DC than other groups. Conclusion IL-12p35-silenced DC suppressed proliferation of allo-gene T cells,which polarized the immune response toward a TH2 cytokine.

  9. Silencing of Ror-1 and Fibromodulin with siRNA results in apoptosis of CLL cells

    OpenAIRE

    Choudhury, Aniruddha; Derkow, Katja; Daneshmanesh, Amir Hossein; Mikaelsson, Eva; Kiaii, Shahryar; Kokhaei, Parviz; Österborg, Anders; Mellstedt, Håkan

    2010-01-01

    Abstract We have previously demonstrated that Ror1 and fibromodulin (FMOD) are two genes upregulated in CLL cells compared to normal blood B cells. In this study, we have used siRNAs to specifically silence Ror1 and fibromodulin gene expression in CLL cells, healthy B cells and human fibroblast cell lines. siRNA treatment induced a specific reduction (75?95%) in fibromodulin and Ror1 mRNA. Western blot analysis with specific antibodies for fibromodulin and Ror1 demonstrated that th...

  10. [Protection of corneal endothelium from apoptosis by gene and cell therapy].

    Science.gov (United States)

    Fuchsluger, T A

    2016-06-01

    Protection of corneal endothelium from apoptosis using gene and cell therapy is in a translational phase. This approach offers advantages for eye banking and after transplantation. Safe vehicles for gene or cell therapeutic transduction of corneal endothelium with nucleic acids are available. This strategy will be further developed in consultation with the Paul Ehrlich Institute and European regulatory authorities.

  11. Orally delivered thioketal nanoparticles loaded with TNF-α-siRNA target inflammation and inhibit gene expression in the intestines

    Science.gov (United States)

    Wilson, D. Scott; Dalmasso, Guillaume; Wang, Lixin; Sitaraman, Shanthi V.; Merlin, Didier; Murthy, Niren

    2010-11-01

    Small interfering RNAs (siRNAs) directed against proinflammatory cytokines have the potential to treat numerous diseases associated with intestinal inflammation; however, the side-effects caused by the systemic depletion of cytokines demands that the delivery of cytokine-targeted siRNAs be localized to diseased intestinal tissues. Although various delivery vehicles have been developed to orally deliver therapeutics to intestinal tissue, none of these strategies has demonstrated the ability to protect siRNA from the harsh environment of the gastrointestinal tract and target its delivery to inflamed intestinal tissue. Here, we present a delivery vehicle for siRNA, termed thioketal nanoparticles (TKNs), that can localize orally delivered siRNA to sites of intestinal inflammation, and thus inhibit gene expression in inflamed intestinal tissue. TKNs are formulated from a polymer, poly-(1,4-phenyleneacetone dimethylene thioketal), that degrades selectively in response to reactive oxygen species (ROS). Therefore, when delivered orally, TKNs release siRNA in response to the abnormally high levels of ROS specific to sites of intestinal inflammation. Using a murine model of ulcerative colitis, we demonstrate that orally administered TKNs loaded with siRNA against the proinflammatory cytokine tumour necrosis factor-alpha (TNF-α) diminish TNF-α messenger RNA levels in the colon and protect mice from ulcerative colitis.

  12. Nucleotide Sequence of the Protective Antigen Gene of Bacillus Anthracis

    Science.gov (United States)

    1988-02-02

    Montie, S. Kadis, and S. I. Ajl (ed.), Microbial toxins, vol. 3. Academic Press, Inc., New York. 23. Little, S. F., and G. B. Knudaon. 1986...Takkinen, and L. Kaariainen. 1981. Nucleotide sequence of the promoter and NHa-terminal signal peptide region of the a- amylase gene from Bacillus

  13. Assessment of Nanobiotechnology-Targeted siRNA Designed to inhibit NF-kappaB Classical and Alternative Signaling in Breast Tumor Macrophages

    Science.gov (United States)

    2013-07-01

    Centrifugation Whole blood compatibility PAT/FA NPs Lipo - fectamine 6 Free siRNA (left), relative to siRNA protected by PAT-SPNs from degradation...and angiogenesis in the absence of VEGF (14). Accordingly, a monoclonal antibody (mAb) against galec- tin-1 inhibited growth of Kaposi sarcoma and B16

  14. Splicing stimulates siRNA formation at Drosophila DNA double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Karin Merk

    2017-06-01

    Full Text Available DNA double-strand breaks trigger the production of locus-derived siRNAs in fruit flies, human cells and plants. At least in flies, their biogenesis depends on active transcription running towards the break. Since siRNAs derive from a double-stranded RNA precursor, a major question is how broken DNA ends can generate matching sense and antisense transcripts. We performed a genome-wide RNAi-screen in cultured Drosophila cells, which revealed that in addition to DNA repair factors, many spliceosome components are required for efficient siRNA generation. We validated this observation through site-specific DNA cleavage with CRISPR-cas9 followed by deep sequencing of small RNAs. DNA breaks in intron-less genes or upstream of a gene's first intron did not efficiently trigger siRNA production. When DNA double-strand breaks were induced downstream of an intron, however, this led to robust siRNA generation. Furthermore, a downstream break slowed down splicing of the upstream intron and a detailed analysis of siRNA coverage at the targeted locus revealed that unspliced pre-mRNA contributes the sense strand to the siRNA precursor. Since splicing factors are stimulating the response but unspliced transcripts are entering the siRNA biogenesis, the spliceosome is apparently stalled in a pre-catalytic state and serves as a signaling hub. We conclude that convergent transcription at DNA breaks is stimulated by a splicing dependent control process. The resulting double-stranded RNA is converted into siRNAs that instruct the degradation of cognate mRNAs. In addition to a potential role in DNA repair, the break-induced transcription may thus be a means to cull improper RNAs from the transcriptome of Drosophila melanogaster. Since the splicing factors identified in our screen also stimulated siRNA production from high copy transgenes, it is possible that this surveillance mechanism serves in genome defense beyond DNA double-strand breaks.

  15. Delivery of kinesin spindle protein targeting siRNA in solid lipid nanoparticles to cellular models of tumor vasculature

    Energy Technology Data Exchange (ETDEWEB)

    Ying, Bo; Campbell, Robert B., E-mail: robert.campbell@mcphs.edu

    2014-04-04

    Highlights: • siRNA-lipid nanoparticles are solid particles not lipid bilayers with aqueous core. • High, but not low, PEG content can prevent nanoparticle encapsulation of siRNA. • PEG reduces cellular toxicity of cationic nanoparticles in vitro. • PEG reduces zeta potential while improving gene silencing of siRNA nanoparticles. • Kinesin spindle protein can be an effective target for tumor vascular targeting. - Abstract: The ideal siRNA delivery system should selectively deliver the construct to the target cell, avoid enzymatic degradation, and evade uptake by phagocytes. In the present study, we evaluated the importance of polyethylene glycol (PEG) on lipid-based carrier systems for encapsulating, and delivering, siRNA to tumor vessels using cellular models. Lipid nanoparticles containing different percentage of PEG were evaluated based on their physical chemical properties, density compared to water, siRNA encapsulation, toxicity, targeting efficiency and gene silencing in vitro. siRNA can be efficiently loaded into lipid nanoparticles (LNPs) when DOTAP is included in the formulation mixture. However, the total amount encapsulated decreased with increase in PEG content. In the presence of siRNA, the final formulations contained a mixed population of particles based on density. The major population which contains the majority of siRNA exhibited a density of 4% glucose, and the minor fraction associated with a decreased amount of siRNA had a density less than PBS. The inclusion of 10 mol% PEG resulted in a greater amount of siRNA associated with the minor fraction. Finally, when kinesin spindle protein (KSP) siRNA was encapsulated in lipid nanoparticles containing a modest amount of PEG, the proliferation of endothelial cells was inhibited due to the efficient knock down of KSP mRNA. The presence of siRNA resulted in the formation of solid lipid nanoparticles when prepared using the thin film and hydration method. LNPs with a relatively modest amount of

  16. Thermo-sensitive nanoparticles for triggered release of siRNA.

    Science.gov (United States)

    Yang, Zheng; Cheng, Qiang; Jiang, Qian; Deng, Liandong; Liang, Zicai; Dong, Anjie

    2015-01-01

    Efficient delivery of small interfering RNA (siRNA) is crucially required for cancer gene therapy. Herein, a thermo-sensitive copolymer with a simple structure, poly (ethylene glycol) methyl ether acrylate-b-poly (N-isopropylacrylamide) (mPEG-b-PNIPAM) was developed. A novel kind of thermo-sensitive nanoparticles (DENPs) was constructed for the cold-shock triggered release of siRNA by double emulsion-solvent evaporation method using mPEG-b-PNIPAM and a cationic lipid, 3β [N-(N', N'-dimethylaminoethane)-carbamoyl] cholesterol [DC-Chol]. DENPs were observed by transmission electron microscopy and dynamical light scattering before and after 'cold shock' treatment. The encapsulation efficiency (EE) of siRNA in DENPs, which was measured by fluorescence spectrophotometer was 96.8% while it was significantly reduced to be 23.2% when DC-Chol was absent. DENPs/siRNA NPs exhibited a thermo-sensitive siRNA release character that the cumulatively released amount of siRNA from cold shock was approximately 2.2 folds higher after 7 days. In vitro luciferase silencing experiments indicated that DENPs showed potent gene silencing efficacy in HeLa-Luc cells (HeLa cells steadily expressed luciferase), which was further enhanced by a cold shock. Furthermore, MTT assay showed that cell viability with DENPs/siRNA up to 200 nM remained above 80%. We also observed that most of siRNA was accumulated in kidney mediated by DENPs instead of liver and spleen in vivo experiments. Thus, DENPs as a cold shock responsive quick release model for siRNA or hydrophilic macromolecules delivery provide a new way to nanocarrier design and clinic therapy.

  17. Hybrid pulmonary surfactant-coated nanogels mediate efficient in vivo delivery of siRNA to murine alveolar macrophages.

    Science.gov (United States)

    De Backer, Lynn; Naessens, Thomas; De Koker, Stefaan; Zagato, Elisa; Demeester, Jo; Grooten, Johan; De Smedt, Stefaan C; Raemdonck, Koen

    2015-11-10

    The local delivery of small interfering RNA (siRNA) to the lungs may provide a therapeutic solution to a range of pulmonary disorders. Resident alveolar macrophages (rAM) in the bronchoalveolar lumen play a critical role in lung inflammatory responses and therefore constitute a particularly attractive target for siRNA therapeutics. However, achieving efficient gene silencing in the lung while avoiding pulmonary toxicity requires appropriate formulation of siRNA in functional nanocarriers. In this study, we evaluated pulmonary surfactant-coated dextran nanogels for the delivery of siRNA to rAM upon pharyngeal aspiration in BALB/c mice. Both the surfactant-coated and uncoated nanogels achieved high levels of siRNA uptake in rAM, yet only the surfactant-coated formulation could significantly reduce gene expression on the protein level. Surfactant-coated nanogels induced a profound downregulation of target mRNA levels, reaching 70% knockdown with ~1mgkg(-1) siRNA dose. In addition, only mild acute pro-inflammatory cytokine and chemokine responses were detected one day after nanoparticle aspiration, accompanied by a moderate neutrophil infiltration in the bronchoalveolar lumen. The latter could be substantially reduced by removal of excess surfactant from the formulation. Overall, our hybrid core-shell nanoparticles have demonstrated safe and effective siRNA delivery to rAM, providing a new therapeutic approach for treatment of inflammatory pathologies in the lung.

  18. Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling.

    Science.gov (United States)

    Sahay, Gaurav; Querbes, William; Alabi, Christopher; Eltoukhy, Ahmed; Sarkar, Sovan; Zurenko, Christopher; Karagiannis, Emmanouil; Love, Kevin; Chen, Delai; Zoncu, Roberto; Buganim, Yosef; Schroeder, Avi; Langer, Robert; Anderson, Daniel G

    2013-07-01

    Despite efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain unclear. Here we examine cellular uptake of short interfering RNA (siRNA) delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy. We also employed defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann-Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene. Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.

  19. First siRNA library screening in hard-to-transfect HUVEC cells.

    Science.gov (United States)

    Zumbansen, Markus; Altrogge, Ludger M; Spottke, Nicole Ue; Spicker, Sonja; Offizier, Sheila M; Domzalski, Sandra Bs; St Amand, Allison L; Toell, Andrea; Leake, Devin; Mueller-Hartmann, Herbert A

    2009-10-29

    Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa(R) Nucleofector(R) 96-well Shuttle(R) System for siRNA screening in primary cells. Lonza's Clonetics(R) HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME(R) siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector(R) 96-well Shuttle(R) System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection.

  20. A genome-wide siRNA screen to identify modulators of insulin sensitivity and gluconeogenesis.

    Directory of Open Access Journals (Sweden)

    Ruojing Yang

    Full Text Available BACKGROUND: Hepatic insulin resistance impairs insulin's ability to suppress hepatic glucose production (HGP and contributes to the development of type 2 diabetes (T2D. Although the interests to discover novel genes that modulate insulin sensitivity and HGP are high, it remains challenging to have a human cell based system to identify novel genes. METHODOLOGY/PRINCIPAL FINDINGS: To identify genes that modulate hepatic insulin signaling and HGP, we generated a human cell line stably expressing beta-lactamase under the control of the human glucose-6-phosphatase (G6PC promoter (AH-G6PC cells. Both beta-lactamase activity and endogenous G6PC mRNA were increased in AH-G6PC cells by a combination of dexamethasone and pCPT-cAMP, and reduced by insulin. A 4-gene High-Throughput-Genomics assay was developed to concomitantly measure G6PC and pyruvate-dehydrogenase-kinase-4 (PDK4 mRNA levels. Using this assay, we screened an siRNA library containing pooled siRNA targeting 6650 druggable genes and identified 614 hits that lowered G6PC expression without increasing PDK4 mRNA levels. Pathway analysis indicated that siRNA-mediated knockdown (KD of genes known to positively or negatively affect insulin signaling increased or decreased G6PC mRNA expression, respectively, thus validating our screening platform. A subset of 270 primary screen hits was selected and 149 hits were confirmed by target gene KD by pooled siRNA and 7 single siRNA for each gene to reduce G6PC expression in 4-gene HTG assay. Subsequently, pooled siRNA KD of 113 genes decreased PEPCK and/or PGC1alpha mRNA expression thereby demonstrating their role in regulating key gluconeogenic genes in addition to G6PC. Last, KD of 61 of the above 113 genes potentiated insulin-stimulated Akt phosphorylation, suggesting that they suppress gluconeogenic gene by enhancing insulin signaling. CONCLUSIONS/SIGNIFICANCE: These results support the proposition that the proteins encoded by the genes identified in

  1. siRNA for Influenza Therapy

    Science.gov (United States)

    Barik, Sailen

    2010-01-01

    Influenza virus is one of the most prevalent and ancient infections in humans. About a fifth of world’s population is infected by influenza virus annually, leading to high morbidity and mortality, particularly in infants, the elderly and the immunocompromised. In the US alone, influenza outbreaks lead to roughly 30,000 deaths each year. Current vaccines and anti-influenza drugs are of limited use due to high mutation rate of the virus and side effects. In recent years, RNA interference, triggered by synthetic short interfering RNA (siRNA), has rapidly evolved as a potent antiviral regimen. Properly designed siRNAs have been shown to function as potent inhibitors of influenza virus replication. The siRNAs outperform traditional small molecule antivirals in a number of areas, such as ease of design, modest cost, and fast turnaround. Although specificity and tissue delivery remain major bottlenecks in the clinical applications of RNAi in general, intranasal application of siRNA against respiratory viruses including, but not limited to influenza virus, has experienced significant success and optimism, which is reviewed here. PMID:21994689

  2. siRNA for Influenza Therapy

    Directory of Open Access Journals (Sweden)

    Sailen Barik

    2010-07-01

    Full Text Available Influenza virus is one of the most prevalent and ancient infections in humans. About a fifth of world's population is infected by influenza virus annually, leading to high morbidity and mortality, particularly in infants, the elderly and the immunocompromised. In the US alone, influenza outbreaks lead to roughly 30,000 deaths each year. Current vaccines and anti-influenza drugs are of limited use due to high mutation rate of the virus and side effects. In recent years, RNA interference, triggered by synthetic short interfering RNA (siRNA, has rapidly evolved as a potent antiviral regimen. Properly designed siRNAs have been shown to function as potent inhibitors of influenza virus replication. The siRNAs outperform traditional small molecule antivirals in a number of areas, such as ease of design, modest cost, and fast turnaround. Although specificity and tissue delivery remain major bottlenecks in the clinical applications of RNAi in general, intranasal application of siRNA against respiratory viruses including, but not limited to influenza virus, has experienced significant success and optimism, which is reviewed here.

  3. In vitro inhibition of field isolates of feline calicivirus with short interfering RNAs (siRNAs).

    Science.gov (United States)

    McDonagh, Phillip; Sheehy, Paul A; Fawcett, Anne; Norris, Jacqueline M

    2015-05-15

    Feline calicivirus (FCV) is a common infection of domestic cats. Most infections are mild and self-limiting; however more severe disease manifestations, such as FCV-associated virulent systemic disease, may be associated with significant morbidity and mortality. There is currently a lack of effective antiviral treatments for these disease manifestations. In this study, a panel of eight siRNAs were designed to target four conserved regions of the FCV genome. siRNAs were screened for in vitro antiviral efficacy against the reference strain FCV F9 by determination of extracellular virus titres and morphological assessment of protection from cytopathic effect. Three of the siRNA (FCV3.7, FCV4.1, and FCV4.2) demonstrated a marked antiviral effect with a greater than 99% reduction in extracellular viral titre. Titration of these effective siRNAs demonstrated a clear concentration-response relationship, with IC50 values of approximately 1 nM, and combination treatment with multiple siRNAs demonstrated additive or synergistic effects. To assess the potential usefulness of the compounds in a clinical setting, siRNAs were screened against a panel of six recent Australian FCV isolates from cats with FCV-related disease. The siRNAs shown to be effective against the reference strain FCV F9 were active against the majority of the isolates tested, although some variability was noted. Taken together these data suggest potential therapeutic application of antiviral RNAi for treating FCV-associated disease in cats. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Mx1 gene protects mice against the highly lethal human H5N1 influenza virus.

    Science.gov (United States)

    Salomon, Rachelle; Staeheli, Peter; Kochs, Georg; Yen, Hui-Ling; Franks, John; Rehg, Jerold E; Webster, Robert G; Hoffmann, Erich

    2007-10-01

    We investigated the importance of the host Mx1 gene in protection against highly pathogenic H5N1 avian influenza virus. Mice expressing the Mx1 gene survived infection with the lethal human H5N1 isolate A/Vietnam/1203/04 and with reassortants combining its genes with those of the non-lethal virus A/chicken/Vietnam/C58/04, while all Mx1-/- mice succumbed. Mx1-expressing mice showed lower organ virus titers, fewer lesions, and less pulmonary inflammation. Our data support the hypothesis that Mx1 expression protects mice against the high pathogenicity of H5N1 virus through inhibition of viral polymerase activity ultimately resulting in reduced viral growth and spread. Drugs that mimic this mechanism may be protective in humans.

  5. Novel polyacrylate-based cationic nanoparticles for survivin siRNA delivery combined with mitoxantrone for treatment of breast cancer.

    Science.gov (United States)

    Arami, Sanam; Mahdavi, Majid; Rashidi, Mohammad Reza; Fathi, Marziyeh; Hejazi, Mohammad-Saeid; Samadi, Nasser

    2016-11-01

    As a gene delivery method in breast cancer therapy, knocking down the undesired genes in the cancerous cells would be promising. Inhibitors of Apoptosis Protein (IAP) family genes are some of the genes whose responsibility is inhibition of apoptosis in cells. Silencing these genes seems to be helpful directing the tumor cells to death. siRNA sequence designed against survivin anti-apoptotic gene can play this role if carried to the cytoplasm. Here we prepared a positive charged biocompatible nano-sized particle made up of a Fe3O4 core covered respectively by polyacrylate (PA) and polyethyleneimine (PEI) layer, which could successfully deliver the siRNA into the MCF-7 cells. The particle structure was checked and having less than 50 nm diameter in size, positive charge and, safety towards MCF-7 cells besides being able to form nanoplexes with the siRNA strand helps it entering into the biologic assays part. The siRNA delivery evaluated via flowcytometry. Apoptosis induction was determined by DAPI staining. The efficiency of survivin gene knockdown was evaluated in mRNA and protein levels using Real time PCR and western blotting methods. Overall, the Fe3O4-PA-PEI nanoparticles can deliver siRNA effectively into the cytoplasm of the MCF-7 breast cancer cells and induce apoptosis.

  6. Gene Transfer to Dendritic Cells Induced a Protective Immunity against Melanoma

    Institute of Scientific and Technical Information of China (English)

    Pat Metharom; Kay A.O. Ellem; Ming Q. Wei

    2005-01-01

    Lentiviral vectors have shown promises for efficient gene transfer to dividing as well as nondividing cells. In this study, we explored lentiviral vector-mediated, the entire mTRP-2 gene transfer and expression in dendritic cells (DCs). Adoptive transfer of DCs-expressing mTRP-2 (DC-HR'CmT2) into C57BL/6 mouse was also assessed.Dendritic cells were harvested from bone marrow and functional DCs were proved by allogeneic mixed lymphocyte reaction. Lentiviral vectors were produced by transient transfection of 293T cells. Transduction of DCs was proved by marker gene expression and PCR and RT-PCR amplification. Implantation of the transduced DCs, depletion of immune cells as well as the survival of the mice after tumour challenge were investigated. High efficiency of gene transfer into mature DCs was achieved. The high level expression of the functional antigen (TRP-2) and induction of protective immunity by adoptive transfer of TRP-2 gene modified DCs were demonstrated. In vivo study showed a complete protection of mice from further melanoma cell challenge. In comparison, only 83% of mice survived when mTRP-2 peptide-pulsed DCs were administered, suggesting the generation of specific protection. Together, these results demonstrated the usefulness of this gene transfer to DC approach for immunotherapy of cancer and indicated that using tumour associated antigens (TAAs) for gene transfer may be potentially beneficial for the therapy of melanoma.

  7. Conjugated polymer nanoparticles for effective siRNA delivery to tobacco BY-2 protoplasts

    Directory of Open Access Journals (Sweden)

    Verchot Jeanmarie

    2010-12-01

    Full Text Available Abstract Background Post transcriptional gene silencing (PTGS is a mechanism harnessed by plant biologists to knock down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Plant biologists have employed electroporation to deliver artificial siRNAs to plant protoplasts to study gene expression mechanisms at the single cell level. One drawback of electroporation is the extensive loss of viable protoplasts that occurs as a result of the transfection technology. Results We employed fluorescent conjugated polymer nanoparticles (CPNs to deliver siRNAs and knockdown a target gene in plant protoplasts. CPNs are non toxic to protoplasts, having little impact on viability over a 72 h period. Microscopy and flow cytometry reveal that CPNs can penetrate protoplasts within 2 h of delivery. Cellular uptake of CPNs/siRNA complexes were easily monitored using epifluorescence microscopy. We also demonstrate that CPNs can deliver siRNAs targeting specific genes in the cellulose biosynthesis pathway (NtCesA-1a and NtCesA-1b. Conclusions While prior work showed that NtCesA-1 is a factor involved in cell wall synthesis in whole plants, we demonstrate that the same gene plays an essential role in cell wall regeneration in isolated protoplasts. Cell wall biosynthesis is central to cell elongation, plant growth and development. The experiments presented here shows that NtCesA is also a factor in cell viability. We show that CPNs are valuable vehicles for delivering siRNAs to plant protoplasts to study vital cellular pathways at the single cell level.

  8. Protection and synergism by recombinant fowl pox vaccines expressing multiple genes from Marek's disease virus.

    Science.gov (United States)

    Lee, Lucy E; Witter, R L; Reddy, S M; Wu, P; Yanagida, N; Yoshida, S

    2003-01-01

    Recombinant fowl poxviruses (rFPVs) were constructed to express genes from serotype 1 Marek's disease virus (MDV) coding for glycoproteins B, E, I, H, and UL32 (gB1, gE, gI, gH, and UL32). An additional rFPV was constructed to contain four MDV genes (gB1, gE, gI, and UL32). These rFPVs were evaluated for their ability to protect maternal antibody-positive chickens against challenge with highly virulent MDV isolates. The protection induced by a single rFPV/gB1 (42%) confirmed our previous finding. The protection induced by rFPV/gI (43%), rFPV/gB1UL32 (46%), rFPV/gB1gEgI (72%), and rFPV/gB1gEgIUL32 (70%) contributed to additional knowledge on MDV genes involved in protective immunity. In contrast, the rFPV containing gE, gH, or UL32 did not induce significant protection compared with turkey herpesvirus (HVT). Levels of protection by rFPV/gB1 and rFPV/gl were comparable with that of HVT. Only gB1 and gI conferred synergism in rFPV containing these two genes. Protection by both rFPV/gB1gEgI (72%) and rFPV/gB1gEgIUL32(70%) against Marek's disease was significantly enhanced compared with a single gB1 or gI gene (40%). This protective synergism between gB1 and gI in rFPVs may be the basis for better protection when bivalent vaccines between serotypes 2 and 3 were used. When rFPV/gB1gIgEUL32 + HVT were used as vaccine against Md5 challenge, the protection was significantly enhanced (94%). This synergism between rFPV/gB1gIgEUL32 and HVT indicates additional genes yet to be discovered in HVT may be responsible for the enhancement.

  9. Triple combination of siRNAs targeting TGFβ1, TGFβR2, and CTGF enhances reduction of collagen I and smooth muscle actin in corneal fibroblasts.

    Science.gov (United States)

    Sriram, Sriniwas; Robinson, Paulette; Pi, Liya; Lewin, Alfred S; Schultz, Gregory

    2013-12-17

    Transforming growth factor β1 (TGFβ1), TGFβ receptor (TGFβR2), and connective tissue growth factor (CTGF) are key regulators of fibrosis in the cornea and in other tissues, including liver, skin, and kidney. We developed an antifibrotic treatment targeting these three critical scarring genes by using a combination of small interfering RNAs (siRNAs) and assessed its effect on downstream scarring genes, collagen I, and α smooth muscle actin (SMA). Up to six individual siRNAs for each of the three target gene mRNAs were transfected into cultures of rabbit corneal fibroblasts at concentrations from 15 to 90 nM. The knockdown of target gene proteins was measured by ELISA, and the two most effective siRNAs were tested in dual combinations. Knockdown percentages of both individual and dual siRNA combinations were analyzed for synergy by using combination index to predict "effective" and "ineffective" triple siRNA combinations. Effects of both triple siRNA combinations on target and downstream mRNAs were measured by using quantitative RT-PCR, and levels of SMA protein were assessed by immunohistochemistry. Single and dual siRNA combinations produced a wide range of protein knockdown of target genes (5%-80%). The effective triple siRNA combination significantly reduced mRNA levels of target genes (>80%) and downstream scarring genes (>85%), and of SMA protein (>95%), and significantly reduced cell migration without reducing cell viability. Simultaneous targeting of TGFβ1, TGFβR2, and CTGF genes by effective triple siRNA combination produced high knockdown of target and downstream scarring genes without cell toxicity, which may have clinical applications in reducing corneal fibrosis and scarring in other tissues.

  10. Thermoresponsive pegylated bubble liposome nanovectors for efficient siRNA delivery via endosomal escape

    KAUST Repository

    Alamoudi, Kholod

    2017-05-19

    Improving the delivery of siRNA into cancer cells via bubble liposomes. Designing a thermoresponsive pegylated liposome through the introduction of ammonium bicarbonate salt into liposomes so as to control their endosomal escape for gene therapy.A sub-200 nm nanovector was fully characterized and examined for cellular uptake, cytotoxicity, endosomal escape and gene silencing.The siRNA-liposomes were internalized into cancer cells within 5 min and then released siRNAs in the cytosol prior to lysosomal degradation upon external temperature elevation. This was confirmed by confocal bioimaging and gene silencing reaching up to 90% and further demonstrated by the protein inhibition of both target genes.The thermoresponsiveness of ammonium bicarbonate containing liposomes enabled the rapid endosomal escape of the particles and resulted in an efficient gene silencing.

  11. Dermato-protective properties of ergothioneine through induction of Nrf2/ARE-mediated antioxidant genes in UVA-irradiated Human keratinocytes.

    Science.gov (United States)

    Hseu, You-Cheng; Lo, Heng-Wei; Korivi, Mallikarjuna; Tsai, Yu-Cheng; Tang, Meng-Ju; Yang, Hsin-Ling

    2015-09-01

    UVA irradiation-induced skin damage and redox imbalance have been shown to be ameliorated by ergothioneine (EGT), a naturally occurring sulfur-containing amino acid. However, the responsible molecular mechanism with nanomolar concentrations of EGT remains unclear. We investigated the dermato protective efficacies of EGT (125-500nM) against UVA irradiation (15J/cm(2)), and elucidated the underlying molecular mechanism in human keratinocyte-derived HaCaT cells. We found that EGT treatment prior to UVA exposure significantly increased the cell viability and prevented lactate dehydrogenase release into the medium. UVA-induced ROS and comet-like DNA formation were remarkably suppressed by EGT with a parallel inhibition of apoptosis, as evidenced by reduced DNA fragmentation (TUNEL), caspase-9/-3 activation, and Bcl-2/Bax dysregulation. Furthermore, EGT alleviated UVA-induced mitochondrial dysfunction. Dose-dependent increases of antioxidant genes, HO-1, NQO-1, and γ-GCLC and glutathione by EGT were associated with upregulated Nrf2 and downregulated Keap-1 expressions. This was confirmed by increased nuclear accumulation of Nrf2 and inhibition of Nrf2 degradation. Notably, augmented luciferase activity of ARE may explain Nrf2/ARE-mediated signaling pathways behind EGT dermato-protective properties. We further demonstrated that Nrf2 translocation was mediated by PI3K/AKT, PKC, or ROS signaling cascades. This phenomenon was confirmed with suppressed nuclear Nrf2 activation, and consequently diminished antioxidant genes in cells treated with respective pharmacological inhibitors (LY294002, GF109203X, and N-acetylcysteine). Besides, increased basal ROS by EGT appears to be crucial for triggering the Nrf2/ARE signaling pathways. Silencing of Nrf2 or OCTN1 (EGT carrier protein) signaling with siRNA showed no such protective effects of EGT against UVA-induced cell death, ROS, and apoptosis, which is evidence of the vitality of Nrf2 translocation and protective efficacy of EGT

  12. CDE-1 affects chromosome segregation through uridylation of CSR-1-bound siRNAs.

    Science.gov (United States)

    van Wolfswinkel, Josien C; Claycomb, Julie M; Batista, Pedro J; Mello, Craig C; Berezikov, Eugene; Ketting, René F

    2009-10-02

    We have studied the function of a conserved germline-specific nucleotidyltransferase protein, CDE-1, in RNAi and chromosome segregation in C. elegans. CDE-1 localizes specifically to mitotic chromosomes in embryos. This localization requires the RdRP EGO-1, which physically interacts with CDE-1, and the Argonaute protein CSR-1. We found that CDE-1 is required for the uridylation of CSR-1 bound siRNAs, and that in the absence of CDE-1 these siRNAs accumulate to inappropriate levels, accompanied by defects in both meiotic and mitotic chromosome segregation. Elevated siRNA levels are associated with erroneous gene silencing, most likely through the inappropriate loading of CSR-1 siRNAs into other Argonaute proteins. We propose a model in which CDE-1 restricts specific EGO-1-generated siRNAs to the CSR-1 mediated, chromosome associated RNAi pathway, thus separating it from other endogenous RNAi pathways. The conserved nature of CDE-1 suggests that similar sorting mechanisms may operate in other animals, including mammals.

  13. Intracellular localization of lipoplexed siRNA in vascular endothelial cells of different mouse tissues.

    Science.gov (United States)

    Aleku, Manuela; Fisch, Gerald; Möpert, Kristin; Keil, Oliver; Arnold, Wolfgang; Kaufmann, Jörg; Santel, Ansgar

    2008-05-01

    Liposomally formulated siRNA can be used for RNAi applications in vivo. Intravenous bolus administration of lipoplexed siRNA has been shown to reduce gene expression in the vascular endothelium. Here, we applied immunofluorescence staining for different endothelial markers (PECAM-1, CD34, laminin) on paraffin sections to compare the respective expression pattern with the intracellular localization of intravenously administered, fluorescently labeled siRNA (siRNA-Cy3-lipoplex). By confocal microscopy, lipoplexed siRNA-Cy3 was detected inside vascular endothelial cells in vivo, which where identified with co-staining of endothelial markers. Consequently, the finding of intracellular siRNA uptake by vascular endothelial cells correlated with RNAi based specific protein reduction in situ as revealed by PECAM-1 specific immunofluorescence staining in lung tissue sections. Therefore, by using a cell biological approach these in situ data emphasize the functional uptake of liposomal siRNA molecules in vascular endothelial cells of different mouse tissues as indicated in our previous molecular study.

  14. 非病毒载体在siRNA体内递送中的应用%Application of non-viral vehicles in siRNA delivery in vivo

    Institute of Scientific and Technical Information of China (English)

    姜扩; 裘秀春

    2011-01-01

    RNAi is a specific gene-silencing mechanism triggered by siRNA. Since its discovery, the RNAi technology has rapidly developed as a new strategy for drug target validation and the study of functional genomics, and it is currently being evaluated as a potential therapy for diseases of a genetic etiology. However, due to the instable physicochemical properties of siRNAs in vivo, the major impediment of RNAi application is the delivery vehicles. Therefore, the research of efficient siRNA delivery for siRNA protection, cellular uptake, correct intracellular localization and endosomal release, combination with high biocompatibility, devoid of nonspecific knockdown and toxic side effects is crucial for the success of RNAi-based therapeutics. In this review, the recent advances of siRNA non-viral vehicles are summarized.%RNAi是一种由siRNA触发的特异基因沉默机制.自其发现以来,RNAi技术迅速发展为药物靶标确认和功能基因组学研究的新策略,并有望成为一种疾病基因治疗学方法.然而,由于siRNA体内不稳定的理化性质,递送siRNA成为RNAi发展的最大障碍.因此研发能够保护siRNA、促进细胞吸收、正确细胞定位和包内体逃逸,结合具有高生物相容性与减少非特异沉默和毒性反应的体内递送系统是RNAi成功用于临床治疗的关键.本文就体内非病毒载体方式递送siRNA的研究进展进行综述.

  15. Development of a simple, biocompatible and cost-effective Inulin-Diethylenetriamine based siRNA delivery system.

    Science.gov (United States)

    Sardo, C; Farra, R; Licciardi, M; Dapas, B; Scialabba, C; Giammona, G; Grassi, M; Grassi, G; Cavallaro, G

    2015-07-30

    Small interfering RNAs (siRNAs) have the potential to be of therapeutic value for many human diseases. So far, however, a serious obstacle to their therapeutic use is represented by the absence of appropriate delivery systems able to protect them from degradation and to allow an efficient cellular uptake. In this work we developed a siRNA delivery system based on inulin (Inu), an abundant and natural polysaccharide. Inu was functionalized via the conjugation with diethylenetriamine (DETA) residues to form the complex Inu-DETA. We studied the size, surface charge and the shape of the Inu-DETA/siRNA complexes; additionally, the cytotoxicity, the silencing efficacy and the cell uptake-mechanisms were studied in the human bronchial epithelial cells (16HBE) and in the hepatocellular carcinoma derived cells (JHH6). The results presented here indicate that Inu-DETA copolymers can effectively bind siRNAs, are highly cytocompatible and, in JHH6, can effectively deliver functional siRNAs. Optimal delivery is observed using a weight ratio Inu-DETA/siRNA of 4 that corresponds to polyplexes with an average size of 600nm and a slightly negative surface charge. Moreover, the uptake and trafficking mechanisms, mainly based on micropinocytosis and clatrin mediated endocytosis, allow the homogeneous diffusion of siRNA within the cytoplasm of JHH6. Notably, in 16 HBE where the trafficking mechanism (caveolae mediated endocytosis) does not allow an even distribution of siRNA within the cell cytoplasm, no significant siRNA activity is observed. In conclusion, we developed a novel inulin-based siRNA delivery system able to efficiently release siRNA in JHH6 with negligible cytotoxicity thus opening the way for further testing in more complex in vivo models.

  16. Characterization and vectorization of siRNA targeting RET/PTC1 in human papillary thyroid carcinoma cells

    Directory of Open Access Journals (Sweden)

    Massade L.

    2011-10-01

    Full Text Available RET/PTC1 fusion oncogene is the most common genetic alteration identified to date in thyroid papillary carcinomas (PTC and represents a good target for small interfering RNA (siRNA. Our aim was: i to target the RET/PTC1 oncogene by siRNAs, ii to assess the knockdown effects on cell growth and cell cycle regulation and iii to vectorize it in order to protect it from degradation. Methods. Human cell lines expressing RET/PTC1 were transfected by siRNA RET/PTC1, inhibition of the oncogene expression was assessed by qRT-PCR and by Western blot. Conjugation of siRNA RET/PTC1 to squalene was performed by coupling it to squalene. In vivo studies are performed in nude mice. Conclusion. In this short communication, we report the main published results obtained during last years.

  17. Screening nylon-3 polymers, a new class of cationic amphiphiles, for siRNA delivery.

    Science.gov (United States)

    Nadithe, Venkatareddy; Liu, Runhui; Killinger, Bryan A; Movassaghian, Sara; Kim, Na Hyung; Moszczynska, Anna B; Masters, Kristyn S; Gellman, Samuel H; Merkel, Olivia M

    2015-02-02

    Amphiphilic nucleic acid carriers have attracted strong interest. Three groups of nylon-3 copolymers (poly-β-peptides) possessing different cationic/hydrophobic content were evaluated as siRNA delivery agents in this study. Their ability to condense siRNA was determined in SYBR Gold assays. Their cytotoxicity was tested by MTT assays, their efficiency of delivering Alexa Fluor-488-labeled siRNA intracellularly in the presence and absence of uptake inhibitors was assessed by flow cytometry, and their transfection efficacies were studied by luciferase knockdown in a cell line stably expressing luciferase (H1299/Luc). Endosomal release was determined by confocal laser scanning microscopy and colocalization with lysotracker. All polymers efficiently condensed siRNA at nitrogen-to-phosphate (N/P) ratios of 5 or lower, as reflected in hydrodynamic diameters smaller than that at N/P 1. Although several formulations had negative zeta potentials at N/P 1, G2C and G2D polyplexes yielded >80% uptake in H1299/Luc cells, as determined by flow cytometry. Luciferase knockdown (20-65%) was observed after transfection with polyplexes made of the high molecular weight polymers that were the most hydrophobic. The ability of nylon-3 polymers to deliver siRNA intracellularly even at negative zeta potential implies that they mediate transport across cell membranes based on their amphiphilicity. The cellular uptake route was determined to strongly depend on the presence of cholesterol in the cell membrane. These polymers are, therefore, very promising for siRNA delivery at reduced surface charge and toxicity. Our study identified nylon-3 formulations at low N/P ratios for effective gene knockdown, indicating that nylon-3 polymers are a new, promising type of gene delivery agent.

  18. Polymers modified with double-tailed fluorous compounds for efficient DNA and siRNA delivery.

    Science.gov (United States)

    He, Bingwei; Wang, Yitong; Shao, Naimin; Chang, Hong; Cheng, Yiyun

    2015-08-01

    Cationic polymers are widely used as gene carriers, however, these polymers are usually associated with low transfection efficacy and non-negligible toxicity. Fluorination on polymers significantly improves their performances in gene delivery, but a high density of fluorous chains must be conjugated on a single polymer. Here we present a new strategy to construct fluorinated polymers with minimal fluorous chains for efficient DNA and siRNA delivery. A double-tailed fluorous compound 2-chloro-4,6-bis[(perfluorohexyl)propyloxy]-1,3,5-triazine (CBT) was conjugated on dendrimers of different generations and low molecular weight polyethylenimine via a facile synthesis. The yielding products with average numbers of 1-2 conjugated CBT moieties showed much improved EGFP and luciferase transfection efficacy compared to unmodified polymers. In addition, these polymers show high siRNA delivery efficacy on different cell lines. Among the synthesized polymers, generation 1 (G1) dendrimer modified with an average number of 1.9 CBT moieties (G1-CBT1.9) shows the highest efficacy when delivering both DNA and siRNA and its efficacy approaches that of Lipofectamine 2000. G1-CBT1.9 also shows efficient gene silencing in vivo. All of the CBT-modified polymers exhibit minimal toxicity on the cells at their optimal transfection conditions. This study provides a new strategy to design efficient fluorous polymers for DNA and siRNA delivery.

  19. PEGylated poly(ethylene imine) copolymer-delivered siRNA inhibits HIV replication in vitro.

    Science.gov (United States)

    Weber, Nick D; Merkel, Olivia M; Kissel, Thomas; Muñoz-Fernández, María Ángeles

    2012-01-10

    RNA interference is increasingly being utilized for the specific targeting and down-regulation of disease-causing genes, including targeting viral infections such as HIV. T lymphocytes, the primary target for HIV, are very difficult to treat with gene therapy applications such as RNA interference because of issues with drug delivery. To circumvent these problems, we investigated poly(ethylene imine) (PEI) as a method of improving transfection efficiency of siRNA to T lymphocytes. Additionally, polyethylene glycol (PEG) moieties were engrafted to the PEI polymers with the goals of improving stability and reducing cytotoxicity. Initial studies on PEG-PEI/siRNA polyplex formation, size and their interaction with cell membranes demonstrated their feasibility as drug delivery agents. Assays with lymphocytes revealed low cytotoxicity profiles of the polyplexes at pharmacologically relevant concentrations with PEGylated copolymers obtaining the best results. Successful transfection of a T cell line or primary T cells with siRNA was observed via flow cytometry and confocal microscopy. Finally, the biological effect of copolymer-delivered siRNA was measured. Of particular significance, siRNA targeted to the HIV gene nef and delivered by one of the PEG-PEI copolymers in repetitive treatments every 2-3 days was observed to inhibit HIV replication to the same extent as azidothymidine over the course of 15 days. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Water-soluble lipopolymer delivery of N-methyl-D-aspartic acid receptor 2B siRNA relieves chronic neuropathic pain in rats

    Institute of Scientific and Technical Information of China (English)

    Jianhua Lu; Yuanxiang Tao; Xue Yang; Weifeng Tu; Hao Chen; Jiaxiang Xiong; Chungui Hu

    2011-01-01

    Spinal dorsal horn N-Methyl-D-aspartic acid receptor 2B (NR2B) overexpression plays an important role in the production and maintenance of neuropathic pain. Because small interfering RNA (siRNA) can inhibit NR2B expression, siRNA may provide a novel approach to treat neuropathic pain and possibly nerve injury. However, an efficient and safe vector for NR2B siRNA has not been discovered. This study shows that a water soluble lipopolymer (WSLP) comprised of low molecular weight polyethyleneimine (PEI) and cholesterol can deliver siRNA targeting NR2B for the treatment of neuropathic pain. Results show that intrathecal injection of WSLP/siRNA complexes for 3 days inhibit NR2B gene expression with reductions in mRNA and protein levels by 59% and 54%, respectively, compared with control rats (P < 0.01). Injection of WSLP complexed with scrambled siRNA, or PEI with siRNA did not show this inhibitory effect. Moreover, injection of WSLP/siRNA complexes significantly relieved neuropathic pain at 3, 7, 12, and 21 days, while injection of WSLP with scrambled siRNA or PEI with siRNA did not. These results demonstrate that WSLP can efficiently deliver siRNA targeting NR2B in vivo and relieve neuropathic pain.

  1. Role of generation, architecture, pH and ionic strength on successful siRNA delivery and transfection by hybrid PPV-PAMAM dendrimers.

    Science.gov (United States)

    Pavan, G M; Monteagudo, S; Guerra, J; Carrión, B; Ocaña, V; Rodríguez-Lopez, J; Danani, A; Pérez-Martínez, F C; Ceña, V

    2012-01-01

    Small interfering RNA (siRNA) constitutes an excellent way of knocking down genes. However, it requires the use of delivery systems to reach the target cells, especially to neuronal cells. Dendrimers are one of the most widely used synthetic nanocarriers for siRNA delivery. However, due to the complexity of the dendrimer-siRNA interactions, when a new dendritic carrier is designed it is difficult to predict its efficiency to bind and to deliver siRNA. At the same time it is not easy to understand the origin of eventual limited functionalities. We have modeled the interactions between two dendrimers (TDG-G1 and TDG-G2) and siRNA using molecular dynamics (MD) simulation. The results were compared to experimental physico-chemical parameters such as siRNA complexation, complex stability, size, and zeta potentials and biological effects such as down-regulation of a specific RNA expression in cortical neurons in culture. Data indicate that the combination of rigid core and flexible branches guarantees strong siRNA binding, which is important to have a good transfection profile. However, the successful nanocarrier for siRNA delivery (TDG-G1) is identified not only by a high affinity for siRNA, but by a favorable equilibrium between a strong binding and the ability to release siRNA to exert its biological action. The conditions under which the dendriplex is formed are also relevant for transfection efficiency and biological activity.

  2. Protection of Mice from Lethal Endotoxemia by Chimeric Human BPI-Fcγ1 Gene Delivery

    Institute of Scientific and Technical Information of China (English)

    Chen Li; Jing Li; Zhe Lv; Xinghua Guo; Qinghua Chen; Qingli Kong; Yunqing An

    2006-01-01

    To evaluate the potentiality of applying gene therapy to endotoxemia in high-risk patients, we investigated the effects of transferring an adeno-associated virus serotype 2 (AAV2)-mediated BPI-Fcγ1 gene on protecting mice from challenge of lethal endotoxin. The chimeric BPI-Fcγ1 gene consists of two parts, one encods functional N-terminus (1 to 199 amino acidic residues) of human BPI, which is a bactericidal/permeability-increasing protein,and the other encodes Fc segment of human immunoglobulin G1 (Fcγ1). Our results indicated that the target protein could be expressed and secreted into the serum of the gene-transferred mice. After lethal endotoxin challenge, the levels of endotoxin and TNF-α in the gene-transferred mice were decreased. The survival rate of the BPI-Fcγ1 gene-transferred mice was markedly increased. Our data suggest that AAV2-mediated chimeric BPI-Fcγ1 gene delivery can potentially be used clinically for the protection and treatment of endotoxemia and endotoxic shock in high-risk individuals.

  3. Protective Effect of Gwakhyangjeonggisan Herbal Acupuncture Solution in Glioblastoma Cells: Microarray Analysis of Gene Expression

    Directory of Open Access Journals (Sweden)

    Hong-Seok Lee

    2005-12-01

    Full Text Available Objectives : Neurological disorders have been one of main therapeutic targets of acupuncture. The present study investigated the protective effects of Gwakhyangjeonggisan herbal acupuncture solution (GHAS. Methods : We performed 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay in glioblastoma cells, and did microarray analysis with cells exposed to reactive oxigen species (ROS of hydrogen peroxide by 8.0 k Human cDNA, with cut-off level of 2-fold changes in gene expression. Results : MTT assay showed protective effect of GHAS on the glioblastoma cells exposed to hydrogen peroxide. When glioblastoma cells were exposed to hydrogen peroxide, 24 genes were downregulated. When the cells were pretreated with GHAS before exposure to hydrogen peroxide, 46 genes were downregulated. Many of the genes downregulated by hydrogen peroxide stimulation were decreased in the amount of downregulation or reversed to upregulation. Conclusions : The gene expression changes observed in the present study are supposed to be related to the protective molecular mechanism of GHAS in the glioblastoma cells exposed to ROS stress.

  4. PEG-detachable cationic polyaspartamide derivatives bearing stearoyl moieties for systemic siRNA delivery toward subcutaneous BxPC3 pancreatic tumor.

    Science.gov (United States)

    Kim, Hyun Jin; Oba, Makoto; Pittella, Frederico; Nomoto, Takahiro; Cabral, Horacio; Matsumoto, Yu; Miyata, Kanjiro; Nishiyama, Nobuhiro; Kataoka, Kazunori

    2012-01-01

    For systemic siRNA delivery into tumor tissues, a safe and efficient vehicle is strongly required. Therefore, we designed a block copolymer of detachable poly(ethylene glycol) (PEG) polycation bearing low pKa amines and hydrophobic moieties in the side chain to develop a smart siRNA complex possessing biocompatibility, high complex stability, and endosomal escaping functionality. A disulfide linkage (-SS-) was inserted as a linker between PEG and a cationic polyaspartamide derivative, poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)), with a flanking stearoyl moiety, where PAsp(DET) segment provides the excellent ability of endosome destabilization by direct interaction with the membrane. The resulting polymer, stearoyl PEG-SS-PAsp(DET), was confirmed to form the siRNA complex with an environment-responsive PEG palisade, which was detached from the complex surface under reductive conditions mimicking tumor tissues and cytoplasm because of the disulfide cleavage. The smart siRNA complex allowed significant gene silencing against cultured pancreatic cancer cells without considerable cytotoxicity and erythrocyte disruption, whereas such significant gene silencing was not observed in a control siRNA complex without the disulfide linkage. This enhanced gene silencing activity might be because of the enhanced cellular uptake and subsequent translocation of siRNA into cytoplasm facilitated by PEG detachment around and/or in the cancer cells. Further, intravital real-time confocal laser scanning microscopic observation revealed the effect of hydrophobic stearoyl modification on the stabilization of the siRNA complex for longevity in the blood. Significant in vivo gene silencing of the smart siRNA complex was achieved by systemic administration of vascular endothelial growth factor siRNA in a mouse model bearing a subcutaneous pancreatic tumor, leading to 40% regression in tumor growth. These results demonstrate the strong potential of stearoyl PEG

  5. SiRNA In Vivo-Targeted Delivery to Murine Dendritic Cells by Oral Administration of Recombinant Yeast.

    Science.gov (United States)

    Xu, Kun; Liu, Zhongtian; Zhang, Long; Zhang, Tingting; Zhang, Zhiying

    2016-01-01

    SiRNA therapeutics promise a future where any target in the transcriptome could be potentially addressed. However, the delivery of SiRNAs and targeting of particular cell types or organs are major challenges. A novel, efficient, and safe delivery system for promising the introduction of SiRNAs into particular cell types within living organisms is of great significance. Our previous studies have proved that recombinant protein (MSTN) and exogenous gene (EGFP) as vaccines, and furthermore functional CD40 shRNA expression can be delivered into dendritic cells (DCs) in mouse by oral administration of recombinant yeast (Saccharomyces cerevisiae). Here, we describe the details of the promising and innovative approach based on oral administration of recombinant yeast that allows in vivo-targeted delivery of functional SiRNA to murine intestinal DCs.

  6. Application of Target Peptide in siRNA Delivery 
for the Research of Lung Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Honglin GAO

    2014-09-01

    Full Text Available Lung cancer is considered a kind of malignant tumors of the world highest incidence. As it is not sensitive to chemotherapy and easy to produce drug resistance, improving effect of anticancer drug becomes a research focus recent years. siRNA, small interfering RNA, can silence complemenary mRNA which is a kind of gene therapy. Target peptides are small molecular peptides which specifically bind to tumor surface materials. When used with siRNA, target peptides can increase accumulation of siRNA in tumor cells and enhance the silencing effect. As result, drug resistance of lung cancer reduced and the effect of therapy can be improved. This method provides new direction and strategy for targeted therapy of lung cancer. This article will make a brief overview of target peptides applying in siRNA dilivery for the research of lung cancer treatment.

  7. Enhanced suppression of adenovirus replication by triple combination of anti-adenoviral siRNAs, soluble adenovirus receptor trap sCAR-Fc and cidofovir.

    Science.gov (United States)

    Pozzuto, Tanja; Röger, Carsten; Kurreck, Jens; Fechner, Henry

    2015-08-01

    Adenoviruses (Ad) generally induce mild self-limiting respiratory or intestinal infections but can also cause serious disease with fatal outcomes in immunosuppressed patients. Antiviral drug therapy is an important treatment for adenoviral infections but its efficiency is limited. Recently, we have shown that gene silencing by RNA interference (RNAi) is a promising new approach to inhibit adenoviral infection. In the present in vitro study, we examined whether the efficiency of an RNAi-based anti-adenoviral therapy can be further increased by combination with a virus receptor trap sCAR-Fc and with the antiviral drug cidofovir. Initially, three siRNAs, siE1A_4, siIVa2_2 and Pol-si2, targeting the adenoviral E1A, IVa2 and DNA polymerase mRNAs, respectively, were used for gene silencing. Replication of the Ad was inhibited in a dose dependent manner by each siRNA, but the efficiency of inhibition differed (Pol-si2>siIVa2_2>siE1A_4). Double or triple combinations of the siRNAs compared with single siRNAs did not result in a measurably higher suppression of Ad replication. Combination of the siRNAs (alone or mixes of two or three siRNAs) with sCAR-Fc markedly increased the suppression of adenoviral replication compared to the same siRNA treatment without sCAR-Fc. Moreover, the triple combination of a mix of all three siRNAs, sCAR-Fc and cidofovir was about 23-fold more efficient than the combination of siRNAs mix/sCAR-Fc and about 95-fold more efficient than the siRNA mix alone. These data demonstrate that co-treatment of cells with sCAR-Fc and cidofovir is suitable to increase the efficiency of anti-adenoviral siRNAs.

  8. Reductively Responsive Hydrogel Nanoparticles with Uniform Size, Shape, and Tunable Composition for Systemic siRNA Delivery in Vivo.

    Science.gov (United States)

    Ma, Da; Tian, Shaomin; Baryza, Jeremy; Luft, J Christopher; DeSimone, Joseph M

    2015-10-01

    To achieve the great potential of siRNA based gene therapy, safe and efficient systemic delivery in vivo is essential. Here we report reductively responsive hydrogel nanoparticles with highly uniform size and shape for systemic siRNA delivery in vivo. "Blank" hydrogel nanoparticles with high aspect ratio were prepared using continuous particle fabrication based on PRINT (particle replication in nonwetting templates). Subsequently, siRNA was conjugated to "blank" nanoparticles via a disulfide linker with a high loading ratio of up to 18 wt %, followed by surface modification to enhance transfection. This fabrication process could be easily scaled up to prepare large quantity of hydrogel nanoparticles. By controlling hydrogel composition, surface modification, and siRNA loading ratio, siRNA conjugated nanoparticles were highly tunable to achieve high transfection efficiency in vitro. FVII-siRNA conjugated nanoparticles were further stabilized with surface coating for in vivo siRNA delivery to liver hepatocytes, and successful gene silencing was demonstrated at both mRNA and protein levels.

  9. siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection

    Science.gov (United States)

    2016-05-20

    pools targeting each of 140 human trafficking genes were 89 TR-16-094 DISTRIBUTION STATEMENT A: Approved for public release; distribution is...remodeling in alphavirus-infected cells might 492 be important for trafficking of E1/E2. To test this hypothesis, primary human astrocytes were 493...An arrayed library targeting 140 trafficking genes (Dharmacon Human ON-TARGETplus 620 siRNA Library - Membrane Trafficking - SMARTpool, G-105500-05

  10. Clustering siRNA conjugates for MMP-responsive therapeutics in chronic wounds of diabetic animals

    Science.gov (United States)

    Kim, Hye Sung; Son, Young Ju; Yoo, Hyuk Sang

    2016-07-01

    The MMP-responsive breakdown of siRNA clusters was translated to site-specific gene transfection and enhanced wound healing in diabetic ulcers. MMP-2 siRNA was chemically tethered to the end of multi-armed PEG via MMP-cleavable linkers (4PEG-siRNA) and subsequently clustered into submicron particles complexed with LPEI. 4PEG-siRNA was more tightly complexed with LPEI and the associated cluster showed higher resistance against RNase attack, in comparison to naked siRNA. Because the size of the clusters increased depending on the increase in charge ratio of LPEI to siRNA, cellular uptake of the 4PEG-siRNA/LPEI cluster was significantly attenuated due to the huge size of the cluster. However, upon MMP treatment, the cluster dissociated into smaller particles and was efficiently endocytosed by cells. An in vivo fluorescence resonance energy transfer (FRET) study also revealed that the clusters were effectively dissociated in MMP-rich environments of dorsal wounds in diabetic animals. In addition, diabetic ulcers treated with the clusters showed a faster wound closure rate and the recovered tissue expressed a larger amount of cytokeratin along with a lower expression level of MMP-2 compared to the other groups.The MMP-responsive breakdown of siRNA clusters was translated to site-specific gene transfection and enhanced wound healing in diabetic ulcers. MMP-2 siRNA was chemically tethered to the end of multi-armed PEG via MMP-cleavable linkers (4PEG-siRNA) and subsequently clustered into submicron particles complexed with LPEI. 4PEG-siRNA was more tightly complexed with LPEI and the associated cluster showed higher resistance against RNase attack, in comparison to naked siRNA. Because the size of the clusters increased depending on the increase in charge ratio of LPEI to siRNA, cellular uptake of the 4PEG-siRNA/LPEI cluster was significantly attenuated due to the huge size of the cluster. However, upon MMP treatment, the cluster dissociated into smaller particles and was

  11. Mechanistic investigation of poly(ethylene imine)-based siRNA delivery: disulfide bonds boost intracellular release of the cargo.

    Science.gov (United States)

    Breunig, Miriam; Hozsa, Constantin; Lungwitz, Uta; Watanabe, Kazuo; Umeda, Isao; Kato, Hiroyuki; Goepferich, Achim

    2008-08-25

    Poly(ethylene imine) (PEI) has gained increasing attention in the delivery of small interfering RNAs (siRNAs) into cells. In order to further optimize PEI for this application, the first goal of this study was to examine particular steps of siRNA delivery with various PEI derivatives as carriers. Furthermore, the hypothesis that disulfide cleavable carrier systems are favorable for the release of siRNA into the cell cytoplasm was investigated. Flow cytometry and confocal microscopy were used to assess the cellular uptake and intracellular distribution of siRNA, which were then related to gene silencing efficacy. We observed a strong correlation between cellular uptake and RNAi activity. The cellular uptake of siRNA was more efficient with increasing branching of the polymer, i.e. linear PEI (lPEI) 5 kDa < lPEI cross-linked via disulfide bonds (ssPEI) < branched PEI (bPEI) 25 kDa. However, it was also evident that the siRNA release from the carrier, which was promoted by ssPEI, played an important role in the accessibility of siRNA for the gene silencing complex. Therefore, we suggest that a combination of a high branching density and reductively cleavable bonds within the PEI-based carrier system could be one possible step towards improving siRNA delivery.

  12. Perivascular delivery of Notch 1 siRNA inhibits injury-induced arterial remodeling.

    Directory of Open Access Journals (Sweden)

    Eileen M Redmond

    Full Text Available To determine the efficacy of perivascular delivery of Notch 1 siRNA in preventing injury-induced arterial remodeling.Carotid artery ligation was performed to induce arterial remodeling. After 14 days, morphometric analysis confirmed increased vSMC growth and subsequent media thickening and neointimal formation. Laser capture microdissection, quantitative qRT-PCR and immunoblot analysis of medial tissue revealed a significant increase in Notch1 receptor and notch target gene, Hrt 1 and 2 expression in the injured vessels. Perivascular delivery of Notch 1 siRNA by pluronic gel inhibited the injury-induced increase in Notch 1 receptor and target gene expression when compared to scrambled siRNA controls while concomitantly reducing media thickening and neointimal formation to pre-injury, sham-operated levels. Selective Notch 1 knockdown also reversed the injury-induced inhibition of pro-apoptotic Bax expression while decreasing injury-induced anti-apoptotic Bcl-XL expression to sham-operated control levels. In parallel experiments, proliferative cyclin levels, as measured by PCNA expression, were reversed to sham-operated control levels following selective Notch 1 knockdown.These results suggest that injury-induced arterial remodeling can be successfully inhibited by localized perivascular delivery of Notch 1 siRNA.

  13. An siRNA pathway prevents transgenerational retrotransposition in plants subjected to stress.

    Science.gov (United States)

    Ito, Hidetaka; Gaubert, Hervé; Bucher, Etienne; Mirouze, Marie; Vaillant, Isabelle; Paszkowski, Jerzy

    2011-04-01

    Eukaryotic genomes consist to a significant extent of retrotransposons that are suppressed by host epigenetic mechanisms, preventing their uncontrolled propagation. However, it is not clear how this is achieved. Here we show that in Arabidopsis seedlings subjected to heat stress, a copia-type retrotransposon named ONSEN (Japanese 'hot spring') not only became transcriptionally active but also synthesized extrachromosomal DNA copies. Heat-induced ONSEN accumulation was stimulated in mutants impaired in the biogenesis of small interfering RNAs (siRNAs); however, there was no evidence of transposition occurring in vegetative tissues. After stress, both ONSEN transcripts and extrachromosomal DNA gradually decayed and were no longer detected after 20-30 days. Surprisingly, a high frequency of new ONSEN insertions was observed in the progeny of stressed plants deficient in siRNAs. Insertion patterns revealed that this transgenerational retrotransposition occurred during flower development and before gametogenesis. Therefore in plants with compromised siRNA biogenesis, memory of stress was maintained throughout development, priming ONSEN to transpose during differentiation of generative organs. Retrotransposition was not observed in the progeny of wild-type plants subjected to stress or in non-stressed mutant controls, pointing to a crucial role of the siRNA pathway in restricting retrotransposition triggered by environmental stress. Finally, we found that natural and experimentally induced variants in ONSEN insertions confer heat responsiveness to nearby genes, and therefore mobility bursts may generate novel, stress-responsive regulatory gene networks.

  14. Combination Anticancer Nanopreparations of Novel Proapoptotic Drug, TRAIL and siRNA

    Science.gov (United States)

    Riehle, Robert D.

    Development of drugs for the treatment of cancer is a challenging endeavor often hindered by the solubility and distribution of the drug in the body. Drug delivery systems have been used for many years to overcome these issues. Polyethylene glycol-phosphatidylethanolamine (PEG-PE) micelles in particular have shown utility as a nanosized drug delivery vehicle capable of incorporating poorly soluble drugs and preferentially delivering them to the tumor. Addition of PEG polymers to the surface prolongs the half-life of the particle in the blood by evading clearance by the reticuloendothelial system (RES) and increases tumor accumulation through the utilization of the enhanced permeability and retention (EPR) effect. Micelles have also been shown to successfully incorporate and protect modified siRNA, a notoriously challenging therapeutic to deliver. Additionally, co-delivery of multiple therapeutics in multifunctional micelles has emerged as an important area in combination therapy research. The main goal of this project was to develop a multifunctional PEG-PE micellar delivery system capable of delivering multiple therapeutics for increased anti-tumor activity. Previous studies have indicated the utility of a DM-PIT-1, a member of a class of novel PIP3-PH inhibitors, and its potential in the treatment of cancer. The PIP3-kinase (PI3K) pathway has been shown to have serious implications in cancer. Inhibiting this pathway has been shown to sensitize the cell to apoptosis. A second generation of more potent and druggable compounds has been developed based on the structure of DM- PIT-1. However, it has been difficult to develop successful compounds inhibiting PIP3 signaling while maintaining the physicochemical properties necessary for an effective drug. Many of these compounds are limited by their poor solubility and rapid clearance in vivo. Incorporating these compounds into PEG-PE micelles allows for increased solubility, prolonged half-life and tumor accumulation

  15. Diatomite biosilica nanocarriers for siRNA transport inside cancer cells.

    Science.gov (United States)

    Rea, Ilaria; Martucci, Nicola M; De Stefano, Luca; Ruggiero, Immacolata; Terracciano, Monica; Dardano, Principia; Migliaccio, Nunzia; Arcari, Paolo; Taté, Rosarita; Rendina, Ivo; Lamberti, Annalisa

    2014-12-01

    Diatomite is a natural porous biomaterial of sedimentary origin, formed by fragments of diatom siliceous skeletons, called "frustules". Due to large availability in many areas of the world, chemical stability, and non-toxicity, these fossil structures have been widespread used in lot of industrial applications, such as food production, water extracting agent, production of cosmetics and pharmaceutics. However, diatomite is surprisingly still rarely used in biomedical applications. In this work, we exploit diatomite nanoparticles for small interfering ribonucleic acid (siRNA) transport inside human epidermoid cancer cells (H1355). Morphology and composition of diatomite microfrustules (average size lower than 40μm) are investigated by scanning electron microscopy equipped by energy dispersive X-ray spectroscopy, Fourier transform infrared analysis, and photoluminescence measurements. Nanometric porous particles (average size lower than 450nm) are obtained by mechanical crushing, sonication, and filtering of micrometric frustules. siRNA bioconjugation is performed on both micrometric and nanometric fragments by silanization. In-vitro experiments show very low toxicity on exposure of the cells to diatomite nanoparticle concentration up to 300μg/ml for 72h. Confocal microscopy imaging performed on cancer cells incubated with siRNA conjugated nanoparticles demonstrates a cytoplasmatic localization of vectors. Gene silencing by delivered siRNA is also demonstrated. Our studies endorse diatomite nanoparticles as non-toxic nanocarriers for siRNA transport in cancer cells. siRNA is a powerful molecular tool for cancer treatment but its delivery is inefficient due to the difficulty to penetrate the cell membrane. siRNA-diatomite nanoconjugate may be well suited for delivery of therapeutic to cancer cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Surface engineering of gold nanoparticles for in vitro siRNA delivery

    Science.gov (United States)

    Zhao, Enyu; Zhao, Zhixia; Wang, Jiancheng; Yang, Chunhui; Chen, Chengjun; Gao, Lingyan; Feng, Qiang; Hou, Wenjie; Gao, Mingyuan; Zhang, Qiang

    2012-07-01

    Cellular uptake, endosomal/lysosomal escape, and the effective dissociation from the carrier are a series of hurdles for specific genes to be delivered both in vitro and in vivo. To construct siRNA delivery systems, poly(allylamine hydrochloride) (PAH) and siRNA were alternately assembled on the surface of 11.8 +/- 0.9 nm Au nanoparticles (GNP), stabilized by denatured bovine serum albumin, by the ionic layer-by-layer (LbL) self-assembly method. By manipulating the outmost PAH layer, GNP-PAH vectors with different surface electric potentials were prepared. Then, the surface potential-dependent cytotoxicity of the resultant GNP-PAH particles was evaluated via sulforhodamine B (SRB) assay, while the surface potential-dependent cellular uptake efficiency was quantitatively analyzed by using the flow cytometry method based on carboxyfluorescein (FAM)-labeled siRNA. It was revealed that the GNP-PAH particles with surface potential of +25 mV exhibited the optimal cellular uptake efficiency and cytotoxicity for human breast cancer MCF-7 cells. Following these results, two more positively charged polyelectrolytes with different protonating abilities in comparison with PAH, i.e., polyethylenimine (PEI), and poly(diallyl dimethyl ammonium chloride) (PDDA), were chosen to fabricate similarly structured vectors. Confocal fluorescence microscopy studies indicated that siRNA delivered by GNP-PAH and GNP-PEI systems was better released than that delivered by the GNP-PDDA system. Further flow cytometric assays based on immunofluorescence staining of the epidermal growth factor receptor (EGFR) revealed that EGFR siRNA delivered by GNP-PAH and GNP-PEI exhibited similar down-regulation effects on EGFR expression in MCF-7 cells. The following dual fluorescence flow cytometry assays by co-staining phosphatidylserine and DNA suggested the EGFR siRNA delivered by GNP-PAH exhibited an improved silencing effect in comparison with that delivered by the commercial transfection reagent

  17. Nose-to-brain delivery of BACE1 siRNA loaded in solid lipid nanoparticles for Alzheimer's therapy.

    Science.gov (United States)

    Rassu, Giovanna; Soddu, Elena; Posadino, Anna Maria; Pintus, Gianfranco; Sarmento, Bruno; Giunchedi, Paolo; Gavini, Elisabetta

    2017-04-01

    We designed a delivery system to obtain an efficient and optimal nose-to-brain transport of BACE1 siRNA, potentially useful in the treatment of Alzheimer's disease. We selected a cell-penetrating peptide, the short peptide derived from rabies virus glycoprotein known as RVG-9R, to increase the transcellular pathway in neuronal cells. The optimal molar ratio between RVG-9R and BACE1 siRNA was elucidated. The complex between the two was then encapsulated. We propose chitosan-coated and uncoated solid lipid nanoparticles (SLNs) as a nasal delivery system capable of exploiting both olfactory and trigeminal nerve pathways. The coating process had an effect on the zeta potential, obtaining positively-charged nanoparticles, and on siRNA protection. The positive charge of the coating formulation ensured mucoadhesiveness to the particles and also prolonged residence time in the nasal cavity. We studied the cellular transport of siRNA released from the SLNs using Caco-2 as a model of epithelial-like phenotypes. We found that siRNA permeates the monolayer to a greater extent when released from any of the studied formulations than from bare siRNA, and primarily from chitosan-coated SLNs.

  18. Stable Expression of Hantavirus H8205 Strain G1/IL-2 Gene and Immune Protection of the Fusion Gene

    Institute of Scientific and Technical Information of China (English)

    XIONG Ying; YUAN Yuan; JIA Min; YU Bing; HUANG Hanju

    2007-01-01

    To explore the feasibility of stable expression of Hantavirus H8205 strain G1 segment and human IL-2 fusion gene in Vero cells, and to examine the immune protection effects on mice vaccinated with this recombinant eukaryotic expression vector containing Hantavirus G1 gene and IL-2 gene. With the help of lipofectamine, the Vero cells were transfected with pcDNA3.1/HisB-IL-2-G1 and the positive cells were selected by G418. IFAT and SDS-PAGE electrophoresis were used to determine the stable transfection and expression of recombinant protein.Each mouse was inoculated with plasmids intramuscularly (i.m.) three times, 2 boosts were given at 2-week intervals, serum anti-hantavirus antibodies were detected by ELISA and neutralizing antibodies (NAb) were detected by Plaque Reduction Neutralization Test. The fusion protein expressed in Vero cells was 78 kD, corresponding to the estimated molecular size. The neutralizing antibody titers of mice with pcDNA3.1/HisB-IL-2-G1 were 1:20-1:80. IL-2/G1 fusion gene could be transferred in Vero cells and stably express the fusion protein. Specific humeral immune responses in mice can be induced with the recombinant eukaryotic expression vector containing the fusion gene, which lays the foundation for further development of therapeutic HTNV vaccine.

  19. VRP09 Reduction of Corneal Scarring Following Blast and Burn Injuries to Cornea Using siRNAs Targeting TGFb and CTGF

    Science.gov (United States)

    2013-03-01

    TGFbRII and CTGF, using cultures of rabbit corneal fibroblasts (RCF). We then would test the optimal siRNA for each gene when formulated into double and...the extensive data below. Objective 1a. Establish primary cultures of rabbit corneal fibroblasts for use in assessing effects of siRNAs on...expression of the three target genes. We successfully established cultures of rabbit corneal fibroblasts using a procedure we described previously in

  20. Utility of MicroRNAs and siRNAs in Cervical Carcinogenesis

    Science.gov (United States)

    Díaz-González, Sacnite del Mar; Benítez-Boijseauneau, Odelia; Gómez-Cerón, Claudia; Bermúdez-Morales, Victor Hugo; Rodríguez-Dorantes, Mauricio; Pérez-Plasencia, Carlos; Peralta-Zaragoza, Oscar

    2015-01-01

    MicroRNAs and siRNAs belong to a family of small noncoding RNAs which bind through partial sequence complementarity to 3′-UTR regions of mRNA from target genes, resulting in the regulation of gene expression. MicroRNAs have become an attractive target for genetic and pharmacological modulation due to the critical function of their target proteins in several signaling pathways, and their expression profiles have been found to be altered in various cancers. A promising technology platform for selective silencing of cell and/or viral gene expression using siRNAs is currently in development. Cervical cancer is the most common cancer in women in the developing world and sexually transmitted infection with HPV is the cause of this malignancy. Therefore, a cascade of abnormal events is induced during cervical carcinogenesis, including the induction of genomic instability, reprogramming of cellular metabolic pathways, deregulation of cell proliferation, inhibition of apoptotic mechanisms, disruption of cell cycle control mechanisms, and alteration of gene expression. Thus, in the present review article, we highlight new research on microRNA expression profiles which may be utilized as biomarkers for cervical cancer. Furthermore, we discuss selective silencing of HPV E6 and E7 with siRNAs which represents a potential gene therapy strategy against cervical cancer. PMID:25874209

  1. Utility of MicroRNAs and siRNAs in Cervical Carcinogenesis

    Directory of Open Access Journals (Sweden)

    Sacnite del Mar Díaz-González

    2015-01-01

    Full Text Available MicroRNAs and siRNAs belong to a family of small noncoding RNAs which bind through partial sequence complementarity to 3′-UTR regions of mRNA from target genes, resulting in the regulation of gene expression. MicroRNAs have become an attractive target for genetic and pharmacological modulation due to the critical function of their target proteins in several signaling pathways, and their expression profiles have been found to be altered in various cancers. A promising technology platform for selective silencing of cell and/or viral gene expression using siRNAs is currently in development. Cervical cancer is the most common cancer in women in the developing world and sexually transmitted infection with HPV is the cause of this malignancy. Therefore, a cascade of abnormal events is induced during cervical carcinogenesis, including the induction of genomic instability, reprogramming of cellular metabolic pathways, deregulation of cell proliferation, inhibition of apoptotic mechanisms, disruption of cell cycle control mechanisms, and alteration of gene expression. Thus, in the present review article, we highlight new research on microRNA expression profiles which may be utilized as biomarkers for cervical cancer. Furthermore, we discuss selective silencing of HPV E6 and E7 with siRNAs which represents a potential gene therapy strategy against cervical cancer.

  2. Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles.

    Science.gov (United States)

    Kooijmans, Sander A A; Stremersch, Stephan; Braeckmans, Kevin; de Smedt, Stefaan C; Hendrix, An; Wood, Matthew J A; Schiffelers, Raymond M; Raemdonck, Koen; Vader, Pieter

    2013-11-28

    Extracellular vesicles (EVs) are specialised endogenous carriers of proteins and nucleic acids and are involved in intercellular communication. EVs are therefore proposed as candidate drug delivery systems for the delivery of nucleic acids and other macromolecules. However, the preparation of EV-based drug delivery systems is hampered by the lack of techniques to load the vesicles with nucleic acids. In this work we have now characterised in detail the use of an electroporation method for this purpose. When EVs were electroporated with fluorescently labelled siRNA, siRNA retention was comparable with previously published results (20-25% based on fluorescence spectroscopy and fluorescence fluctuation spectroscopy), and electroporation with unlabelled siRNA resulted in significant siRNA retention in the EV pellet as measured by RT-PCR. Remarkably, when siRNA was electroporated in the absence of EVs, a similar or even greater siRNA retention was measured. Nanoparticle tracking analysis and confocal microscopy showed extensive formation of insoluble siRNA aggregates after electroporation, which could be dramatically reduced by addition of EDTA. Other strategies to reduce aggregate formation, including the use of cuvettes with conductive polymer electrodes and the use of an acidic citrate electroporation buffer, resulted in a more efficient reduction of siRNA precipitation than EDTA. However, under these conditions, siRNA retention was below 0.05% and no significant differences in siRNA retention could be measured between samples electroporated in the presence or absence of EVs. Our results show that electroporation of EVs with siRNA is accompanied by extensive siRNA aggregate formation, which may cause overestimation of the amount of siRNA actually loaded into EVs. Moreover, our data clearly illustrate that electroporation is far less efficient than previously described, and highlight the necessity for alternative methods to prepare siRNA-loaded EVs.

  3. Attenuation of fibrosis with selective inhibition of c-Abl by siRNA in systemic sclerosis dermal fibroblasts.

    Science.gov (United States)

    Karimizadeh, Elham; Motamed, Nasrin; Mahmoudi, Mahdi; Jafarinejad-Farsangi, Saeideh; Jamshidi, Ahmadreza; Faridani, Habibeh; Gharibdoost, Farhad

    2015-03-01

    Cellular abelson (c-Abl), a non-receptor tyrosine kinase, is an important molecule in the pathogenesis of systemic sclerosis. There have been reports of beneficial effects of pharmacological tyrosine kinase inhibitors, such as imatinib mesylate, on fibrosis. However, these inhibitors affect multiple tyrosine kinases including c-Abl, c-kit, and platelet-derived growth factor receptor. The effects of selective inhibition of c-Abl using small interfering RNA (siRNA) on dermal fibrosis have not yet been explored. The aim of this study is to evaluate whether specific inhibition of c-Abl by siRNA can influence the transforming growth factor-β1 (TGF-β1)-induced fibrotic responses. Dermal fibroblasts from systemic sclerosis patients and healthy controls were transfected with c-Abl siRNA. The expression levels of collagen type I, fibronectin, connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA) were measured at both the mRNA and protein levels in the absence or presence of TGF-β1 pro-fibrotic cytokine. In healthy dermal fibroblasts, the expression of collagen type 1, fibronectin, α-SMA, and CTGF mRNAs and proteins that were upregulated after stimulation with TGF-β1 was markedly decreased by c-Abl siRNA. Silencing of c-Abl via siRNA efficiently reduced the basal synthesis of collagen type I, fibronectin, α-SMA, and CTGF mRNAs and proteins in systemic sclerosis fibroblasts, but it had no effect on the baseline expression of these genes and proteins in healthy dermal fibroblasts. In conclusion, specific c-Abl gene silencing using siRNA effectively reduced fibrosis-related gene expression. Inhibition of c-Abl by siRNA may be a potential therapeutic approach for fibrotic diseases such as systemic sclerosis.

  4. Inhibitory Effect of siRNA Targeting hTERT Genes on the Transformed Mesenchymal Cells%小干扰RNA抑制人端粒酶逆转录酶基因对恶性转化后骨髓间充质干细胞的影响

    Institute of Scientific and Technical Information of China (English)

    周晓岗; 李熙雷; 郭常安; 周健; 戴文达; 董健; 陈峥嵘

    2011-01-01

    了端粒酶的活性,从而增加了TMCs的凋亡、抑制了TMCs的生长,并降低了TMCs在裸鼠体内的成瘤能力.端粒酶在MSCs恶性转化的过程中可能起了重要的作用.%Objective:To investigate the effect of inhibiting human telomerase reverse transcriptase (hTERT) gene by small interfering RNA (siRNA) interference on the proliferation and the tumorigenicity ability of transformed mesenchymal stem cells (TMCs). Methods:Small interference RNA (siRNA) targeting hTERT sequences were synthesized chemically in vitro. TMCs were transfected with three different concentrations of siRNA (50nmol/L, 100nmol/L and 200nmol/L). The proliferation activity of TMCs was determined using methylthiazol tetrazolium (MTT). The expression of hTERT was detected by Reverse transcript polymerase chain reaction (RT-PCR). The content and the activity of the telomerase were tested by western blot and telomeric repeat amplification protocolo(TRAP)-enzyme-linked immunosorbent assay(ELISA) staining. The ability of the tumorigenicity in nude mice of TMCs was also measured. Results: The content of temolerase of TMCs transfected by three different concentrations of siRNA (50nmol/L, 100nmol/L and 200nmol/L) was obviously reduced than that of untransfected control group (46. 2%, 69. 5% and 91.5%). As detected by RT-PCR,mRNA level of hTERT in TMCs transfeeted with siRNA was reduced 19. 2% ,47.2% and 86. 4% than that of control group, respectively. Telomerase activities of TMCs transfeeted by siRNA in all groups were also decreased. The proliferation and the abilities of tumorigenesis of TMCs transfected by siRNA in all groups were significantly reduced. Conclusions:hTERT expression in TMCs can be inhibited significantly with siRNA. Downregulation of hTERT expression can cause the reduction of the proliferation of TMCs in vitro and inhibit their abilities of tumorigenesis in vivo. Telomerase is involved in the malignant transformation of MSCs.

  5. siRNA epidermal growth factor receptor silencing in U251 glioma cells

    Institute of Scientific and Technical Information of China (English)

    Chunsheng Kang; Zhiyong Zhang; Zhifan Jia; Qiang Huang; Guangxiu Wang; Mingzhe Qiu; Peiyu Pu

    2008-01-01

    BACKGROUND: Dicer, a large multidomain ribonuclease, is responsible for processing double-stranded RNAs (dsRNAs) to 20-bp-long small interfering RNAs (siRNAs), which act as effectors during RNA interference (RNAi). OBJECTIVE: To observe the efficacy of siRNA cocktails generated by recombinant human Dicer on the down-regulation of epidermal growth factor receptor (EGFR) expression in human glioma cells. DESIGN, TIME AND SETTING: The following in vitro experiment was performed at the Department of Neurosurgery, Tianjin Medical University General Hospital and Laboratory of Neuro-Oncology, Tianjin Neurological Institute. MATERIALS: Mini-RNA isolation kit, human placenta complimentary DNA (cDNA) was produced by Tiangen Biotech (Beijing, China), human glioblastoma U251-MG cells were produced by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. METHODS: A PCR product from the human EGFR, which corresponded to the tyrosine kinase domain of the 3'-end fragment, was used as the T7-promotor for in vitro transcription, siRNA cocktails were generated by in vitro dicing of double stranded RNA. A total of 500, 250 and 125 μg siRNA cocktails were transiently transfected into U251 glioma cells through the use of the GeneSilencer. MAIN OUTCOME MEASURE: Expression of EGFR was detected by real-time PCR. RESULTS: The total PCR product of the human EGFR, corresponding to the tyrosine kinase domain, is approximately 680 bp in length. The PCR transcriptants included GCC leader sequences and a T7 promoter sequence, with a fragment of EGFR cDNA at the center. The T7 promoter was prepared for in vitro transcription of dsRNA. After dicing for 24 hours, the 21-nt siRNA cocktails were verified by 4% agarose gel. The difference between threshold cycle of a sample assay and threshold cycle of the corresponding endogenous reference (△ Ct) among parental U251 cells and cells transfected with different doses of siRNA cocktails were determined to be 3.06, 7.35, and 10

  6. Bach1 siRNA attenuates bleomycin-induced pulmonary fibrosis by modulating oxidative stress in mice

    Science.gov (United States)

    Liu, Yuan; Zheng, Yi

    2017-01-01

    Oxidative stress plays an essential role in inflammation and fibrosis. Bach1 is an important transcriptional repressor that acts by modulating oxidative stress and represents a potential target in the treatment of pulmonary fibrosis (PF). In this study, we knocked down Bach1 using adenovirus-mediated small interfering RNA (siRNA) to determine whether the use of Bach1 siRNA is an effective therapeutic strategy in mice with bleomycin (BLM)-induced PF. Mouse lung fibroblasts (MLFs) were incubated with transforming growth factor (TGF)-β1 (5 ng/ml) and subsequently infected with recombined adenovirus-like Bach1 siRNA1 and Bach1 siRNA2, while an empty adenovirus vector was used as the negative control. The selected Bach1 siRNA with higher interference efficiency was used for the animal experiments. A mouse model of BLM-induced PF was established, and Bach1 siRNA (1×109 PFU) was administered to the mice via the tail vein. The results revealed that the Bach1 mRNA and protein levels were significantly downregulated by Bach1 siRNA. Furthermore, the MLFs infected with Bach1 siRNA exhibited increased mRNA and protein expression levels of heme oxygenase-1 and glutathione peroxidase 1, but decreased levels of TGF-β1 and interleukin-6 in the cell supernatants compared with the cells exposed to TGF-β1 alone. Bach1 knockdown by siRNA also enhanced the expression of antioxidant factors, but suppressed that of fibrosis-related cytokines in mice compared with the BLM group. Finally, the inflammatory infiltration of alveolar and interstitial cells and the destruction of lung structure were significantly attenuated in the mide administered Bach1 siRNA compared with those in the BLM group. On the whole, our findings demonstrate that Bach1 siRNA exerts protective effects against BLM-induced PF in mice. Our data may provide the basis for the development of novel targeted therapeutic strategies for PF. PMID:27959382

  7. siRNA and cancer immunotherapy.

    Science.gov (United States)

    Ghafouri-Fard, Soudeh; Ghafouri-Fard, Somayyeh

    2012-09-01

    Immunotherapeutic approaches have been gaining attention in the field of cancer treatment because of their possible ability to eradicate cancer cells as well as metastases by recruiting the host immune system. On the other hand, RNA-based therapeutics with the ability to silence expression of specific targets are currently under clinical investigation for various disorders including cancer. As the mechanisms of tumor evasion from the host immune system are versatile, different molecules have the capacity to be targeted by RNAi technology in order to enhance the immune response against tumors. This technology has been used to silence specific targets in tumor cells, as well as immune cells in cancer cell lines, animal models and clinical trials. siRNAs can also stimulate innate immune responses through activation of Toll-like receptors. Although currently clinical trials of the application of siRNA in cancer immunotherapy are few, it is predicted that in future this technology will be used broadly in cancer treatment.

  8. Gene and Drug delivery system and potential treatment into inner ear for protection and regeneration

    Directory of Open Access Journals (Sweden)

    Sho eKanzaki

    2014-10-01

    Full Text Available The most common type of hearing loss results from damage to the cochlea including lost hair cells (HCs and spiral ganglion neurons (SGNs. In mammals, cochlear HC loss causes irreversible hearing impairment because this type of sensory cell cannot regenerate. The protection of SGN degeneration has implications for cochlear implant to patients with severe deafness. This review summarizes the several treatments for HC regeneration based on experiments. We discuss how the neurotrophic factor transgene expression can protect SGN degeneration and describe potential new therapeutic interventions to reduce hearing loss.  We also summarized viral vectors and introduced the gene and drug delivery system for cochlear hair cells regeneration and protection. Finally, we introduce the novel endoscopy we developed for local injection into cochlea.

  9. The effect of deafness duration on neurotrophin gene therapy for spiral ganglion neuron protection.

    Science.gov (United States)

    Wise, Andrew K; Tu, Tian; Atkinson, Patrick J; Flynn, Brianna O; Sgro, Beatrice E; Hume, Cliff; O'Leary, Stephen J; Shepherd, Robert K; Richardson, Rachael T

    2011-08-01

    A cochlear implant can restore hearing function by electrically exciting spiral ganglion neurons (SGNs) in the deaf cochlea. However, following deafness SGNs undergo progressive degeneration ultimately leading to their death. One significant cause of SGN degeneration is the loss of neurotrophic support that is normally provided by cells within the organ of Corti (OC). The administration of exogenous neurotrophins (NTs) can protect SGNs from degeneration but the effects are short-lived once the source of NTs has been exhausted. NT gene therapy, whereby cells within the cochlea are transfected with genes enabling them to produce NTs, is one strategy for providing a cellular source of NTs that may provide long-term support for SGNs. As the SGNs normally innervate sensory cells within the OC, targeting residual OC cells for gene therapy in the deaf cochlea may provide a source of NTs for SGN protection and targeted regrowth of their peripheral fibers. However, the continual degeneration of the OC over extended periods of deafness may deplete the cellular targets for NT gene therapy and hence limit the effectiveness of this method in preventing SGN loss. This study examined the effects of deafness duration on the efficacy of NT gene therapy in preventing SGN loss in guinea pigs that were systemically deafened with aminoglycosides. Adenoviral vectors containing green fluorescent protein (GFP) with or without genes for Brain Derived Neurotrophic Factor (BDNF) and Neurotrophin-3 (NT3) were injected into the scala media (SM) compartment of cochleae that had been deafened for one, four or eight weeks prior to the viral injection. The results showed that viral transfection of cells within the SM was still possible even after severe degeneration of the OC. Supporting cells (pillar and Deiters' cells), cells within the stria vascularis, the spiral ligament, endosteal cells lining the scala compartments and interdental cells in the spiral limbus were transfected. However, the

  10. Enhanced endosomal/lysosomal escape by distearoyl phosphoethanolamine-polycarboxybetaine lipid for systemic delivery of siRNA.

    Science.gov (United States)

    Li, Yan; Cheng, Qiang; Jiang, Qian; Huang, Yuanyu; Liu, Hongmei; Zhao, Yuliang; Cao, Weipeng; Ma, Guanghui; Dai, Fengying; Liang, Xingjie; Liang, Zicai; Zhang, Xin

    2014-02-28

    Cationic liposome based siRNA delivery system has improved the efficiencies of siRNA. However, cationic liposomes are prone to be rapidly cleared by the reticuloendothelial system (RES). Although modification of cationic liposomes with polyethylene glycol (PEG) could prolong circulation lifetime, PEG significantly inhibits siRNA entrapment efficiency, cellular uptake and endosomal/lysosomal escape process, resulting in low gene silencing efficiency of siRNA. In this study, we report the synthesis of zwitterionic polycarboxybetaine (PCB) based distearoyl phosphoethanolamine-polycarboxybetaine (DSPE-PCB) lipid for cationic liposome modification. The DSPE-PCB20 cationic liposome/siRNA complexes (lipoplexes) show an excellent stability in serum medium. The siRNA encapsulation efficiency of DSPE-PCB20 lipoplexes could reach 92% at N/P ratio of 20/1, but only 73% for DSPE-PEG lipoplexes. The zeta potential of DSPE-PCB20 lipoplexes is 8.19±0.53mV at pH 7.4, and increases to 24.6±0.87mV when the pH value is decreased to 4.5, which promotes the endosomal/lysosomal escape of siRNA. The DSPE-PCB20 modification could enhance the silencing efficiency of siRNA by approximately 20% over the DSPE-PEG 2000 lipoplexes at the same N/P ratio in vitro. Furthermore, DSPE-PCB20 lipoplexes could efficiently mediate the down-regulation of Apolipoprotein B (ApoB) mRNA in the liver and consequently decrease the total cholesterol in the serum in vivo, suggesting therapeutic potentials for siRNA delivery in hypercholesterolemia-related diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Albumin pre-coating enhances intracellular siRNA delivery of multifunctional amphiphile/siRNA nanoparticles

    Directory of Open Access Journals (Sweden)

    Kummitha CM

    2012-10-01

    Full Text Available China M Kummitha, Anthony S Malamas, Zheng-Rong LuDepartment of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USAAbstract: Nonspecific association of serum molecules with short-interfering RNA (siRNA nanoparticles can change their physiochemical characteristics, and results in reduced cellular uptake in the target tissue during the systemic siRNA delivery process. Serum albumin is the most abundant protein in the body and has been used to modify the surface of nanoparticles, to inhibit association of other serum molecules. Here, we hypothesized that surface modification of lipid-based nanoparticular siRNA delivery systems with albumin could prevent their interaction with serum proteins, and improve intracellular uptake. In this study, we investigated the influence of albumin on the stability and intracellular siRNA delivery of the targeted siRNA nanoparticles of a polymerizable and pH-sensitive multifunctional surfactant N-(1-aminoethyliminobis[N-(oleoylcysteinylhistinyl-1-aminoethylpropionamide] (EHCO in serum. Serum resulted in a significant increase in the size of targeted EHCO/siRNA nanoparticles and inhibited cellular uptake of the nanoparticles. Coating of targeted EHCO/siRNA nanoparticles with bovine serum albumin at 9.4 µM prior to cell transfection improved cellular uptake and gene silencing efficacy of EHCO/siRNA targeted nanoparticles in serum-containing media, as compared with the uncoated nanoparticles. At a proper concentration, albumin has the potential to minimize interactions of serum proteins with siRNA nanoparticles for effective systemic in vivo siRNA delivery.Keywords: multifunctional, lipid nanoparticles, RNA interference, pH-sensitive amphiphile, siRNA

  12. Hydrolytic charge-reversal of PEGylated polyplexes enhances intracellular un-packaging and activity of siRNA.

    Science.gov (United States)

    Werfel, Thomas A; Swain, Corban; Nelson, Christopher E; Kilchrist, Kameron V; Evans, Brian C; Miteva, Martina; Duvall, Craig L

    2016-04-01

    Hydrolytically degrading nano-polyplexes (HDG-NPs) that reverse charge through conversion of tertiary amines to carboxylic acids were investigated to improve intracellular un-packaging of siRNA and target gene silencing compared to a non-degradable analog (non-HDG-NPs). Both NP types comprised reversible addition-fragmentation chain-transfer (RAFT) synthesized diblock copolymers of a poly(ethylene glycol) (PEG) corona-forming block and a cationic block for nucleic acid packaging that incorporated butyl methacrylate (BMA) and either dimethylaminoethyl methacrylate (DMAEMA, non-HDG-NPs) or dimethylaminoethyl acrylate (DMAEA, HDG-NPs). HDG-NPs decreased significantly in size and released significantly more siRNA (∼40%) than non-HDG-NPs after 24 h in aqueous solution. While both HDG-NPs and non-HDG-NPs had comparable uptake and cytotoxicity up to 150 nM siRNA doses, HDG-NPs achieved significantly higher target gene silencing of the model gene luciferase in vitro. High resolution FRET confocal microscopy was used to monitor the intracellular un-packaging of siRNA. Non-HDG-NPs had significantly higher FRET efficiency than HDG-NPs, indicating that siRNA delivered from HDG-NPs was more fully un-packaged and therefore had improved intracellular bioavailability.

  13. Photoluminescent and biodegradable polycitrate-polyethylene glycol-polyethyleneimine polymers as highly biocompatible and efficient vectors for bioimaging-guided siRNA and miRNA delivery.

    Science.gov (United States)

    Wang, Min; Guo, Yi; Yu, Meng; Ma, Peter X; Mao, Cong; Lei, Bo

    2017-02-20

    Development of biodegradable and biocompatible non-viral vectors with intrinsical multifunctional properties such as bioimaging ability for highly efficient nucleic acids delivery still remains a challenge. Here, a biodegradable poly (1,8-octanedio-citric acid)-co-polyethylene glycol grafted with polyethyleneimine (PEI) (POCG-PEI) polymers with the photoluminescent capacity were synthesized for nucleic acids delivery (siRNA and miRNA). POCG-PEI polymers can efficiently bind various nucleic acids, protect them against enzymatic degradation and release the genes in the presence of polyanionic heparin. POCG-PEI also showed a significantly low cytotoxicity, enhanced cellular uptake and high transfection efficiency of nucleic acids, as compared to commercial transfection agents, lipofectamine 2000 (Lipo) and polyethylenimine (PEI 25K). POCG-PEI polymers demonstrate an excellent photostability, which allows for imaging the cells and real-time tracking the nucleic acids delivery. The photoluminescent property, low cytotoxicity, biodegradation, good gene binding and protection ability and high genes delivery efficiency make POCG-PEI highly competitive as a non-virus vector for genes delivery and real-time bioimaging applications. Our results may be also an important step for designing biodegradable biomaterials with multifunctional properties towards bioimaging-guided genes therapeutic applications.

  14. Tumor priming enhances siRNA delivery and transfection in intraperitoneal tumors.

    Science.gov (United States)

    Wang, Jie; Lu, Ze; Yeung, Bertrand Z; Wientjes, M Guillaume; Cole, David J; Au, Jessie L-S

    2014-03-28

    Cancers originating from the digestive system account for 290,000 or ~20% of all new cancer cases annually in the US. We previously developed paclitaxel-loaded tumor-penetrating microparticles (TPM) for intraperitoneal (IP) treatment of peritoneal tumors (Lu et al., 2008; Tsai et al., 2007; Tsai et al., 2013). TPM is undergoing NIH-supported IND-enabling studies for clinical evaluation. The present study evaluated the hypothesis that TPM, via inducing apoptosis and expanding the interstitial space, promotes the delivery and transfection of lipid vectors containing siRNA. The in vivo model was the metastatic human Hs766T pancreatic tumor that, upon IP injection, produced widely distributed solid tumors and ascites in the peritoneal cavity in 100% of animals. The target gene was survivin, an anti-apoptotic protein induced by chemotherapy and associated with metastases and poor prognosis of patients with gastric and colorectal cancers. The siRNA carrier was pegylated liposomes comprising cationic and neutral lipids plus a fusogenic lipid (PCat). PCat-loaded with survivin siRNA (PCat-siSurvivin) was active in cultured cells (decreased survivin mRNA and protein levels, reduced cell clonogenicity, enhanced paclitaxel activity), but lost its activity in vivo; this difference is consistent with the well-known problem of inadequate delivery and transfection of siRNA in vivo. In comparison, single agent TPM prolonged animal survival and, as expected, induced survivin expression in tumors. Addition of PCat-siSurvivin reversed the TPM-induced survivin expression and enhanced the antitumor activity of TPM. The finding that in vivo survivin knockdown by PCat-siSurvivin was successful only when it was given in combination with TPM provides the proof-of-concept that tumor priming promotes the delivery and transfection of liposomal siRNA. The data further suggest the TPM/PCat-siSurvivin combination as a potentially useful chemo-gene therapy for peritoneal cancer.

  15. Improvement of cytomegalovirus pp65 DNA vaccine efficacy by co-administration of siRNAs targeting BAK and BAX.

    Science.gov (United States)

    Liu, Jixiao; Feng, Keke; Zhao, Lu; Luo, Haining; Zhu, Yingjun

    2017-06-01

    The efficacy of DNA vaccines may be improved by small interfering (si)RNA adjuvants targeting pro-apoptotic genes. The aim of the present study was to investigate the capacity of siRNAs targeting B-cell lymphoma 2 homologous antagonist killer (BAK) and B-cell lymphoma 2-associated X protein (BAX) to improve the efficacy of a cytomegalovirus (CMV) vaccine. BALB/c mice were divided into four groups (n=18 in each): unimmunized and immunized with pcDNA 3.1-pp65 expressing CMV 65 kDa matrix phosphoprotein and BAK + BAX siRNAs, pcDNA 3.1-pp65 and control siRNA, or control pcDNA 3.1 and BAK + BAX siRNAs. Immunizations were performed twice with an interval of 3 weeks. CMV-specific mouse splenocyte interferon (IFN)-γ secretion was assessed by ELISPOT; furthermore, an in vivo cytotoxic T lymphocyte assay was performed 2 weeks after the last immunization. After lethal CMV challenge of the mice, body weight, virus titers in the spleens and salivary glands as well as survival were recorded. The amount of splenocytes secreting IFN-γ in response to CMV pp65 peptides and specific lysis of peptide-pulsed target cells were significantly higher in mice administered pcDNA3.1-pp65 and BAK + BAX siRNAs than those in mice administered pcDNA3.1-pp65 and control siRNA (PBAX siRNAs were significantly lower than those in mice immunized with pcDNA3.1-pp65 and control siRNA (PBAX siRNAs survived for longer, and at 21 days after lethal CMV challenge, 66 and 100% of these mice survived, respectively. These mice also experienced less weight loss compared with mice immunized with pcDNA3.1-pp65 and control siRNA (PBAX improved the efficacy of CMV pp65 DNA vaccine.

  16. Transgenic Tobacco Expressing a Modified VP6 Gene Protects Mice Against Rotavirus Infection

    Institute of Scientific and Technical Information of China (English)

    Jiang-Li DONG; Bo ZHOU; Gang SHENG; Tao WANG

    2005-01-01

    Elevated expression of the rotavirus VP6 antigen in transgenic plants is a critical factor in the development of a safe and effective rotavirus vaccine. Using codon optimization, a gene that encodes the inner capsid protein VP6 of the human group A rotavirus was synthesized (sVP6). The VP6 and sVp6genes were transformed into tobacco (Nicotiana tabacum L.) plants using Agrobacterium tumefaciens. The expression level of the sVP6 gene in transgenic plants was 3.8-34-fold higher than that of controls containing the non-modified VP6 gene, accounting for up to 0.34% of the total soluble protein (TSP). Then, BALB/c female mice that had been gavaged weekly with 10 mg TSP containing 34 μg VP6 protein, in which VP6-specific serum IgG and mucosal IgA antibodies were investigated. The severity and duration of diarrhea caused by simian rotavirus SA-11 challenge were reduced significantly in passively immunized pups, which indicates that anti-VP6 antibodies generated in orally immunized female mice can be passed onto pups and provide heterotypic protection. An edible vaccine based on the VP6 of human rotavirus group A could provide a means to protect children and young animals from severe acute diarrhea.

  17. Direct transfer of A20 gene into pancreas protected mice from streptozotocin-induced diabetes

    Institute of Scientific and Technical Information of China (English)

    Lu-yang YU; Bo LIN; Zhen-lin ZHANG; Li-he GUO

    2004-01-01

    AIM: To investigate the efficiency of transfer of A20 gene into pancreas against STZ-induced diabetes. METHODS:PVP-plasmid mixture was directly transferred into the pancreatic parenchyma 2 d before STZ injection. The uptake of plasmid pcDNA3-LacZ or pcDNA3-A20 was detected by PCR and the expression of LacZ was confirmed by histological analysis with X-gal. A20 expression in the pancreas of pcDNA3-A20 transgenic mice was measured by RT-PCR and Westem blots. Urine amylase, NO generation, and histological examination were examined. RESULTS:Injection of PVP-plasmid mixture directly into the pancreatic parenchyma increased urine amylase concentration 16 h after operation and reversed it to nearly normal 36 h later. On d 33 LacZ expression could be found in spleen,duodenum, and islets. The development of diabetes was prevented by direct A20 gene transferring into the pancreas and A20-mediated protection was correlated with suppression of NO production. The insulitis was ameliorated in A20-treated mice. CONCLUSION: Injection of PVP-plasmid mixture directly into the pancreatic parenchyma led to target gene expression in islets. Direct transfer of A20 gene into the pancreas protected mice from STZ-induced diabetes.

  18. Profiling of genes related to cross protection and competition for NbTOM1 by HLSV and TMV.

    Science.gov (United States)

    Wen, Yi; Lim, Grace Xiao-Yun; Wong, Sek-Man

    2013-01-01

    Cross protection is the phenomenon through which a mild strain virus suppresses symptoms induced by a closely related severe strain virus in infected plants. Hibiscus latent Singapore virus (HLSV) and Tobacco mosaic virus (TMV) are species within the genus tobamovirus. HLSV can protect Nicotianabenthamiana against TMV-U1 strain, resulting in mild symptoms instead of severe systemic necrosis. The mechanism of cross protection between HLSV and TMV is unknown. In the past, some researchers suggest that the protecting virus strain might occupy virus-specific replication sites within a cell leaving no room for the challenge virus. Quantitative real-time RT-PCR was performed to detect viral RNA levels during cross protection. HLSV accumulation increased in cross protected plants compared with that of single HLSV infected plants, while TMV decreased in cross protected plants. This suggests that there is a competition for host factors between HLSV and TMV for replication. To investigate the mechanism under the cross protection between HLSV and TMV, microarray analysis was conducted to examine the transcriptional levels of global host genes during cross protection, using Tobacco Gene Expression Microarray, 4 x 44 k slides. The transcriptional level of some host genes corresponded to accumulation level of TMV. Some host genes were up-regulated only by HLSV. Tobamovirus multiplication gene 1 (TOM1), essential for tobamovirus multiplication, was involved in competition for replication by HLSV and TMV during cross protection. Both HLSV and TMV accumulation decreased when NbTOM1 was silenced. A large quantity of HLSV resulted in decreased TMV accumulation in HLSV+TMV (100:1) co-infection. These results indicate that host genes involved in the plant defense response and virus multiplication are up-regulated by challenge virus TMV but not by protecting virus HLSV during cross protection.

  19. ANTI-INFLAMMATORY EFFECT OF ANTI-TNF-ALPHA siRNA CATIONIC PHOSPHOROUS DENDRIMERS NANOCOMPLEXES ADMINISTERED INTRANASALLY IN A MURINE ACUTE LUNG INJURY MODEL

    DEFF Research Database (Denmark)

    Bohr, Adam; Tsapis, Nicolas; Andreana, Ilaria

    2017-01-01

    was attributed to their higher pKa value leading to a stronger siRNA complexation and improved protection against enzymatic degradation resulting in a higher cellular uptake. The superior silencing effect of the pyrrolidinium dendriplexes, compared to non-complexed siRNA, was confirmed in vivo in an LPS......-induced murine model of short term acute lung injury upon lung delivery via nasal administration. These data suggest that phosphorous dendriplexes have a strong potential in lung delivery of siRNA for treating inflammatory lung diseases....

  20. Topoisomerase 1 inhibition suppresses inflammatory genes and protects from death by inflammation.

    Science.gov (United States)

    Rialdi, Alex; Campisi, Laura; Zhao, Nan; Lagda, Arvin Cesar; Pietzsch, Colette; Ho, Jessica Sook Yuin; Martinez-Gil, Luis; Fenouil, Romain; Chen, Xiaoting; Edwards, Megan; Metreveli, Giorgi; Jordan, Stefan; Peralta, Zuleyma; Munoz-Fontela, Cesar; Bouvier, Nicole; Merad, Miriam; Jin, Jian; Weirauch, Matthew; Heinz, Sven; Benner, Chris; van Bakel, Harm; Basler, Christopher; García-Sastre, Adolfo; Bukreyev, Alexander; Marazzi, Ivan

    2016-05-27

    The host innate immune response is the first line of defense against pathogens and is orchestrated by the concerted expression of genes induced by microbial stimuli. Deregulated expression of these genes is linked to the initiation and progression of diseases associated with exacerbated inflammation. We identified topoisomerase 1 (Top1) as a positive regulator of RNA polymerase II transcriptional activity at pathogen-induced genes. Depletion or chemical inhibition of Top1 suppresses the host response against influenza and Ebola viruses as well as bacterial products. Therapeutic pharmacological inhibition of Top1 protected mice from death in experimental models of lethal inflammation. Our results indicate that Top1 inhibition could be used as therapy against life-threatening infections characterized by an acutely exacerbated immune response.

  1. Protection of rat islet viability following heme oxygenase-1 gene transfection via adenoviral vector in vitro

    Institute of Scientific and Technical Information of China (English)

    Xiaobo Chen; Yongxiang Li; Weiping Dong; Yang Jiao; Jianming Tan

    2007-01-01

    Objective: To investigate the effect of Heme oxygenase-1 (HO-1) gene transfection on the viability of cultured rat islets, and to explore the potential value of HO-1 gene in islet transplantation. Methods:Recombinant adenovirus vector containing human HO-1 gene(Ad-HO-1 ) or enhanced green fluorescent protein gene(Ad-EGFP) was generated by using AdEasy system respectively.The rat islets were transfected with Ad-HO-1, Ad-EGFP or blank vector and then cultured for 7 days. Transfection was confirmed by expression of EGFP and human HO-1 protein detected by fluorescence photographs and western blot, respectively. The insulin release upon different concentration of glucose stimulation was detected using insulin radioimmunoassay kit, and stimulation index (SI) was calculated. Glucose-stimulated insulin release was usedto assess islet viability. Results:Adenovirus vector successfully transferred HO-1 gene to rat islet cells in vitro, and the insulin release upon high level of glucose stimulation and stimulation index(SI) of Ad-HO-1-infected islets were significantly higher than those of Ad-EGFP-infected islets and control islets(P < 0.05).Conclusion: Adenovirus-mediated HO-1 gene transfection is a feasible strategy to confer cytoprotection and therefore protect the viability of cultured rat islets.

  2. Construction and identification of replication-competent adenovirus expressing siRNA targeting CD133 gene regulated by survivin promoter and its inhibition of liver cancer cell growth%survivin 启动子调控肿瘤干细胞标记 CD133基因 siRNA增殖型溶瘤腺病毒的构建及对肝癌细胞生长的抑制作用

    Institute of Scientific and Technical Information of China (English)

    牛坚; 王月; 刘斌; 王人颢; 朱志军; 申海莲

    2016-01-01

    目的:构建 survivin 启动子调控的靶向 CD133基因的 siRNA 增殖型溶瘤腺病毒,研究其对肝癌细胞生长的影响。方法RT-PCR 法扩增 survivin 启动子,测序鉴定,双酶切连接,获得 pH-XC2-survivin。酶切 pH-XC2-survivin、pZD55-CD133-siRNA 获得 survivin 启动子表达框的亚克隆和CD133-siRNA 基因表达框的亚克隆,连接获得 survivin 启动子调控的 siRNA 增殖型溶瘤腺病毒表达载体质粒 pT-ZD55-CD133-siRNA。增殖型溶瘤腺病毒 survivin-T-ZD55-CD133-siRNA 经 PCR 和测序鉴定。 qRT-PCR 法检测 CD133表达, Western blot 法检测 E1A,CCK-8法检测细胞生长,流式细胞术检测细胞凋亡。结果成功构建增殖型溶瘤腺病毒 sur-vivin-T-ZD55-CD133-siRNA。 qRT-PCR 法检测 CD133 mRNA明显下降, Western blot 证实 survivin-T-ZD55-CD133-siRNA在肿瘤细胞中表达 E1A 能抑制肝癌细胞 CD133表达及生长。结论构建的增殖型溶瘤腺病毒可有效降低肝癌细胞CD133的表达,用于肝癌基因治疗的进一步研究。%Objective To construct a replication-competent adenovirus expressing siRNA targeting CD133 gene regulated by survivin promoter and investigate its inhibitory effect on Hep 3B cells.Methods The fragment of the survivin promoter was amplified by PCR and inserted into pH -XC2 to reconstruct a recombinant plasmid pH -XC2-survivin.Complete digestion pH-XC2-survivin and pZD55-CD133-siRNA, combinational joining the subclones, then getting replication-competent adenovirus expressing short interference RNA targeting CD 133 gene regulated by survivin promoter, replication-competent adenovirus was constructed .The recombined adenoviruses ( T-ZD55-CD133-siRNA) were verified by PCR and sequencing .The effect of T-ZD55-CD133-siRNA on CD133 expression in Hep3B cells was detected by qRT-PCR.The expression of E1A was detected by Western blot.The antitumor po-tential of replication

  3. Protective gene expression changes elicited by an inherited defect in photoreceptor structure.

    Directory of Open Access Journals (Sweden)

    Yagya V Sharma

    Full Text Available Inherited defects in retinal photoreceptor structure impair visual transduction, disrupt relationship with the retinal pigment epithelium (RPE, and compromise cell viability. A variety of progressive retinal degenerative diseases can result, and knowledge of disease etiology remains incomplete. To investigate pathogenic mechanisms in such instances, we have characterized rod photoreceptor and retinal gene expression changes in response to a defined insult to photoreceptor structure, using the retinal degeneration slow (rds mouse model. Global gene expression profiling was performed on flow-sorted rds and wild-type rod photoreceptors immediately prior and subsequent to times at which OSs are normally elaborated. Dysregulated genes were identified via microarray hybridization, and selected candidates were validated using quantitative PCR analyses. Both the array and qPCR data revealed that gene expression changes were generally modest and dispersed amongst a variety of known functional networks. Although genes showing major (>5-fold differential expression were identified in a few instances, nearly all displayed transient temporal profiles, returning to WT levels by postnatal day (P 21. These observations suggest that major defects in photoreceptor cell structure may induce early homeostatic responses, which function in a protective manner to promote cell viability. We identified a single key gene, Egr1, that was dysregulated in a sustained fashion in rds rod photoreceptors and retina. Egr1 upregulation was associated with microglial activation and migration into the outer retina at times subsequent to the major peak of photoreceptor cell death. Interestingly, this response was accompanied by neurotrophic factor upregulation. We hypothesize that activation of Egr1 and neurotrophic factors may represent a protective immune mechanism which contributes to the characteristically slow retinal degeneration of the rds mouse model.

  4. Does the KIR2DS5 gene protect from some human diseases?

    Directory of Open Access Journals (Sweden)

    Izabela Nowak

    Full Text Available BACKGROUND: KIR2DS5 gene encodes an activating natural killer cell receptor whose ligand is not known. It was recently reported to affect the outcome of hematopoietic stem cell transplantation. METHODOLOGY/PRINCIPAL FINDINGS: In our studies on KIR2DS5 gene associations with human diseases, we compared the frequencies of this gene in patients and relevant controls. Typing for KIR2DS5 gene was performed by either individual or multiplex polymerase chain reactions which, when compared in the same samples, gave concordant results. We noted an apparently protective effect of KIR2DS5 gene presence in several clinical conditions, but not in others. Namely, this effect was observed in ankylosing spondylitis (p=0.003, odds ratio [OR]=0.47, confidence interval [CI]=0.28-0.79, endometriosis (p=0.03, OR=0.25, CI = 0.07-0.82 and acute rejection of kidney graft (p=0.0056, OR=0.44, CI=0.24-0.80, but not in non-small-cell lung carcinoma, rheumatoid arthritis, spontaneous abortion, or leukemia (all p>0.05. In addition, the simultaneous presence of KIR2DS5 gene and HLA-C C1 allotype exhibited an even stronger protective effect on ankylosing spondylitis (p=0.0003, OR=0.35, CI=0.19-0.65, whereas a lack of KIR2DS5 and the presence of the HLA-C C2 allotype was associated with ankylosing spondylitis (p=0.0017, OR=1.92, CI=1.28-2.89, whereas a lack of KIR2DS5 and presence of C1 allotype was associated with rheumatoid arthritis (p=0.005, OR=1.47, CI=1.13-1.92. The presence of both KIR2DS5 and C1 seemed to protect from acute kidney graft rejection (p=0.017, OR=0.47, CI=0.25-0.89, whereas lack of KIR2DS5 and presence of C2 seemed to favor rejection (p=0.0015, OR=2.13, CI=1.34-3.37. CONCLUSIONS/SIGNIFICANCE: Our results suggest that KIR2DS5 may protect from endometriosis, ankylosing spondylitis, and acute rejection of kidney graft.

  5. New tools to study RNA interference to fish viruses: Fish cell lines permanently expressing siRNAs targeting the viral polymerase of viral hemorrhagic septicemia virus

    DEFF Research Database (Denmark)

    Ruiz, S.; Schyth, Brian Dall; Encinas, P.

    2009-01-01

    Previous studies have indicated that low transfection efficiency can be a major problem when gene inhibition by the use of small interfering RNAs (siRNAs) is attempted in fish cells. This may especially be true when targeting genes of viruses which are fast replicating and which can still infect ...... stably expressing rhabdoviral specific siRNA sequences could be a strategy to further investigate the use of RNA interference for targeting costly fish pathogenic viruses.......Previous studies have indicated that low transfection efficiency can be a major problem when gene inhibition by the use of small interfering RNAs (siRNAs) is attempted in fish cells. This may especially be true when targeting genes of viruses which are fast replicating and which can still infect...... cells that have not been transfected with the antiviral siRNAs. To increase the amount of antiviral siRNAs per cell a different strategy than transfection was taken here. Thus, we describe carp epithelioma papulosum cyprinid (EPC) cell clones expressing siRNAs designed to target the L polymerase gene...

  6. Inefficient cationic lipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelial cells in vivo

    Directory of Open Access Journals (Sweden)

    Hu Jim

    2006-02-01

    Full Text Available Abstract Background The cationic lipid Genzyme lipid (GL 67 is the current "gold-standard" for in vivo lung gene transfer. Here, we assessed, if GL67 mediated uptake of siRNAs and asODNs into airway epithelium in vivo. Methods Anti-lacZ and ENaC (epithelial sodium channel siRNA and asODN were complexed to GL67 and administered to the mouse airway epithelium in vivo Transfection efficiency and efficacy were assessed using real-time RT-PCR as well as through protein expression and functional studies. In parallel in vitro experiments were carried out to select the most efficient oligonucleotides. Results In vitro, GL67 efficiently complexed asODNs and siRNAs, and both were stable in exhaled breath condensate. Importantly, during in vitro selection of functional siRNA and asODN we noted that asODNs accumulated rapidly in the nuclei of transfected cells, whereas siRNAs remained in the cytoplasm, a pattern consistent with their presumed site of action. Following in vivo lung transfection siRNAs were only visible in alveolar macrophages, whereas asODN also transfected alveolar epithelial cells, but no significant uptake into conducting airway epithelial cells was seen. SiRNAs and asODNs targeted to β-galactosidase reduced βgal mRNA levels in the airway epithelium of K18-lacZ mice by 30% and 60%, respectively. However, this was insufficient to reduce protein expression. In an attempt to increase transfection efficiency of the airway epithelium, we increased contact time of siRNA and asODN using the in vivo mouse nose model. Although highly variable and inefficient, transfection of airway epithelium with asODN, but not siRNA, was now seen. As asODNs more effectively transfected nasal airway epithelial cells, we assessed the effect of asODN against ENaC, a potential therapeutic target in cystic fibrosis; no decrease in ENaC mRNA levels or function was detected. Conclusion This study suggests that although siRNAs and asODNs can be developed to inhibit

  7. Efficient siRNA delivery and tumor accumulation mediated by ionically cross-linked folic acid-poly(ethylene glycol)-chitosan oligosaccharide lactate nanoparticles: for the potential targeted ovarian cancer gene therapy.

    Science.gov (United States)

    Li, Tony Shing Chau; Yawata, Toshio; Honke, Koichi

    2014-02-14

    For effective ovarian cancer gene therapy, systemic administrated tumor-targeting siRNA/folic acid-poly(ethylene glycol)-chitosan oligosaccharide lactate (FA-PEG-COL) nanoparticles is vital for delivery to cancer site(s). siRNA/FA-PEG-COL nanoparticles were prepared by ionic gelation for effective FA receptor-expressing ovarian cancer cells transfection and in vivo accumulation. The chemical structure of FA-PEG-COL conjugate was characterized by MALDI-TOF-MS, FT-IR and (1)H NMR. The average size of siRNA/FA-PEG-COL nanoparticles was approximately 200 nm, and the surface charge was +8.4 mV compared to +30.5 mV with siRNA/COL nanoparticles. FA-PEG-COL nanoparticles demonstrated superior compatibility with erythrocytes in terms of degree of aggregation and haemolytic activity and also effects on cell viability was lower when compared with COL nanoparticles. FA grafting significantly facilitated the uptake of nanoparticles via receptor mediated endocytosis as demonstrated by flow cytometry. The in vitro transfection and gene knockdown efficiency of HIF-1α were superior to COL nanoparticles (76-62%, respectively) and was comparable to Lipofectamine 2000 (79%) as demonstrated by RT-qPCR and Western blot. Gene knockdown at the molecular level translated into effective inhibition of proliferation in vitro. Accumulation efficiency of FA-PEG-COL nanoparticles was investigated in BALB/c mice bearing OVK18 #2 tumor xenograft using in vivo imaging. The active targeting FA-PEG-COL nanoparticles showed significantly greater accumulation than the passive targeting COL nanoparticles. Based on the results obtained, siRNA/FA-PEG-COL nanoparticles show much potential for effective ovarian cancer treatment via gene therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Global Gene Expression Analysis of Cross-Protected Phenotype of Pectobacterium atrosepticum

    Science.gov (United States)

    Gorshkov, Vladimir; Kwenda, Stanford; Petrova, Olga; Osipova, Elena; Gogolev, Yuri; Moleleki, Lucy N.

    2017-01-01

    The ability to adapt to adverse conditions permits many bacterial species to be virtually ubiquitous and survive in a variety of ecological niches. This ability is of particular importance for many plant pathogenic bacteria that should be able to exist, except for their host plants, in different environments e.g. soil, water, insect-vectors etc. Under some of these conditions, bacteria encounter absence of nutrients and persist, acquiring new properties related to resistance to a variety of stress factors (cross-protection). Although many studies describe the phenomenon of cross-protection and several regulatory components that induce the formation of resistant cells were elucidated, the global comparison of the physiology of cross-protected phenotype and growing cells has not been performed. In our study, we took advantage of RNA-Seq technology to gain better insights into the physiology of cross-protected cells on the example of a harmful phytopathogen, Pectobacterium atrosepticum (Pba) that causes crop losses all over the world. The success of this bacterium in plant colonization is related to both its virulence potential and ability to persist effectively under various stress conditions (including nutrient deprivation) retaining the ability to infect plants afterwards. In our previous studies, we showed Pba to be advanced in applying different adaptive strategies that led to manifestation of cell resistance to multiple stress factors. In the present study, we determined the period necessary for the formation of cross-protected Pba phenotype under starvation conditions, and compare the transcriptome profiles of non-adapted growing cells and of adapted cells after the cross-protective effect has reached the maximal level. The obtained data were verified using qRT-PCR. Genes that were expressed differentially (DEGs) in two cell types were classified into functional groups and categories using different approaches. As a result, we portrayed physiological features

  9. Global Gene Expression Analysis of Cross-Protected Phenotype of Pectobacterium atrosepticum.

    Science.gov (United States)

    Gorshkov, Vladimir; Kwenda, Stanford; Petrova, Olga; Osipova, Elena; Gogolev, Yuri; Moleleki, Lucy N

    2017-01-01

    The ability to adapt to adverse conditions permits many bacterial species to be virtually ubiquitous and survive in a variety of ecological niches. This ability is of particular importance for many plant pathogenic bacteria that should be able to exist, except for their host plants, in different environments e.g. soil, water, insect-vectors etc. Under some of these conditions, bacteria encounter absence of nutrients and persist, acquiring new properties related to resistance to a variety of stress factors (cross-protection). Although many studies describe the phenomenon of cross-protection and several regulatory components that induce the formation of resistant cells were elucidated, the global comparison of the physiology of cross-protected phenotype and growing cells has not been performed. In our study, we took advantage of RNA-Seq technology to gain better insights into the physiology of cross-protected cells on the example of a harmful phytopathogen, Pectobacterium atrosepticum (Pba) that causes crop losses all over the world. The success of this bacterium in plant colonization is related to both its virulence potential and ability to persist effectively under various stress conditions (including nutrient deprivation) retaining the ability to infect plants afterwards. In our previous studies, we showed Pba to be advanced in applying different adaptive strategies that led to manifestation of cell resistance to multiple stress factors. In the present study, we determined the period necessary for the formation of cross-protected Pba phenotype under starvation conditions, and compare the transcriptome profiles of non-adapted growing cells and of adapted cells after the cross-protective effect has reached the maximal level. The obtained data were verified using qRT-PCR. Genes that were expressed differentially (DEGs) in two cell types were classified into functional groups and categories using different approaches. As a result, we portrayed physiological features

  10. Small interfering RNA targeting the nonstructural gene 1 transcript inhibits influenza A virus replication in experimental mice.

    Science.gov (United States)

    Rajput, Roopali; Khanna, Madhu; Kumar, Prashant; Kumar, Binod; Sharma, Sonal; Gupta, Neha; Saxena, Latika

    2012-12-01

    Nonstructural protein 1 (NS1) of influenza A viruses counteracts the host immune response against the influenza viruses by not only inhibiting the nuclear export and maturation of host cell messenger RNA (mRNA), but by also blocking the double-stranded RNA-activated protein kinase-mediated inhibition of viral RNA translation. Reduction of NS1 gene product in the host cell may be a potent antiviral strategy to provide protection against the influenza virus infection. We used small interfering RNAs (siRNAs) synthesized against the viral mRNA to down regulate the NS1 gene and observed its effect on inhibition of virus replication. When NS1 gene-specific siRNA were transfected in Madin Darby canine kidney (MDCK) cells followed by influenza A virus infection, approximately 60% inhibition in intracellular levels of NS1 RNA was observed. When siRNA was administered in BALB/c mice, 92% reduction in the levels of NS1 gene expression in mice lungs was observed. A significant reduction in the lung virus titers and cytokine levels was also detected in the presence of siRNAs as compared with the untreated control. The study was validated by the use of selectively disabled mutants of each set of siRNA. Our findings suggest that siRNA targeted against NS1 gene of influenza A virus can provide considerable protection to the virus-infected host cells and may be used as potential candidates for nucleic acid-based antiviral therapy for prevention of influenza A virus infection.

  11. Protective Effect of CRHR1 Gene Variants on the Development of Adult Depression Following Childhood Maltreatment

    Science.gov (United States)

    Polanczyk, Guilherme; Caspi, Avshalom; Williams, Benjamin; Price, Thomas S.; Danese, Andrea; Sugden, Karen; Uher, Rudolf; Poulton, Richie; Moffitt, Terrie E.

    2013-01-01

    Context A previous study reported a gene × environment interaction in which a haplotype in the corticotropin-releasing hormone receptor 1 gene (CRHR1) was associated with protection against adult depressive symptoms in individuals who were maltreated as children (as assessed by the Childhood Trauma Questionnaire [CTQ]). Objective To replicate the interaction between childhood maltreatment and a TAT haplotype formed by rs7209436, rs110402, and rs242924 in CRHR1, predicting adult depression. Design Two prospective longitudinal cohort studies. Setting England and New Zealand. Participants Participants in the first sample were women in the E-Risk Study (N= 1116), followed up to age 40 years with 96% retention. Participants in the second sample were men and women in the Dunedin Study (N= 1037), followed up to age 32 years with 96% retention. Main Outcome Measure Research diagnoses of pastyear and recurrent major depressive disorder. Results In the E-Risk Study, the TAT haplotype was associated with a significant protective effect. In this effect, women who reported childhood maltreatment on the CTQ were protected against depression. In the Dunedin Study, which used a different type of measure of maltreatment, this finding was not replicated. Conclusions A haplotype in CRHR1 has been suggested to exert a protective effect against adult depression among research participants who reported maltreatment on the CTQ, a measure that elicits emotional memories. This suggests the hypothesis that CRHR1’s protective effect may relate to its function in the consolidation of memories of emotionally arousing experiences. PMID:19736354

  12. In-depth sequencing of the siRNAs associated with peach latent mosaic viroid infection

    Directory of Open Access Journals (Sweden)

    Perreault Jean-Pierre

    2010-02-01

    Full Text Available Abstract Background It has been observed that following viroid infection, there is an accumulation of viroid-derived siRNAs in infected plants. Some experimental results suggest that these small RNAs may be produced by the plant defense system to protect it from infection, indicating that viroids can elicit the RNA-silencing pathways. The objective of this study is to identify in the peach latent mosaic viroid (PLMVd, a model RNA genome, the regions that are most susceptible to RNA interference machinery. Results The RNA isolated from an infected tree have been used to sequence in parallel viroid species and small non-coding RNA species. Specifically, PLMVd RNAs were amplified, cloned and sequenced according to a conventional approach, while small non-coding RNAs were determined by high-throughput sequencing. The first led to the typing of 18 novel PLMVd variants. The second provided a library of small RNAs including 880 000 sequences corresponding to PLMVd-derived siRNAs, which makes up 11.2% of the sequences of the infected library. These siRNAs contain mainly 21-22 nucleotide RNAs and are equivalently distributed between the plus and the minus polarities of the viroid. They cover the complete viroid genome, although the amount varies depending on the regions. These regions do not necessarily correlate with the double-stranded requirement to be a substrate for Dicer-like enzymes. We noted that some sequences encompass the hammerhead self-cleavage site, indicating that the circular conformers could be processed by the RNA-silencing machinery. Finally, a bias in the relative abundance of the nature of the 5' nucleotides was observed (A, U >> G, C. Conclusions The approach used provided us a quantitative representation of the PLMVd-derived siRNAs retrieved from infected peach trees. These siRNAs account for a relatively large proportion of the small non-coding RNAs. Surprisingly, the siRNAs from some regions of the PLMVd genome appear over

  13. HIV-1 TAR miRNA protects against apoptosis by altering cellular gene expression

    Directory of Open Access Journals (Sweden)

    Meiri Eti

    2009-02-01

    Full Text Available Abstract Background RNA interference is a gene regulatory mechanism that employs small RNA molecules such as microRNA. Previous work has shown that HIV-1 produces TAR viral microRNA. Here we describe the effects of the HIV-1 TAR derived microRNA on cellular gene expression. Results Using a variation of standard techniques we have cloned and sequenced both the 5' and 3' arms of the TAR miRNA. We show that expression of the TAR microRNA protects infected cells from apoptosis and acts by down-regulating cellular genes involved in apoptosis. Specifically, the microRNA down-regulates ERCC1 and IER3, protecting the cell from apoptosis. Comparison to our cloned sequence reveals possible target sites for the TAR miRNA as well. Conclusion The TAR microRNA is expressed in all stages of the viral life cycle, can be detected in latently infected cells, and represents a mechanism wherein the virus extends the life of the infected cell for the purpose of increasing viral replication.

  14. Synthesis and characterization of Fe3O4-PEG-LAC-chitosan-PEI nanoparticle as a survivin siRNA delivery system.

    Science.gov (United States)

    Arami, S; Rashidi, M R; Mahdavi, M; Fathi, M; Entezami, A A

    2017-03-01

    The limited effectiveness of the conventional methods for cancer treatment makes the researchers to find novel safe and effective therapeutic strategies. One of these strategies is to use small interfering RNAs (siRNAs). A major challenge here is the siRNA delivery into the cells. The purpose of this study was to design and prepare a biocompatible, biodegradable, and safe nanosized particle for siRNA delivery into human breast cancer MCF-7 and leukemia K562 cells. Chemically synthesized magnetic nanoparticles containing polyethyleneglycol-lactate polymer (PEG-LAC), chitosan, and polyethyleneimine (PEI) were successfully prepared and used as a gene delivery vehicle. The nanoparticles were characterized by Fourier transform infrared spectroscopy and zeta potential. The Fe3O4-PEG-LAC-chitosan-PEI nanoparticle showed efficient and stable survivin siRNA loading in gel retardation assay. The cytotoxicity of the prepared nanoparticle was studied using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and was compared with that of mitoxantrone (MTX) in combination with the prepared siRNA delivery system to evaluate the possible synergic effect of MTX and survivin siRNA. The nanoparticles with and without noncomplementary siRNA showed low toxicity against both cell lines; however, a twofold decrease was observed in cell survival percent after MTX addition to MCF-7 cells treated with either nanoparticle itself or complexed with noncomplementary siRNA. While survivin siRNA nanoplex caused threefold decrease in the cell survival percent, its combination with MTX did not result in a significant increase in the cytotoxic effect. Therefore, Fe3O4-PEG-LAC-chitosan-PEI nanoparticle should be considered as a potential carrier for enhanced survivin siRNA delivery into MCF-7 and K562 cells.

  15. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles

    Science.gov (United States)

    Davis, Mark E.; Zuckerman, Jonathan E.; Choi, Chung Hang J.; Seligson, David; Tolcher, Anthony; Alabi, Christopher A.; Yen, Yun; Heidel, Jeremy D.; Ribas, Antoni

    2010-01-01

    Therapeutics that are designed to engage RNA interference (RNAi) pathways have the potential to provide new, major ways of imparting therapy to patients.1,2 Fire et al. first demonstrated that long, double stranded RNAs mediate RNAi in Caenorhabditis elegans,3 and Elbashir et al. opened the pathway to the use of RNAi for human therapy by showing that small interfering RNAs (siRNAs: ca. 21 base pair double stranded RNA) can elicit RNAi in mammalian cells without producing an interferon response.4 We are currently conducting the first-in-human Phase I clinical trial involving the systemic administration of siRNA to patients with solid cancers using a targeted, nanoparticle delivery system. Here we provide evidence of inducing an RNAi mechanism of action in a human from the delivered siRNA. Tumor biopsies from melanoma patients obtained after treatment reveal: (i) the presence of intracellularly-localized nanoparticles in amounts that correlate with dose levels of the nanoparticles administered (this is a first for systemically delivered nanoparticles of any kind), and (ii) reduction in both the specific mRNA (M2 subunit of ribonucleotide reductase (RRM2)) and the protein (RRM2) when compared to pre-dosing tissue. Most importantly, we detect the presence of an mRNA fragment that demonstrates siRNA mediated mRNA cleavage occurs specifically at the site predicted for an RNAi mechanism from a patient who received the highest dose of the nanoparticles. These data when taken in total demonstrate that siRNA administered systemically to a human can produce a specific gene inhibition (reduction in mRNA and protein) by an RNAi mechanism of action. PMID:20305636

  16. Spray-dried powders enhance vaginal siRNA delivery by potentially modulating the mucus molecular sieve structure

    Directory of Open Access Journals (Sweden)

    Wu N

    2015-08-01

    Full Text Available Na Wu,1,2,* Xinxin Zhang,2,* Feifei Li,2 Tao Zhang,2 Yong Gan,2 Juan Li1 1School of Pharmacy, China Pharmaceutical University, Nanjing, People’s Republic of China; 2Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: Vaginal small interfering RNA (siRNA delivery provides a promising strategy for the prevention and treatment of vaginal diseases. However, the densely cross-linked mucus layer on the vaginal wall severely restricts nanoparticle-mediated siRNA delivery to the vaginal epithelium. In order to overcome this barrier and enhance vaginal mucus penetration, we prepared spray-dried powders containing siRNA-loaded nanoparticles. Powders with Pluronic F127 (F127, hydroxypropyl methyl cellulose (HPMC, and mannitol as carriers were obtained using an ultrasound-assisted spray-drying technique. Highly dispersed dry powders with diameters of 5–15 µm were produced. These powders showed effective siRNA protection and sustained release. The mucus-penetrating properties of the powders differed depending on their compositions. They exhibited different potential of opening mesh size of molecular sieve in simulated vaginal mucus system. A powder formulation with 0.6% F127 and 0.1% HPMC produced the maximum increase in the pore size of the model gel used to simulate vaginal mucus by rapidly extracting water from the gel and interacting with the gel; the resulting modulation of the molecular sieve effect achieved a 17.8-fold improvement of siRNA delivery in vaginal tract and effective siRNA delivery to the epithelium. This study suggests that powder formulations with optimized compositions have the potential to alter the steric barrier posed by mucus and hold promise for effective vaginal siRNA delivery. Keywords: siRNA delivery, vaginal administration, spray-dried powders, mucus penetration, molecular sieve effect

  17. Silencage du gene MDR1 et resensibilisation des cellules MCF-7 MDR a la doxorubicine en utilisant les nanoparticules chitosane/MDR1-siARN

    Science.gov (United States)

    El-Ariss, Mohamad

    ARN_2M are complementary to a sequence shifted slightly downstream in the same gene (583-607 nucleic acids). RNA duplexes siRNA_1 and siARN_2 consist exclusively of DNA while "modifed" siRNA_1M and si RNA_2M consist of RNA overhangs. siRNA duplexes (siRNA_1 and siRNA_2) were chosen from the work published by Hao Wu et al. (2003), Stege et al. (2004) and Miletti-Gonzalez et al. (2005) which showed that these siRNA sequences are effective to silence MDR1 gene in cancer cells (breast cancer cells MCF-7 / AdrR and MCF-7 / BC-19 and stomach cancer cells: EPG85-257RDB).[3-5] Moreover, Strapps et al. (2010) showed that the use of siRNA having overhangs formed of ribonucleotides leads to a similar silencing but lasting longer in vivo and in vitro compared to the use of siRNA containing deoxyribonucleotides overhangs.[6] Thus siARN_1M and siARN_2M sequences correspond to siARN_1 and siARN_2 sequences but whose overhangs are formed of ribonucleotides. These siRNA specific to the MDR1 gene (MDR1-siRNA) were combined to chitosan to form nanoparticles capable of protecting these MDR1-siRNA and delivering it into the MCF-7 MDR cells. Chitosan used here as a delivery system, is a natural and biodegradable polysaccharide whose biological properties are defined by its average molecular weight (MW) and by its degree of deacetylation (DD). When the positively charged chitosan is added with the negatively charged siRNA, there is formation of nanoparticles by electrostatic attraction. In this project, chitosan 92-10 (DDA- MW) was used as a delivery system with a N:P (ratio chitosan amino groups: RNA phosphate) of 5. Analysis by dynamic light scattering (DLS) demonstrated that the nanoparticles have a diameter between 62.56 and 82.72 nm and a zeta potential ranging from 17.4 to 23.5 mV. Analysis by confocal microscopy showed that chitosan (92-10-5)/labeled siRNA are internalized in MCF-7 MDR cells and that siRNAs are released in the cytoplasm. MCF-7 cells resistant MDR were transfected in

  18. Expressing functional siRNAs in mammalian cells using convergent transcription

    Directory of Open Access Journals (Sweden)

    Dawes Ian W

    2003-11-01

    Full Text Available Abstract Background The use of small interfering RNAs (siRNAs as genetic inhibitors of gene expression has been shown to be an effective way of studying gene function in mammalian cells. Recently, different DNA vectors for expression of small hairpin RNAs (shRNAs or co-expression of sense and antisense RNAs have been developed that direct siRNA-mediated gene silencing. One expression cassette design that has been used to express long sense and antisense RNAs in non-mammalian cell types is symmetric transcription using convergent promoters. However, convergent transcription as a way to generate functional siRNAs in mammalian cells has not been reported. This vector design permits the generation of expression constructs containing no repeat sequences, but capable of inducing RNA interference (RNAi-mediated gene silencing. Results With the aim of simplifying the construction of RNAi expression vectors, we report on the production and application of a novel convergent promoter cassette capable of expressing sense and antisense RNAs, that form double-stranded RNA, and mediate gene silencing in mammalian cells. We use this cassette to inhibit the expression of both the EGFP transgene and the endogenous TP53 gene. The gene silencing effect is Dicer-dependent and the level of gene inactivation achieved is comparable to that produced with synthetic siRNA. Furthermore, this expression system can be used for both short and long-term control of specific gene expression in mammalian cells. Conclusion The experiments performed in this study demonstrate that convergent transcription can be used in mammalian cells to invoke gene-specific silencing via RNAi. This method provides an alternative to expression of shRNAs and co-expression of sense and antisense RNAs from independent cassettes or a divergent promoter. The main advantage of the present vector design is the potential to produce a functional siRNA expression cassette with no repeat sequences

  19. Salmonella enterica serovar Typhimurium lacking hfq gene confers protective immunity against murine typhoid.

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    Uday Shankar Allam

    Full Text Available Salmonella enterica is an important enteric pathogen and its various serovars are involved in causing both systemic and intestinal diseases in humans and domestic animals. The emergence of multidrug-resistant strains of Salmonella leading to increased morbidity and mortality has further complicated its management. Live attenuated vaccines have been proven superior over killed or subunit vaccines due to their ability to induce protective immunity. Of the various strategies used for the generation of live attenuated vaccine strains, focus has gradually shifted towards manipulation of virulence regulator genes. Hfq is a RNA chaperon which mediates the binding of small RNAs to the mRNA and assists in post-transcriptional gene regulation in bacteria. In this study, we evaluated the efficacy of the Salmonella Typhimurium Δhfq strain as a candidate for live oral vaccine in murine model of typhoid fever. Salmonella hfq deletion mutant is highly attenuated in cell culture and animal model implying a significant role of Hfq in bacterial virulence. Oral immunization with the Salmonella hfq deletion mutant efficiently protects mice against subsequent oral challenge with virulent strain of Salmonella Typhimurium. Moreover, protection was induced upon both multiple as well as single dose of immunizations. The vaccine strain appears to be safe for use in pregnant mice and the protection is mediated by the increase in the number of CD4(+ T lymphocytes upon vaccination. The levels of serum IgG and secretory-IgA in intestinal washes specific to lipopolysaccharide and outer membrane protein were significantly increased upon vaccination. Furthermore, hfq deletion mutant showed enhanced antigen presentation by dendritic cells compared to the wild type strain. Taken together, the studies in murine immunization model suggest that the Salmonella hfq deletion mutant can be a novel live oral vaccine candidate.

  20. In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes.

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    Mahmoud ElHefnawi

    Full Text Available RNA interference (RNAi is a post-transcriptional gene silencing mechanism that mediates the sequence-specific degradation of targeted RNA and thus provides a tremendous opportunity for development of oligonucleotide-based drugs. Here, we report on the design and validation of small interfering RNAs (siRNAs targeting highly conserved regions of the hepatitis C virus (HCV genome. To aim for therapeutic applications by optimizing the RNAi efficacy and reducing potential side effects, we considered different factors such as target RNA variations, thermodynamics and accessibility of the siRNA and target RNA, and off-target effects. This aim was achieved using an in silico design and selection protocol complemented by an automated MysiRNA-Designer pipeline. The protocol included the design and filtration of siRNAs targeting highly conserved and accessible regions within the HCV internal ribosome entry site, and adjacent core sequences of the viral genome with high-ranking efficacy scores. Off-target analysis excluded siRNAs with potential binding to human mRNAs. Under this strict selection process, two siRNAs (HCV353 and HCV258 were selected based on their predicted high specificity and potency. These siRNAs were tested for antiviral efficacy in HCV genotype 1 and 2 replicon cell lines. Both in silico-designed siRNAs efficiently inhibited HCV RNA replication, even at low concentrations and for short exposure times (24h; they also exceeded the antiviral potencies of reference siRNAs targeting HCV. Furthermore, HCV353 and HCV258 siRNAs also inhibited replication of patient-derived HCV genotype 4 isolates in infected Huh-7 cells. Prolonged treatment of HCV replicon cells with HCV353 did not result in the appearance of escape mutant viruses. Taken together, these results reveal the accuracy and strength of our integrated siRNA design and selection protocols. These protocols could be used to design highly potent and specific RNAi-based therapeutic

  1. [Evaluation of gene protective effects of some natural anti-mutation substance].

    Science.gov (United States)

    Iamnenko, M I; Berdyshev, G D

    2001-01-01

    Antimutation activity of some bee products was evaluated with a battery of test-systems. Tablets of apilac prepared out of queen bee milk did not demonstrate gene-protective effects in Ames' test. Other bee products, i.e. propolis, beebread, honey preparations 1 and 2 and queen bee milk were able to mitigate to a degree ill effects of some chemical and physical mutagenes. Propolis was found to be toxic to the yeast cell. Antimutation properties of natural queen bee milk revealed in the experiment with test-objects were lost after the product had been processed into the tabloid form or stored at room temperature.

  2. Folic Acid Conjugated Chitosan for Targeted Delivery of siRNA to Activated Macrophages in vitro and in vivo

    DEFF Research Database (Denmark)

    Yang, Chuanxu; Gao, Shan; Kjems, Jørgen

    2014-01-01

    was conjugated to chitosan (FA–CS) and used to formulate siRNA into nanoparticles capable of cell specific delivery. The physiochemical properties of the nanoparticles, including size, zeta-potential and encapsulation efficiency, were characterized and the intracellular uptake and gene silencing efficiency were...

  3. Inhibition of STAT3 expression by siRNA suppresses growth and induces apoptosis in laryngeal cancer cells

    Institute of Scientific and Technical Information of China (English)

    Li-fang GAO; De-qi XU; Lian-ji WEN; Xing-yi ZHANG; Yue-ting SHAO; Xue-jian ZHAO

    2005-01-01

    Aim: To determine the inhibitory effect of the synthetic STAT3 siRNA on the expression of STAT3 gene in human laryngeal cancer cell lines Hep2 and to investigate the effect of STAT3 siRNA on growth and apoptosis in Hep2 cells. Methods:A pair of DNA templates coding siRNA against STAT3-mRNA was synthesized to reconstruct plasmid of pSilencerl.0-U6 siRNA-STAT3. Hep2 cells were transfected with RPMI-1640 media (untreated), plasmid (empty), and STAT3 siRNA,respectively. Northern blot and Western blot analysis of STAT3 and pTyr-STAT3 expression in Hep2 cells and Western blot analysis of Bcl-2 expression in the Hep2 cell was performed 72 h after transfection. MTT, flow cytometry, and AO/EB assay were used for determination of cells proliferation and apoptosis in Hep2 cells. Results: pTyr-STAT3 was markedly expressed in untreated Hep2 cells and the vector-treated Hep2 cells, whereas pTyr-STAT3 expression was significantly reduced in STAT3 siRNA-transfected Hep2 cells, indicating that STAT3 siRNA inhibited the activity of STAT3. Transfection of Hep2 cells with STAT3 siRNA significantly inhibited STAT3 expression at both mRNA and protein level in Hep2 cells and the inhibition was characterized by time-dependent transfection. Treatment of Hep2 cells with STAT3 siRNA resulted in dose-dependent growth inhibition of Hep2, this significantly increased apoptotic cell rate, and decreased Bcl-2 expression level in Hep2 cells. STAT3 siRNA had an effect on induction of either early or late stage apoptosis. Conclusion: This study demonstrates that STAT3 siRNA effectively inhibits STAT3 gene expression in Hep2 cells leading to growth suppression and induction of apoptosis in Hep2 cells. The use of siRNA technique may provide a novel therapeutic approach to treat laryngeal cancer and other malignant tumors expressing constitutively activated STAT3.

  4. Bio-inspired pulmonary surfactant-modified nanogels: A promising siRNA delivery system.

    Science.gov (United States)

    De Backer, Lynn; Braeckmans, Kevin; Stuart, Marc C A; Demeester, Jo; De Smedt, Stefaan C; Raemdonck, Koen

    2015-05-28

    Inhalation therapy with small interfering RNA (siRNA) is a promising approach in the treatment of pulmonary disorders. However, clinical translation is severely limited by the lack of suitable delivery platforms. In this study, we aim to address this limitation by designing a novel bioinspired hybrid nanoparticle with a core-shell nanoarchitecture, consisting of a siRNA-loaded dextran nanogel (siNG) core and a pulmonary surfactant (Curosurf®) outer shell. The decoration of siNGs with a surfactant shell enhances the colloidal stability and prevents siRNA release in the presence of competing polyanions, which are abundantly present in biofluids. Additionally, the impact of the surfactant shell on the biological efficacy of the siNGs is determined in lung cancer cells. The presence of the surfactants substantially reduces the cellular uptake of siNGs. Remarkably, the lowered intracellular dose does not impede the gene silencing effect, suggesting a crucial role of the pulmonary surfactant in the intracellular processing of the nanoparticles. In order to surmount the observed reduction in cellular dose, folate is incorporated as a targeting ligand in the pulmonary surfactant shell to incite receptor-mediated endocytosis. The latter substantially enhances both cellular uptake and gene silencing potential, achieving efficient knockdown at siRNA concentrations in the low nanomolar range.

  5. Functional studies of the PI(3)-kinase signalling pathway employing synthetic and expressed siRNA.

    Science.gov (United States)

    Czauderna, Frank; Fechtner, Melanie; Aygün, Hüseyin; Arnold, Wolfgang; Klippel, Anke; Giese, Klaus; Kaufmann, Jörg

    2003-01-15

    RNA interference (RNAi) is a RNA-mediated sequence-specific gene silencing mechanism. Recently, this mechanism has been used to down-regulate protein expression in mammalian cells by applying synthetic- or vector-generated small interfering RNAs (siRNAs). However, for the evaluation of this new knockdown technology, it is crucial to demonstrate biological consequences beyond protein level reduction. Here, we demonstrate that this new siRNA-based technology is suitable to analyse protein functions using the phosphatidylinositol (PI) 3-kinase signal transduction pathway as a model system. We demonstrate stable and transient siRNA-mediated knockdown of one of the PI 3-kinase catalytic subunits, p110beta, which leads to inhibition of invasive cell growth in vitro as well as in a tumour model system. Importantly, this result is consistent with loss-of-function phenotypes induced by conventional RNase H-dependent antisense molecules or treatment with the PI 3-kinase inhibitor LY294002. RNAi knockdown of the downstream kinases Akt1 and Akt2 does not reduce cell growth on extracellular matrix. Our data show that synthetic siRNAs, as well as vector-based expression of siRNAs, are a powerful new tool to interfere with signal transduction processes for the elucidation of gene function in mammalian cells.

  6. Cell transcytosing poly-arginine coated magnetic nanovector for safe and effective siRNA delivery.

    Science.gov (United States)

    Veiseh, Omid; Kievit, Forrest M; Mok, Hyejung; Ayesh, Joseph; Clark, Cassra; Fang, Chen; Leung, Matthew; Arami, Hamed; Park, James O; Zhang, Miqin

    2011-08-01

    Lack of safe and effective carriers for delivery of RNA therapeutics remains a barrier to its broad clinical application. We report the development of a cell tanscytosing magnetic nanovector engineered as an siRNA carrier. Iron oxide nanoparticles were modified with poly(ethylene glycol) (PEG), small interfering RNA (siRNA), and a cationic polymer layer. Three nanovector formulations with cationic polymer coatings of poly-arginine (pArg), polylysine (pLys), and polyethylenimine (PEI), respectively, were prepared. The three nanovector formulations where evaluated for safety and ability to promote gene silencing in three types of cancer cells C6/GFP(+), MCF7/GFP(+), and TC2/GFP(+), mimicking human cancers of the brain, breast, and prostate, respectively. Cell viability and fluorescence quantification assays revealed that pArg-coated nanovectors were most effective in promoting gene knockdown and least toxic of the three nanovector formulations tested. Transmission electron microscopy (TEM) imaging of nanovector treated cells further demonstrated that pArg-coated nanovectors enter cells through cell transcytosis, while pLys and PEI coated nanovectors enter cells endocytosis. Our findings suggest that NPs engineered to exploit the cell transcytosis intracellular trafficking pathway may offer a more safe and efficient route for siRNA delivery.

  7. Effects of silencing RIP1 with siRNA on the biological behavior of the LoVo human colon cancer cell line.

    Science.gov (United States)

    You, Hong-Xia; Zhou, Yan-Hong; Tan, Shi-Yun; She, Tong-Hui

    2014-06-01

    The present study aimed to investigate the effects of silencing RIP1 by small interfering RNA (siRNA) on the biological behavior of the LoVo human colorectal carcinoma cell line and to provide evidence for the feasibility of colorectal cancer gene therapy. LoVo cells were divided into the RIP1 siRNA group, the blank control group and the negative control group. Chemically synthesized siRNA targeting RIP1 (RIP1 siRNA) was transfected into LoVo cells. Following transfection of the RIP1-targeted siRNA into the LoVo cells, the expression of the RIP1 gene was effectively inhibited. The results demonstrated that RIP1 effectively regulated the malignant biological behavior of the LoVo colon cancer cell line. Furthermore, the proliferation, motility and invasiveness of LoVo cells were inhibited by siRNA knockdown of RIP1. The results revealed that the RIP1 gene has an important role in the regulation of proliferation and apoptosis in colorectal carcinoma cells.

  8. The Escherichia coli SOS gene dinF protects against oxidative stress and bile salts.

    Directory of Open Access Journals (Sweden)

    Jerónimo Rodríguez-Beltrán

    Full Text Available DNA is constantly damaged by physical and chemical factors, including reactive oxygen species (ROS, such as superoxide radical (O(2(-, hydrogen peroxide (H(2O(2 and hydroxyl radical (•OH. Specific mechanisms to protect and repair DNA lesions produced by ROS have been developed in living beings. In Escherichia coli the SOS system, an inducible response activated to rescue cells from severe DNA damage, is a network that regulates the expression of more than 40 genes in response to this damage, many of them playing important roles in DNA damage tolerance mechanisms. Although the function of most of these genes has been elucidated, the activity of some others, such as dinF, remains unknown. The DinF deduced polypeptide sequence shows a high homology with membrane proteins of the multidrug and toxic compound extrusion (MATE family. We describe here that expression of dinF protects against bile salts, probably by decreasing the effects of ROS, which is consistent with the observed decrease in H(2O(2-killing and protein carbonylation. These results, together with its ability to decrease the level of intracellular ROS, suggests that DinF can detoxify, either direct or indirectly, oxidizing molecules that can damage DNA and proteins from both the bacterial metabolism and the environment. Although the exact mechanism of DinF activity remains to be identified, we describe for the first time a role for dinF.

  9. Therapeutic Silencing of KRAS using Systemically Delivered siRNAs

    Science.gov (United States)

    Pecot, Chad V.; Wu, Sherry Y.; Bellister, Seth; Filant, Justyna; Rupaimoole, Rajesha; Hisamatsu, Takeshi; Bhattacharya, Rajat; Maharaj, Anshumaan; Azam, Salma; Rodriguez-Aguayo, Cristian; Nagaraja, Archana S.; Morelli, Maria Pia; Gharpure, Kshipra M.; Waugh, Trent A.; Gonzalez-Villasana, Vianey; Zand, Behrouz; Dalton, Heather J.; Kopetz, Scott; Lopez-Berestein, Gabriel; Ellis, Lee M.; Sood, Anil K.

    2015-01-01

    Despite being amongst the most common oncogenes in human cancer, to date there are no effective clinical options for inhibiting KRAS activity. We investigated whether systemically delivered KRAS siRNAs have therapeutic potential in KRAS mutated cancer models. We identified KRAS siRNA sequences with notable potency in knocking-down KRAS expression. Using lung and colon adenocarcinoma cell lines, we assessed anti-proliferative effects of KRAS silencing in vitro. For in vivo experiments, we used a nano-liposomal delivery platform, DOPC, for systemic delivery of siRNAs. Various lung and colon cancer models were utilized to determine efficacy of systemic KRAS siRNA based on tumor growth, development of metastasis and down-stream signaling. KRAS siRNA sequences induced >90% knock-down of KRAS expression, significantly reducing viability in mutant cell lines. In the lung cancer model, KRAS siRNA treatment demonstrated significant reductions in primary tumor growth and distant metastatic disease, while the addition of CDDP was not additive. Significant reductions in Ki-67 indices were seen in all treatment groups, while significant increases in caspase-3 activity was only seen in the CDDP treatment groups. In the colon cancer model, KRAS siRNA reduced tumor KRAS and pERK expression. KRAS siRNAs significantly reduced HCP1 subcutaneous tumor growth, as well as outgrowth of liver metastases. Our studies demonstrate a proof-of-concept approach to therapeutic KRAS targeting using nanoparticle delivery of siRNA. This study highlights the potential translational impact of therapeutic RNA interference, which may have broad applications in oncology, especially for traditional “undruggable” targets. PMID:25281617

  10. MDR1 siRNA loaded hyaluronic acid-based CD44 targeted nanoparticle systems circumvent paclitaxel resistance in ovarian cancer

    Science.gov (United States)

    Yang, Xiaoqian; Lyer, Arun K.; Singh, Amit; Choy, Edwin; Hornicek, Francis J.; Amiji, Mansoor M.; Duan, Zhenfeng

    2015-02-01

    Development of multidrug resistance (MDR) is an almost universal phenomenon in patients with ovarian cancer, and this severely limits the ultimate success of chemotherapy in the clinic. Overexpression of the MDR1 gene and corresponding P-glycoprotein (Pgp) is one of the best known MDR mechanisms. MDR1 siRNA based strategies were proposed to circumvent MDR, however, systemic, safe, and effective targeted delivery is still a major challenge. Cluster of differentiation 44 (CD44) targeted hyaluronic acid (HA) based nanoparticle has been shown to successfully deliver chemotherapy agents or siRNAs into tumor cells. The goal of this study is to evaluate the ability of HA-PEI/HA-PEG to deliver MDR1 siRNA and the efficacy of the combination of HA-PEI/HA-PEG/MDR1 siRNA with paclitaxel to suppress growth of ovarian cancer. We observed that HA-PEI/HA-PEG nanoparticles can efficiently deliver MDR1 siRNA into MDR ovarian cancer cells, resulting in down-regulation of MDR1 and Pgp expression. Administration of HA-PEI/HA-PEG/MDR1 siRNA nanoparticles followed by paclitaxel treatment induced a significant inhibitory effect on the tumor growth, decreased Pgp expression and increased apoptosis in MDR ovarian cancer mice model. Our findings suggest that CD44 targeted HA-PEI/HA-PEG/MDR1 siRNA nanoparticles can serve as a therapeutic tool with great potentials to circumvent MDR in ovarian cancer.

  11. Identification and characterization of microRNAs and endogenous siRNAs in Schistosoma japonicum

    Directory of Open Access Journals (Sweden)

    Wang Heng

    2010-01-01

    Full Text Available Abstract Background Small endogenous non-coding RNAs (sncRNAs such as small interfering RNA (siRNA, microRNA and other small RNA transcripts are derived from distinct loci in the genome and play critical roles in RNA-mediated gene silencing mechanisms in plants and metazoa. They are approximately 22 nucleotides long; regulate mRNA stability through perfect or imperfect match to the targets. The biological activities of sncRNAs have been related to many biological events, from resistance to microbe infections to cellular differentiation. The development of the zoonotic parasite Schistosoma japonicum parasite includes multiple steps of morphological alterations and biological differentiations, which provide a unique model for studies on the functions of small RNAs. Characterization of the genome-wide transcription of the sncRNAs will be a major step in understanding of the parasite biology. The objective of this study is to investigate the transcriptional profile and potential function of the small non-coding RNAs in the development of S. japanicum. Results The endogenous siRNAs were found mainly derived from transposable elements (TE or transposons and the natural antisense transcripts (NAT. In contrast to other organisms, the TE-derived siRNAs in S. japonicum were more predominant than other sncRNAs including microRNAs (miRNAs. Further, there were distinct length and 3'end variations in the sncRNAs, which were associated with the developmental differentiation of the parasite. Among the identified miRNA transcripts, there were 38 unique to S. japonicum and 16 that belonged to 13 miRNA families are common to other metazoan lineages. These miRNAs were either ubiquitously expressed, or they exhibited specific expression patterns related to the developmental stages or sex. Genes that encoded miRNAs are mainly located in clusters within the genome of S. japonicum. However, genes within one cluster could be differentially transcribed, which suggested

  12. Suppression of TAFI by siRNA inhibits invasion and migration of breast cancer cells.

    Science.gov (United States)

    Yu, Chao; Luan, Yun; Wang, Zejun; Zhao, Jingjie; Xu, Chengwei

    2017-09-01

    Human thrombin activatable fibrinolysis inhibitor (TAFI), also known as carboxypeptidase B2 (CPB2), is a procarboxypeptidase enzyme. The purpose of the present study was to observe the expression of TAFI in breast cancer (BC) and breast cancer cell (BCC) lines and to investigate the effect of TAFI suppression by small interfering (si)RNA gene silencing on invasion and migration of BCC lines. A significant increase in TAFI level was identified by immunohistochemical analysis in BC tissues compared with normal breast tissues. TAFI suppression also inhibited cell viability, invasion and migration ability as demonstrated by MTT, Transwell chamber, and wound scratch assays, respectively (P<0.05). The data suggested that suppression of TAFI by siRNA inhibits invasion and migration of breast cancer cells and that TAFI may be a new target for breast cancer therapy.

  13. siRNA delivery targeting to the lung via agglutination-induced accumulation and clearance of cationic tetraamino fullerene

    Science.gov (United States)

    Minami, Kosuke; Okamoto, Koji; Doi, Kent; Harano, Koji; Noiri, Eisei; Nakamura, Eiichi

    2014-05-01

    The efficient treatment of lung diseases requires lung-selective delivery of agents to the lung. However, lung-selective delivery is difficult because the accumulation of micrometer-sized carriers in the lung often induces inflammation and embolization-related toxicity. Here we demonstrate a lung-selective delivery system of small interfering RNA (siRNA) by controlling the size of carrier vehicle in blood vessels. The carrier is made of tetra(piperazino)fullerene epoxide (TPFE), a water-soluble cationic tetraamino fullerene. TPFE and siRNA form sub-micrometer-sized complexes in buffered solution and these complexes agglutinate further with plasma proteins in the bloodstream to form micrometer-sized particles. The agglutinate rapidly clogs the lung capillaries, releases the siRNA into lung cells to silence expression of target genes, and is then cleared rapidly from the lung after siRNA delivery. We applied our delivery system to an animal model of sepsis, indicating the potential of TPFE-based siRNA delivery for clinical applications.

  14. Primary and secondary siRNA synthesis triggered by RNAs from food bacteria in the ciliate Paramecium tetraurelia.

    Science.gov (United States)

    Carradec, Quentin; Götz, Ulrike; Arnaiz, Olivier; Pouch, Juliette; Simon, Martin; Meyer, Eric; Marker, Simone

    2015-02-18

    In various organisms, an efficient RNAi response can be triggered by feeding cells with bacteria producing double-stranded RNA (dsRNA) against an endogenous gene. However, the detailed mechanisms and natural functions of this pathway are not well understood in most cases. Here, we studied siRNA biogenesis from exogenous RNA and its genetic overlap with endogenous RNAi in the ciliate Paramecium tetraurelia by high-throughput sequencing. Using wild-type and mutant strains deficient for dsRNA feeding we found that high levels of primary siRNAs of both strands are processed from the ingested dsRNA trigger by the Dicer Dcr1, the RNA-dependent RNA polymerases Rdr1 and Rdr2 and other factors. We further show that this induces the synthesis of secondary siRNAs spreading along the entire endogenous mRNA, demonstrating the occurrence of both 3'-to-5' and 5'-to-3' transitivity for the first time in the SAR clade of eukaryotes (Stramenopiles, Alveolates, Rhizaria). Secondary siRNAs depend on Rdr2 and show a strong antisense bias; they are produced at much lower levels than primary siRNAs and hardly contribute to RNAi efficiency. We further provide evidence that the Paramecium RNAi machinery also processes single-stranded RNAs from its bacterial food, broadening the possible natural functions of exogenously induced RNAi in this organism.

  15. Spatially Controlled Delivery of siRNAs to Stem Cells in Implants Generated by Multi-Component Additive Manufacturing

    DEFF Research Database (Denmark)

    Andersen, Morten Østergaard; Le, Dang Quang Svend; Chen, Muwan

    2013-01-01

    Additive manufacturing is a promising technique in tissue engineering, as it enables truly individualized implants to be made to fit a particular defect. As previously shown, a feasible strategy to produce complex multicellular tissues is to deposit different small interfering RNA (siRNA) in porous...... implants that are subsequently sutured together. In this study, an additive manufacturing strategy to deposit carbohydrate hydrogels containing different siRNAs is applied into an implant, in a spatially controlled manner. When the obtained structures are seeded with mesenchymal stem (stromal) cells......, the selected siRNAs are delivered to the cells and induces specific and localized gene silencing. Here, it is demonstrated how to replicate part of a patient's spinal cord from a computed tomography scan, using an additive manufacturing technique to produce an implant with compartmentalized si...

  16. Protection of germline gene expression by the C. elegans Argonaute CSR-1.

    Science.gov (United States)

    Wedeles, Christopher J; Wu, Monica Z; Claycomb, Julie M

    2013-12-23

    In Caenorhabditis elegans, the Piwi-interacting small RNA (piRNA)-mediated germline surveillance system encodes more than 30,000 unique 21-nucleotide piRNAs, which silence a variety of foreign nucleic acids. What mechanisms allow endogenous germline-expressed transcripts to evade silencing by the piRNA pathway? One likely candidate in a protective mechanism is the Argonaute CSR-1, which interacts with 22G-small RNAs that are antisense to nearly all germline-expressed genes. Here, we use an in vivo RNA tethering assay to demonstrate that the recruitment of CSR-1 to a transcript licenses expression of the transcript, protecting it from piRNA-mediated silencing. Licensing occurs mainly at the level of transcription, as we observe changes in pre-mRNA levels consistent with transcriptional activation when CSR-1 is tethered. Furthermore, the recruitment of CSR-1 to a previously silenced locus transcriptionally activates its expression. Together, these results demonstrate a rare positive role for an endogenous Argonaute pathway in heritably licensing and protecting germline transcripts.

  17. Exercise promotes alpha7 integrin gene transcription and protection of skeletal muscle.

    Science.gov (United States)

    Boppart, Marni D; Volker, Sonja E; Alexander, Nicole; Burkin, Dean J; Kaufman, Stephen J

    2008-11-01

    The alpha7beta1 integrin is increased in skeletal muscle in response to injury-producing exercise, and transgenic overexpression of this integrin in mice protects against exercise-induced muscle damage. The present study investigates whether the increase in the alpha7beta1 integrin observed in wild-type mice in response to exercise is due to transcriptional regulation and examines whether mobilization of the integrin at the myotendinous junction (MTJ) is a key determinant in its protection against damage. A single bout of downhill running exercise selectively increased transcription of the alpha7 integrin gene in 5-wk-old wild-type mice 3 h postexercise, and an increased alpha7 chain was detected in muscle sarcolemma adjacent to tendinous tissue immediately following exercise. The alpha7B, but not alpha7A isoform, was found concentrated and colocalized with tenascin-C in muscle fibers lining the MTJ. To further validate the importance of the integrin in the protection against muscle damage following exercise, muscle injury was quantified in alpha7(-/-) mice. Muscle damage was extensive in alpha7(-/-) mice in response to both a single and repeated bouts of exercise and was largely restricted to areas of high MTJ concentration and high mechanical force near the Achilles tendon. These results suggest that exercise-induced muscle injury selectively increases transcription of the alpha7 integrin gene and promotes a rapid change in the alpha7beta integrin at the MTJ. These combined molecular and cellular alterations are likely responsible for integrin-mediated attenuation of exercise-induced muscle damage.

  18. An efficient and low immunostimulatory nanoparticle formulation for systemic siRNA delivery to the tumor.

    Science.gov (United States)

    Chono, Sumio; Li, Shyh-Dar; Conwell, Christine C; Huang, Leaf

    2008-10-01

    We have developed a nanoparticle formulation [liposomes-protamine-hyaluronic acid nanoparticle (LPH-NP)] for systemically delivering siRNA into the tumor. The LPH-NP was prepared in a self-assembling process. Briefly, protamine and a mixture of siRNA and hyaluronic acid were mixed to prepare a negatively charged complex. Then, cationic liposomes were added to coat the complex with lipids via charge-charge interaction to prepare the LPH-NP. The LPH-NP was further modified by DSPE-PEG or DSPE-PEG-anisamide by the post-insertion method. Anisamide is a targeting ligand for the sigma receptor over-expressed in the B16F10 melanoma cells. The particle size, zeta potential and siRNA encapsulation efficiency of the formulation were approximately 115 nm, +25 mV and 90%, respectively. Luciferase siRNA was used to evaluate the gene silencing activity in the B16F10 cells, which were stably transduced with a luciferase gene. The targeted LPH-NP (PEGylated with ligand) silenced 80% of luciferase activity in the metastatic B16F10 tumor in the lung after a single i.v. injection (0.15 mg siRNA/kg). The targeted LPH-NP also showed very little immunotoxicity in a wide dose range (0.15-1.2 mg siRNA/kg), while the previously published formulation, LPD-NP (liposome-protamine-DNA nanoparticle), had a much narrow therapeutic window (0.15-0.45 mg/kg).

  19. 靶向Ang2的RNAi慢病毒载体的构建及其对恶性黑色素瘤细胞中Ang2基因表达的影响%Construction of Recombinant Lentiviral Vector of siRNA for Ang2 and the Effect of the Expression of Ang2 Gene in Malignant Melanoma Cells

    Institute of Scientific and Technical Information of China (English)

    刘照亮; 王彪; 郭国祥; 单秀英; 王美水; 庄福连; 蔡传书; 张明凤; 张彦定

    2011-01-01

    vector systems of three plasmids were constituted using pNL-EGFP-U6-Ang2- I ( pNL-EGFP-U6-Ang2- Ⅱ ), pVSVG and pHelper. The lentiviral vector system was transfected into 293T cell to produce pNL-EGFP-U6-Ang2- I and pNL-EGFP-U6-Ang2- Ⅱ lentivirus. Supernatant was collected to determine the titer. Malignant melanoma cells were transduced with both lentiviruses. Realtime RT-PCR was conducted to examine inhibitory efficiency. Results The recombinant lentiviral vectors of Ang2-RNAi were constructed successfully, as confirmed by restriction enzyme digestion and sequencing. Lentiviral titer was up to 8. 0 x l03/ml. Real-time RT-PCR demonstrated that the lentiviral vectors of Ang2-RNAi infect malignant melanoma cells and inhibit the expression of Ang2 genes in malignant melanoma cells ( P < 0. 05 ) . Conclusion Lentivector carrying Ang2-siRNA was constructed successfully and it was able to inhibit the expression of Ang2 gene in vitro significantly. The study established preliminary research on the inhibition of the growth of malignant melanoma xenografts in nude mice and provides the experimental evidence for tumor gene therapy in future.

  20. THE RISK OF GENE TRANSFERRING IN THE INSURANCE PROTECTION OF AGRICULTERE

    Directory of Open Access Journals (Sweden)

    M. Malik

    2016-03-01

    Full Text Available The paper justified essence of genetic engineering as the object of insurance services. Defines the concept of risk gene transferring. The character features of this specific risk. The influence and consequences for agricultural producers. The description of the possible creation of the concept of insurance services that cover risk of gene transferring. The study reveals of the use of GMOs in agriculture, due to issues of economic security of a particular region or country as a whole. To determined the impact of risks and control for developing and developed countries that are important aspects of farming. Changes in weather, climate, productivity, price values, public policy, the situation on global markets can cause large fluctuations in agricultural production, and consequently affecting the income of agricultural producers. Risk management includes a range of strategies that reduce the social and financial implications of possible changes affecting the production and income of farmers. There is a need for an in-depth study of the theoretical and practical aspects of the impact of the risk of gene transferring in the context of insurance protection.

  1. Dendrosome-based delivery of siRNA against E6 and E7 oncogenes in cervical cancer.

    Science.gov (United States)

    Dutta, Tathagata; Burgess, Melinda; McMillan, Nigel A J; Parekh, Harendra S

    2010-06-01

    Although small interfering RNA (siRNA) treatment holds great promise for the treatment of cancers, the field has been held back by the availability of suitable delivery vehicles. For cervical cancer the E6 and E7 oncogenes are ideal siRNA targets for treatment. The purpose of the present study was to explore the potential of dendrosomes for the delivery of siRNA targeting E6 and E7 proteins of cervical cancer cells in vitro. Optimization of dendrimer generation and nitrogen-to-phosphate (N/P) ratio was carried out using dendrimer-fluorescein isothiocyanate oligo complexes. The optimized N/P ratios were used in formulating complexes between dendrimers and siRNA targeting green fluorescence protein (siGFP). Although formulation 4D100 (dendrimer-siRNA complex) displayed the highest GFP knockdown, it was also found to be highly toxic to cells. In the final formulation 4D100 was encapsulated into dendrosomes so as to mask these toxic effects. The optimized dendrosomal formulation (DF), DF3 was found to possess a siGFP-entrapment efficiency of 49.76% +/- 1.62%, vesicle size of 154 +/- 1.73 nm, and zeta potential of +3.21 +/- 0.07 mV. The GFP knockdown efficiency of DF3 (dendrosome) was found to be almost identical to that of 4D100, but the former was completely nontoxic to the cells. DF3 containing siRNA against E6 and E7 was found to knock down the target genes considerably, as compared with the other formulations tested. Our results imply that dendrosomes hold potential for the delivery of siRNA and that a suitable targeting strategy could be useful for applications in vivo. siRNA treatment holds great promise for the treatment of cancers, but overall, the availability of suitable delivery vehicles remains a major issue. The purpose of this study was to explore the potential of dendrosomes for the delivery of siRNA targeting specific proteins in cervical cancer cells in vitro. The results suggest that dendrosomes hold potential for the delivery of siRNA and a suitable

  2. Well-Defined Degradable Cationic Polylactide as Nanocarrier for the Delivery of siRNA to Silence Angiogenesis in Prostate Cancer

    Science.gov (United States)

    Chen, Chih-Kuang; Law, Wing-Cheung; Aalinkeel, Ravikumar; Nair, Bindukumar; Kopwitthaya, Atcha; Mahajan, Supriya D.; Reynolds, Jessica L.; Zou, Jiong

    2013-01-01

    Well-defined tertiary amine-functionalized cationic polylactides (CPLAs) are synthesized by thiol-ene click functionalization of an allyl-functionalized polylactide, and utilized here for the delivery of interleukin-8 (IL-8) siRNA via CPLA-IL-8 siRNA nanoplexes. The CPLAs possess remarkable hydrolytic degradability, and their cytotoxicity is relatively low. The CPLA-IL-8 siRNA nanoplexes can be readily taken up by prostate cancer cells, resulting in significant IL-8 gene silencing. It is found that the degradability and cytotoxicity of CPLAs, as well as the transfection efficiency of the CPLA-IL-8 siRNA nanoplexes, positively correlate with the amine mol% of CPLAs. PMID:23184827

  3. Local delivery of siRNA using a biodegradable polymer application to enhance BMP-induced bone formation.

    Science.gov (United States)

    Manaka, Tomoya; Suzuki, Akinobu; Takayama, Kazushi; Imai, Yuuki; Nakamura, Hiroaki; Takaoka, Kunio

    2011-12-01

    Small interfering RNA (siRNA) is useful tool for specific and efficient knockdown of disease-related genes. However, in vivo applications of siRNA are limited due to difficulty in its efficient delivery to target cells. In this study, we investigated the efficacy of a biodegradable hydrogel, poly-d,l-lactic acid-p-dioxanone-polyethylene glycol block co-polymer (PLA-DX-PEG), as a siRNA carrier. PLA-DX-PEG pellets with or without fluorescein-labeled dsRNA were implanted into mouse dosal muscle pouches. The cellular uptake of dsRNA surround the polymer was confirmed by fluorescent microscopy. The fluorescence intensity was dose-dependent of the dsRNA, and exhibited a time-dependent decrease. To investigate its biological efficiency, noggin (antagonoist to BMPs) gene-silencing with siRNA (siRNA/Noggin) was examined by the amount of suppression of BMP-2-induced noggin expression and the level of performance of BMP, indicated by ectopic bone formation. Noggin gene expression induced by BMP-2 was suppressed by addition of siRNA/Noggin to the implant, and the ectopic bone formation induced by implants with both BMP-2 and siRNA/Noggin was significantly greater than those induced by implants with BMP-2 alone. These results indicate the efficacy of local delivery of siRNAs by PLA-DX-PEG polymer, which intensified bone-inducing effects of BMP and promoted new bone formation by suppressing gene expression of Noggin.

  4. Construction of Cannabinoid Receptor 1 Gene(CNR1)siRNA Expression Vector and Screening of CNR1-interference Positive L6 Cell Clones%大麻素Ⅰ型受体基因(CNR1)特异性siRNA表达载体的构建及稳定干扰阳性L6细胞克隆的筛选

    Institute of Scientific and Technical Information of China (English)

    徐娥; 任阳; 朱琳娜; 伍婷; 袁章琴; 黄艳娜; 汪以真

    2012-01-01

    The cannabinoid receptor type 1 (CNR1) is a key component of the endocannabinoid system, which has been reported to play a pivotal role in modulating feeding behavior and energy balance. In order to further study on gene function of CNR1, this study was conducted to construct and identify CNR1 gene small interfering (siRNA) expression vectors and screened the stable CNR1- interference positive L6 cell clones. Three pairs of CNR 1-specific double-strand siRNAs were designed and inserted into the pYr-1.1 vector. The CNR1 gene siRNA expression vectors were identified by restriction enzyme digestion and sequencing. After that siRNAs were transfected with L6 cells by LipofectamineTM(Lip)2000. Then, the transfection efficiency was detected by EGFP and FCM. CNR1 gene expression was determined by Real-time PCR and the stable transgenic L6 cell clones were screened by G418. The results revealed that the CNR1 gene siRNA expression vectors have been constructed successfully. The transient transfection efficiencies of L6 cells were 10.45% (P<0.01), 8.57%(P<0.01)and 8.71% (P<0.01) respectively, and the silencing efficacies of the transient transfected L6 cells were 39%(P<0.05), 64%(P<0.01) and 68%(P<0.01), respectively. The optimal selection concentration of G418 for stable transfected L6 cell clones was 800 μg/mL. The silencing efficacies of CNR-1-positive transgenic cell clones were 43%(P<0.05), 18%(P<0.01) and 91%(P<0.01), respectively. The results showed that CNR1-3 expression vector was optimal silencing vector and CNR1-3 stable transgenic cell clones were best silencing cell line. This study successfully provides CNR1 gene silencing method by siRNA and the screening of CNR1-interference positive L6 cell clones renders basic tools for further studying the functions of CNR1 gene.%大麻素Ⅰ型受体(CNR1)是介导内源性大麻素发挥作用的关键分子,在食欲和能量代谢调控中发挥着重要作用.为了更深入研究CNR1的基因功能,本实验旨在

  5. Attenuation of Telomerase Activity by siRNA Targeted Telomerase RNA Leads to Apoptosis and Inhibition of Proliferation in Human Renal Carcinoma Cells

    Institute of Scientific and Technical Information of China (English)

    Rumin Wen; Junjie Liu; Wang Li; Wenfa Yang; Lijun Mao; Junnian Zheng

    2006-01-01

    OBJECTIVE Telomerase is an attractive molecular target for cancer therapy because the activation of telomerase is one of the key steps in cell immortalization and carcinogenesis. RNA interference using small-interfering RNA (siRNA) has been demonstrated to be an effective method for inhibiting the expression of a given gene in human cells. The aim of the present study was to investigate whether inhibition of telomerase activity by siRNA targeted against human telomerase RNA (hTR) can inhibit proliferation and induce apoptotic cell death in human renal carcinoma cells(HRCCs).METHODS The siRNA duplexes for hTR were synthesized and 786-O HRCCs were transfected with different concentrations of hTR-siRNA. The influence on the hTR mRNA level, telomerase activity, as well as the effect on cell proliferation and apoptosis was examined.RESULTS Anti-hTR siRNA treatment of HRCCs resulted in specific reduction of hTR mRNA and inhibition of telomerase activity. Additionally,significant inhibition of proliferation and induction of apoptosis were observed.CONCLUSION siRNA against the hTR gene can inhibit proliferation and induce apoptosis by blocking telomerase activity of HRCCs. Specific hTR inhibition by siRNA represents a promising new option for renal cancer treatment.

  6. Marburg virus infection in nonhuman primates: Therapeutic treatment by lipid-encapsulated siRNA.

    Science.gov (United States)

    Thi, Emily P; Mire, Chad E; Ursic-Bedoya, Raul; Geisbert, Joan B; Lee, Amy C H; Agans, Krystle N; Robbins, Marjorie; Deer, Daniel J; Fenton, Karla A; MacLachlan, Ian; Geisbert, Thomas W

    2014-08-20

    Marburg virus (MARV) and the closely related filovirus Ebola virus cause severe and often fatal hemorrhagic fever (HF) in humans and nonhuman primates with mortality rates up to 90%. There are no vaccines or drugs approved for human use, and no postexposure treatment has completely protected nonhuman primates against MARV-Angola, the strain associated with the highest rate of mortality in naturally occurring human outbreaks. Studies performed with other MARV strains assessed candidate treatments at times shortly after virus exposure, before signs of disease are detectable. We assessed the efficacy of lipid nanoparticle (LNP) delivery of anti-MARV nucleoprotein (NP)-targeting small interfering RNA (siRNA) at several time points after virus exposure, including after the onset of detectable disease in a uniformly lethal nonhuman primate model of MARV-Angola HF. Twenty-one rhesus monkeys were challenged with a lethal dose of MARV-Angola. Sixteen of these animals were treated with LNP containing anti-MARV NP siRNA beginning at 30 to 45 min, 1 day, 2 days, or 3 days after virus challenge. All 16 macaques that received LNP-encapsulated anti-MARV NP siRNA survived infection, whereas the untreated or mock-treated control subjects succumbed to disease between days 7 and 9 after infection. These results represent the successful demonstration of therapeutic anti-MARV-Angola efficacy in nonhuman primates and highlight the substantial impact of an LNP-delivered siRNA therapeutic as a countermeasure against this highly lethal human disease.

  7. Protective effect of the thioltransferase gene on in vivo UVR-300 nm-induced cataract.

    Science.gov (United States)

    Kronschläger, Martin; Galichanin, Konstantin; Ekström, Joakim; Lou, Marjorie F; Söderberg, Per G

    2012-01-25

    To determine the protection factor (PF) for glutaredoxin-1 (Grx1) with regard to UVR-induced cataract by comparison of in vivo ultraviolet radiation (UVR) lens toxicity between double knockout Grx1⁻/⁻ and Grx1⁺/⁺ mice. Twenty Grx1⁺/⁺ mice and 20 Grx1⁻/⁻ mice were unilaterally exposed in vivo to UVR for 15 minutes. Groups of four animals each received 0.0, 2.1, 2.9, 3.6, and 4.1 kJ/m(2) UVR-300 nm. At 48 hours after UVR exposure, light-scattering in the exposed and contralateral nonexposed lenses was measured quantitatively. Macroscopic lens changes were documented with dark-field illumination photography. UVR-300 nm induced subcapsular and cortical cataract in Grx1⁻/⁻ and Grx1⁺/⁺ mice. In both Grx1⁻/⁻ and Grx1⁺/⁺, the light-scattering intensified with increased in vivo exposure doses of UVR-300 nm. The intensity of forward light-scattering was higher in the lenses of Grx1⁻/⁻ mice than in the lenses of Grx1⁺/⁺ mice. The threshold dose for in vivo UVR-300 nm-induced cataract, expressed as MTD(2.3:16), was 3.8 in the Grx1⁺/⁺ group and 3.0 in the Grx1⁻/⁻ group, resulting in a PF of 1.3. The PF is an objective relative measure of protective properties. The Grx1 gene is associated with an in vivo PF of 1.3. This result signifies that the presence of the gene allows a 1.3 times longer in vivo exposure to UVR, at equivalent irradiance, than the absence of the gene before early-onset, UVR-induced cataract occurs. This finding indicates the important role of the Grx1 gene in the oxidation defense system of the lens.

  8. Protective

    Directory of Open Access Journals (Sweden)

    Wessam M. Abdel-Wahab

    2013-10-01

    Full Text Available Many active ingredients extracted from herbal and medicinal plants are extensively studied for their beneficial effects. Antioxidant activity and free radical scavenging properties of thymoquinone (TQ have been reported. The present study evaluated the possible protective effects of TQ against the toxicity and oxidative stress of sodium fluoride (NaF in the liver of rats. Rats were divided into four groups, the first group served as the control group and was administered distilled water whereas the NaF group received NaF orally at a dose of 10 mg/kg for 4 weeks, TQ group was administered TQ orally at a dose of 10 mg/kg for 5 weeks, and the NaF-TQ group was first given TQ for 1 week and was secondly administered 10 mg/kg/day NaF in association with 10 mg/kg TQ for 4 weeks. Rats intoxicated with NaF showed a significant increase in lipid peroxidation whereas the level of reduced glutathione (GSH and the activity of superoxide dismutase (SOD, catalase (CAT, glutathione S-transferase (GST and glutathione peroxidase (GPx were reduced in hepatic tissues. The proper functioning of the liver was also disrupted as indicated by alterations in the measured liver function indices and biochemical parameters. TQ supplementation counteracted the NaF-induced hepatotoxicity probably due to its strong antioxidant activity. In conclusion, the results obtained clearly indicated the role of oxidative stress in the induction of NaF toxicity and suggested hepatoprotective effects of TQ against the toxicity of fluoride compounds.

  9. Biocompatible and colloidally stabilized mPEG-PE/calcium phosphate hybrid nanoparticles loaded with siRNAs targeting tumors.

    Science.gov (United States)

    Gao, Pei; Zhang, Xiangyu; Wang, Hongzhi; Zhang, Qinghong; Li, He; Li, Yaogang; Duan, Yourong

    2016-01-19

    Calcium phosphate nanoparticles are safe and effective delivery vehicles for small interfering RNA (siRNA), as a result of their excellent biocompatibility. In this work, mPEG-PE (polyethylene glycol-L-α-phosphatidylethanolamine) was synthesized and used to prepare nanoparticles composed of mPEG-PE and calcium phosphate for siRNA delivery. Calcium phosphate and mPEG-PE formed the stable hybrid nanoparticles through self-assembly resulting from electrostatic interaction in water. The average size of the hybrid nanoparticles was approximately 53.2 nm with a negative charge of approximately -16.7 mV, which was confirmed by dynamic light scattering (DLS) measurements. The nanoparticles exhibited excellent stability in serum and could protect siRNA from ribonuclease (RNase) degradation. The cellular internalization of siRNA-loaded nanoparticles was evaluated in SMMC-7721 cells using a laser scanning confocal microscope (CLSM) and flow cytometry. The hybrid nanoparticles could efficiently deliver siRNA to cells compared with free siRNA. Moreover, the in vivo distribution of Cy5-siRNA-loaded hybrid nanoparticles was observed after being injected into tumor-bearing nude mice. The nanoparticles concentrated in the tumor regions through an enhanced permeability and retention (EPR) effect based on the fluorescence intensities of tissue distribution. A safety evaluation of the nanoparticles was performed both in vitro and in vivo demonstrating that the hybrid nanoparticle delivery system had almost no toxicity. These results indicated that the mPEG-PE/CaP hybrid nanoparticles could be a stable, safe and promising siRNA nanocarrier for anticancer therapy.

  10. NIR-to-visible upconversion nanoparticles for fluorescent labeling and targeted delivery of siRNA

    Science.gov (United States)

    Jiang, Shan; Zhang, Yong; Lim, Kian Meng; Sim, Eugene K. W.; Ye, Lei

    2009-04-01

    Near-infrared (NIR)-to-visible upconversion fluorescent nanoparticles were synthesized and used for imaging and targeted delivery of small interfering RNA (siRNA) to cancer cells. Silica-coated NaYF4 upconversion nanoparticles (UCNs) co-doped with lanthanide ions (Yb/Er) were synthesized. Folic acid and anti-Her2 antibody conjugated UCNs were used to fluorescently label the folate receptors of HT-29 cells and Her2 receptors of SK-BR-3 cells, respectively. The intracellular uptake of the folic acid and antibody conjugated UCNs was visualized using a confocal fluorescence microscope equipped with an NIR laser. siRNA was attached to anti-Her2 antibody conjugated UCNs and the delivery of these nanoparticles to SK-BR-3 cells was studied. Meanwhile, a luciferase assay was established to confirm the gene silencing effect of siRNA. Upconversion nanoparticles can serve as a fluorescent probe and delivery system for simultaneous imaging and delivery of biological molecules.

  11. NIR-to-visible upconversion nanoparticles for fluorescent labeling and targeted delivery of siRNA

    Energy Technology Data Exchange (ETDEWEB)

    Jiang Shan; Zhang Yong [Division of Bioengineering, Faculty of Engineering, National University of Singapore, 117576 (Singapore); Lim, Kian Meng [Department of Mechanical Engineering, National University of Singapore, 119260 (Singapore); Sim, Eugene K W [Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 117597 (Singapore); Ye Lei [National University Medical Institutes, National University of Singapore, 117597 (Singapore)], E-mail: biezy@nus.edu.sg

    2009-04-15

    Near-infrared (NIR)-to-visible upconversion fluorescent nanoparticles were synthesized and used for imaging and targeted delivery of small interfering RNA (siRNA) to cancer cells. Silica-coated NaYF{sub 4} upconversion nanoparticles (UCNs) co-doped with lanthanide ions (Yb/Er) were synthesized. Folic acid and anti-Her2 antibody conjugated UCNs were used to fluorescently label the folate receptors of HT-29 cells and Her2 receptors of SK-BR-3 cells, respectively. The intracellular uptake of the folic acid and antibody conjugated UCNs was visualized using a confocal fluorescence microscope equipped with an NIR laser. siRNA was attached to anti-Her2 antibody conjugated UCNs and the delivery of these nanoparticles to SK-BR-3 cells was studied. Meanwhile, a luciferase assay was established to confirm the gene silencing effect of siRNA. Upconversion nanoparticles can serve as a fluorescent probe and delivery system for simultaneous imaging and delivery of biological molecules.

  12. Polyspermine imine, a pH responsive polycationic siRNA carrier degradable to endogenous metabolites.

    Science.gov (United States)

    Du, Zixiu; Xiang, Shengnan; Zang, Yi; Zhou, Yi; Wang, Chuandong; Tang, Hailing; Jin, Tuo; Zhang, Xiaoling

    2014-10-06

    Cationic polymers readily degradable in response to cellular environment are especially favored as easy-formulating materials to pack siRNA into a nanoparticle and to release the cargo in the cytoplasm in time. In addition to the efficiency of cytosomal release, the degradation products should best be free of safety concerns, a typical challenge for cationic polymers. To satisfy the two criteria, we report a new cationic polymer, polyspermine imine, named as PSP-Imine, which is formed by condensing two endogenous molecules, spermine and glyoxal, through conjugated π linkage, -N═C-C═N- (Schiff base reaction), a poly linkage structure sufficiently stable under neutral condition but dissociative under the endosomal pH. Cellular assays under a confocal microscope indicated that the polyplex formed of PSP-Imine readily released the loaded siRNA to the cytoplasm after being engulfed in the target cells and efficiently silenced the target genes in different cell lines and xenograft mouse model of human cervical carcinoma, as compared with nondegradable PEI 25 kDa. Cell viability assays confirmed that PSP-Imine showed no visible cytotoxicity within the concentration being tested. The present study suggests that PSP-Imine is an excellent siRNA condensing material for forming the core of a therapeutically feasible synthetic carrier system.

  13. Intraventricular Delivery of siRNA Nanoparticles to the Central Nervous System

    Directory of Open Access Journals (Sweden)

    Rishab Shyam

    2015-01-01

    Full Text Available Alzheimer's disease (AD is a progressive neurodegenerative disease currently lacking effective treatment. Efficient delivery of siRNA via nanoparticles may emerge as a viable therapeutic approach to treat AD and other central nervous system disorders. We report here the use of a linear polyethyleneimine (LPEI-g-polyethylene glycol (PEG copolymer-based micellar nanoparticle system to deliver siRNA targeting BACE1 and APP, two therapeutic targets of AD. Using LPEI-siRNA nanoparticles against either BACE1 or APP in cultured mouse neuroblastoma (N2a cells, we observe selective knockdown, respectively, of BACE1 or APP. The encapsulation of siRNA by LPEI-g-PEG carriers, with different grafting degrees of PEG, leads to the formation of micellar nanoparticles with distinct morphologies, including worm-like, rod-like, or spherical nanoparticles. By infusing these shaped nanoparticles into mouse lateral ventricles, we show that rod-shaped nanoparticles achieved the most efficient knockdown of BACE1 in the brain. Furthermore, such knockdown is evident in spinal cords of these treated mice. Taken together, our findings indicate that the shape of siRNA-encapsulated nanoparticles is an important determinant for their delivery and gene knockdown efficiency in the central nervous system.

  14. Multifunctional polymeric micelles for delivery of drugs and siRNA

    Directory of Open Access Journals (Sweden)

    Aditi M. Jhaveri

    2014-04-01

    Full Text Available Polymeric micelles, self-assembling nano-constructs of amphiphilic copolymers with a core-shell structure have been used as versatile carriers for delivery of drugs as well as nucleic acids. They have gained immense popularity owing to a host of favorable properties including their capacity to effectively solubilize a variety of poorly soluble pharmaceutical agents, biocompatibility, longevity, high stability in vitro and in vivo and the ability to accumulate in pathological areas with compromised vasculature. Moreover, additional functions can be imparted to these micelles by engineering their surface with various ligands and cell-penetrating moieties to allow for specific targeting and intracellular accumulation, respectively, to load them with contrast agents to confer imaging capabilities, and incorporating stimuli-sensitive groups that allow drug release in response to small changes in the environment. Recently, there has been an increasing trend towards designing polymeric micelles which integrate a number of the above functions into a single carrier to give rise to smart, multifunctional polymeric micelles. Such multifunctional micelles can be envisaged as key to improving the efficacy of current treatments which have seen a steady increase not only in hydrophobic small molecules, but also in biologics including therapeutic genes, antibodies and small interfering RNA (siRNA. The purpose of this review is to highlight recent advances in the development of multifunctional polymeric micelles specifically for delivery of drugs and siRNA. In spite of the tremendous potential of siRNA, its translation into clinics has been a significant challenge because of physiological barriers to its effective delivery and the lack of safe, effective and clinically suitable vehicles. To that end, we also discuss the potential and suitability of multifunctional polymeric micelles, including lipid-based micelles, as promising vehicles for both siRNA and drugs.

  15. Targeted in vivo delivery of siRNA and an endosome-releasing agent to hepatocytes.

    Science.gov (United States)

    Sebestyén, Magdolna G; Wong, So C; Trubetskoy, Vladimir; Lewis, David L; Wooddell, Christine I

    2015-01-01

    The discoveries of RNA interference (RNAi) and short interfering RNAs (siRNAs) have provided the opportunity to treat diseases in a fundamentally new way: by co-opting a natural process to inhibit gene expression at the mRNA level. Given that siRNAs must interact with the cells' natural RNAi machinery in order to exert their silencing effect, one of the most fundamental requirements for their use is efficient delivery to the desired cell type and, specifically, into the cytoplasm of those cells. Numerous research efforts involving the testing of a large number of delivery approaches using various carrier molecules and inventing several distinct formulation technologies during the past decade illustrate the difficulty and complexity of this task. We have developed synthetic polymer formulations for in vivo siRNA delivery named Dynamic PolyConjugates™ (DPCs) that are designed to mimic the features viruses possess for efficient delivery of their nucleic acids. These include small size, long half-life in circulation, capability of displaying distinct host cell tropism, efficient receptor binding and cell entry, disassembly in the endosome and subsequent release of the nucleic acid cargo to the cytoplasm. Here we present an example of this delivery platform composed of a hepatocyte-targeted endosome-releasing agent and a cholesterol-conjugated siRNA (chol-siRNA). This delivery platform forms the basis of ARC-520, an siRNA-based therapeutic for the treatment of chronic hepatitis B virus (HBV) infection. In this chapter, we provide a general overview of the steps in developing ARC-520 and detailed protocols for two critical stages of the discovery process: (1) verifying targeted in vivo delivery to hepatocytes and (2) evaluating in vivo drug efficacy using a mouse model of chronic HBV infection.

  16. siRNA delivery into tumor cells by lipid-based nanoparticles composed of hydroxyethylated cholesteryl triamine.

    Science.gov (United States)

    Hattori, Yoshiyuki; Nakamura, Tsukasa; Ohno, Hiroaki; Fujii, Nobutaka; Maitani, Yoshie

    2013-02-25

    Previously, we reported that cationic nanoparticles (NP) composed of cholesteryl diamine (OH-Chol, (3S)-N-(2-(2-hydroxyethylamino)ethyl)cholesteryl-3-carboxamide) and Tween 80 could deliver plasmid DNA (pDNA) and small interfering RNA (siRNA) with high transfection efficiency into various tumor cells. In this study, to facilitate the endosomal escape of siRNA transfected by lipid-based nanoparticles, we synthesized new cationic cholesteryl triamine (OH-N-Chol, (3S)-N-(2-(2-(2-hydroxyethylamino)ethylamino)ethyl)cholesteryl-3-carboxamide) with an ethylenimine extension and prepared cationic nanoparticles (NP-N) composed of cholesteryl triamine and Tween 80. Although NP-N/siRNA complex (NP-N nanoplex) after mixing NP-N with siRNA was >350 nm in size, the vortex-mixing during the nanoplex formation decreased it to about 200 nm, which was an injectable size. NP-N nanoplex was mainly internalized by macropinocytosis-mediated endocytosis, as was NP nanoplex, and showed higher gene knockdown efficiency than NP nanoplex in human cervical carcinoma SiHa cells. From these results, cationic nanoparticles composed of OH-N-Chol and Tween 80 may have potential as a gene vector for siRNA transfection to tumor cells.

  17. Conservation, spillover and gene flow within a network of Northern European marine protected areas.

    Directory of Open Access Journals (Sweden)

    Mats Brockstedt Olsen Huserbråten

    Full Text Available To ensure that marine protected areas (MPAs benefit conservation and fisheries, the effectiveness of MPA designs has to be evaluated in field studies. Using an interdisciplinary approach, we empirically assessed the design of a network of northern MPAs where fishing for European lobster (Homarusgammarus is prohibited. First, we demonstrate a high level of residency and survival (50% for almost a year (363 days within MPAs, despite small MPA sizes (0.5-1 km(2. Second, we demonstrate limited export (4.7% of lobsters tagged within MPAs (N = 1810 to neighbouring fished areas, over a median distance of 1.6 km out to maximum 21 km away from MPA centres. In comparison, median movement distance of lobsters recaptured within MPAs was 164 m, and recapture rate was high (40%. Third, we demonstrate a high level of gene flow within the study region, with an estimated F ST of less than 0.0001 over a ≈ 400 km coastline. Thus, the restricted movement of older life stages, combined with a high level of gene flow suggests that connectivity is primarily driven by larval drift. Larval export from the MPAs can most likely affect areas far beyond their borders. Our findings are of high importance for the design of MPA networks for sedentary species with pelagic early life stages.

  18. RAP1 Protects from Obesity through Its Extratelomeric Role Regulating Gene Expression

    Directory of Open Access Journals (Sweden)

    Paula Martínez

    2013-06-01

    Full Text Available RAP1 is part of shelterin, the protective complex at telomeres. RAP1 also binds along chromosome arms, where it is proposed to regulate gene expression. To investigate the nontelomeric roles of RAP1 in vivo, we generated a RAP1 whole-body knockout mouse. These mice show early onset of obesity, which is more severe in females than in males. Rap1-deficient mice show accumulation of abdominal fat, hepatic steatosis, and high-fasting plasma levels of insulin, glucose, cholesterol, and alanine aminotransferase. Gene expression analyses of liver and visceral white fat from Rap1-deficient mice before the onset of obesity show deregulation of metabolic programs, including fatty acid, glucose metabolism, and PPARα signaling. We identify Pparα and Pgc1α as key factors affected by Rap1 deletion in the liver. We show that RAP1 binds to Pparα and Pgc1α loci and modulates their transcription. These findings reveal a role for a telomere-binding protein in the regulation of metabolism.

  19. The role of prourokinase gene in protecting vein grafts from intimal hyperplasia

    Institute of Scientific and Technical Information of China (English)

    黄志雄; 郭加强; 胡盛寿

    2003-01-01

    Objective To study the duration of prourokinase gene expression in vein grafts and the role of the prourokinase gene in protecting vein grafts from neointimal hyperplasia.Methods Fifty-four Wistar rats were used in this study. In each rat, the jugular vein was excised and distended for 30 minutes using a solution containing either Adv5-CMV (control group) or Adv5-CMV/Pro-UK (treatment group). Next, the jugular vein was reversed and interposed into the divided carotid artery of the same rat. On the 14th day after transfection, vein grafts of the control group were collected in order to perform a fibrinolysis test for prourokinase (Pro-UK) activity. On the 2nd, 7th, 14th, 28th, and 60th day, the vein grafts of the treatment group were likewise collected in order to detect prourokinase activity. On the 28th day, the vein grafts of both groups were explanted to evaluate the 3H-TDR incorporation so that pathologic analysis could be performed.Results Pro-UK activity could not be detected in the control group, while in the treatment group, the Pro-UK activity could be detected from the 2nd day onwards, peaking on the 7th day and declining from the 14th day, but yet persisting at a low level for a further month. The amount of 3H-TDR incorporated in the control group was higher than that in the treatment group. Pathologic analysis demonstrated that vein grafts of both groups exhibited wall thickening, but that the degree of graft neointimal hyperplasia and reduction of the graft lumen was greater in the control group than that in the treatment group. The occlusion rate of grafts in the control group was 20%. All grafts in the treatment group were patent.Conclusions Pro-UK gene transfer before vein grafting in vitro results in a high level of gene expression in the vein graft from the 7th day to 14th day. And its gene expression in the vein graft could reduce neointimal hyperlasia in the vein graft.

  20. Dual expression profile of type VI secretion system immunity genes protects pandemic Vibrio cholerae.

    Directory of Open Access Journals (Sweden)

    Sarah T Miyata

    Full Text Available The Vibrio cholerae type VI secretion system (T6SS assembles as a molecular syringe that injects toxic protein effectors into both eukaryotic and prokaryotic cells. We previously reported that the V. cholerae O37 serogroup strain V52 maintains a constitutively active T6SS to kill other Gram-negative bacteria while being immune to attack by kin bacteria. The pandemic O1 El Tor V. cholerae strain C6706 is T6SS-silent under laboratory conditions as it does not produce T6SS structural components and effectors, and fails to kill Escherichia coli prey. Yet, C6706 exhibits full resistance when approached by T6SS-active V52. These findings suggested that an active T6SS is not required for immunity against T6SS-mediated virulence. Here, we describe a dual expression profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 that provides pandemic V. cholerae strains with T6SS immunity and allows T6SS-silent strains to maintain immunity against attacks by T6SS-active bacterial neighbors. The dual expression profile allows transcription of the three genes encoding immunity proteins independently of other T6SS proteins encoded within the same operon. One of these immunity proteins, TsiV2, protects against the T6SS effector VasX which is encoded immediately upstream of tsiV2. VasX is a secreted, lipid-binding protein that we previously characterized with respect to T6SS-mediated virulence towards the social amoeba Dictyostelium discoideum. Our data suggest the presence of an internal promoter in the open reading frame of vasX that drives expression of the downstream gene tsiV2. Furthermore, VasX is shown to act in conjunction with VasW, an accessory protein to VasX, to compromise the inner membrane of prokaryotic target cells. The dual regulatory profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 permits V. cholerae to tightly control T6SS gene expression while maintaining immunity to T6SS activity.

  1. Triple Combination of siRNAs Targeting TGFβ1, TGFβR2, and CTGF Enhances Reduction of Collagen I and Smooth Muscle Actin in Corneal Fibroblasts

    OpenAIRE

    Sriram, Sriniwas; Robinson, Paulette; Pi, Liya; Lewin, Alfred S.; Schultz, Gregory

    2013-01-01

    An optimized triple combination of siRNAs targeting TGFβ1, TGFβR2, and CTGF genes produced very high knockdown of target and downstream scarring genes (collagen and alpha smooth muscle actin) without cell toxicity, suggesting a new approach for reducing fibrosis in the cornea and other tissues.

  2. Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene.

    Science.gov (United States)

    Melo, Luis G; Agrawal, Reitu; Zhang, Lunan; Rezvani, Mojgan; Mangi, Abeel A; Ehsan, Afshin; Griese, Daniel P; Dell'Acqua, Giorgio; Mann, Michael J; Oyama, Junichi; Yet, Shaw-Fang; Layne, Matthew D; Perrella, Mark A; Dzau, Victor J

    2002-02-05

    Ischemia and oxidative stress are the leading mechanisms for tissue injury. An ideal strategy for preventive/protective therapy would be to develop an approach that could confer long-term transgene expression and, consequently, tissue protection from repeated ischemia/reperfusion injury with a single administration of a therapeutic gene. In the present study, we used recombinant adeno-associated virus (rAAV) as a vector for direct delivery of the cytoprotective gene heme oxygenase-1 (HO-1) into the rat myocardium, with the purpose of evaluating this strategy as a therapeutic approach for long-term protection from ischemia-induced myocardial injury. Human HO-1 gene (hHO-1) was delivered to normal rat hearts by intramyocardial injection. AAV-mediated transfer of the hHO-1 gene 8 weeks before acute coronary artery ligation and release led to a dramatic reduction (>75%) in left ventricular myocardial infarction. The reduction in infarct size was accompanied by decreases in myocardial lipid peroxidation and in proapoptotic Bax and proinflammatory interleukin-1beta protein abundance, concomitant with an increase in antiapoptotic Bcl-2 protein level. This suggested that the transgene exerts its cardioprotective effects in part by reducing oxidative stress and associated inflammation and apoptotic cell death. This study documents the beneficial therapeutic effect of rAAV-mediated transfer, before myocardial injury, of a cytoprotective gene that confers long-term myocardial protection from ischemia/reperfusion injury. Our data suggest that this novel "pre-event" gene transfer approach may provide sustained tissue protection from future repeated episodes of injury and may be beneficial as preventive therapy for patients with or at risk of developing coronary ischemic events.

  3. Cancer-targeted MDR-1 siRNA delivery using self-cross-linked glycol chitosan nanoparticles to overcome drug resistance.

    Science.gov (United States)

    Yhee, Ji Young; Song, Seungyong; Lee, So Jin; Park, Sung-Gurl; Kim, Ki-Suk; Kim, Myung Goo; Son, Sejin; Koo, Heebeom; Kwon, Ick Chan; Jeong, Ji Hoon; Jeong, Seo Young; Kim, Sun Hwa; Kim, Kwangmeyung

    2015-01-28

    P-glycoprotein (Pgp) mediated multi-drug resistance (MDR) is a major cause of failure in chemotherapy. In this study, small interfering RNA (siRNA) for Pgp down-regulation was delivered to tumors to overcome MDR in cancer. To achieve an efficient siRNA delivery in vivo, self-polymerized 5'-end thiol-modified siRNA (poly-siRNA) was incorporated in tumor targeting glycol chitosan nanoparticles. Pgp-targeted poly-siRNA (psi-Pgp) and thiolated glycol chitosan polymers (tGC) formed stable nanoparticles (psi-Pgp-tGC NPs), and the resulting nanoparticles protected siRNA molecules from enzymatic degradation. The psi-Pgp-tGC NPs could release functional siRNA molecules after cellular delivery, and they were able to facilitate siRNA delivery to Adriamycin-resistant breast cancer cells (MCF-7/ADR). After intravenous administration, the psi-Pgp-tGC NPs accumulated in MCF-7/ADR tumors and down-regulated P-gp expression to sensitize cancer cells. Consequently, chemo-siRNA combination therapy significantly inhibited tumor growth without systemic toxicity. These psi-Pgp-tGC NPs showed great potential as a supplementary therapeutic agent for drug-resistant cancer.

  4. Heat shock protein 70 gene transfection protects rat myocardium cell against anoxia-reoxygeneration injury

    Institute of Scientific and Technical Information of China (English)

    LIU Ji-chun; HE Ming; WAN Li; CHENG Xiao-shu

    2007-01-01

    Background A number of studies suggest that the expression of heat shock protein 70 (HSP70) induced by heat stress are associated with protection against ischemia-reperfusion injury. But the protective effects may be contaminated by other factors in the same stress. This study was conducted to explore the protective role of HSP70 expression in acute myocardial anoxia/reoxygeneration (A/R) injury with a liposome-mediated gene transfer technique for the introduction of pCDNA HSP70 into the neonatal rat myocardial cells. In addition, heat shock stress cytoprotection was also investigated for comparison.Methods The cultured primary neonatal rat myocardiocytes with an acute myocardial A/R injury model and the HS-treated rat myocardiocyte model were used. Three-day cultured myocardiocytes were randomly divided into four groups (n=8): control group, A/R group, HS+A/R group and pCDNA HSP70 +A/R group. A liposome-coated HSP70 pCDNA plasmid was transfected into the primary neonatal rat myocardiocytes; HSP70 mRNA and its protein were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The cell viability was assayed by monotetrazolium (MTT) and the lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) activity of cells during incubation and the changes in cells ultrastructure were examined. NF-κB activity in the primary neonatal rat myocardiocytes was measured with flow cytometry.Results Compared with viability in the A/R group ((35.4±6.9)%) the cell viability in the HS+A/R group ((72.8±11.6)%)and the pCDNA HSP70 + A/R group ((76.3±12.2)%) was improved significantly (P<0.05). The activity of LDH and CPK was significantly elevated in the A/R group. However, in the HS+A/R group and pCDNA HSP70 +A/R group, significant decreases in activity were observed. The cell ultrastructure of the A/R group cells was abnormal, whereas nearly normal ultrastructure was observed in HS+A/R group and pCDNA HSP70+A/R group. HSP70 mRNA and protein

  5. Synthesis and characterization of methylated poly(L-histidine) to control the stability of its siRNA polyion complexes for RNAi.

    Science.gov (United States)

    Asayama, Shoichiro; Kumagai, Takao; Kawakami, Hiroyoshi

    2012-07-18

    Poly(L-histidine) (PLH) with dimethylimidazole groups has been synthesized as a pH-sensitive polypeptide to control the stability of its small interfering RNA (siRNA) polyion complexes for RNA interference (RNAi). The resulting methylated PLH (PLH-Me) was water-soluble despite deprotonation of the imidazole groups at physiological pH, as determined by acid-base titration and solution turbidity measurement. Agarose gel retardation assay proved that the quaternary dimethylimidazole groups worked as cationic groups to retain siRNA. The stability of the PLH-Me/siRNA complexes has depended on the content of hydrophobic groups, that is, τ/π-methylimidazole groups as well as deprotonated imidazole groups. PLH-Me exhibited no significant cytotoxicity despite the existence of cationic dimethylimidazole groups. By use of PLH-Me as a pH-sensitive siRNA carrier, the PLH-Me/siRNA complexes mediated efficient siRNA delivery attributed to the dimethylimidazole groups, and the gene silencing depended on the content balance among dimethyl, τ/π-methyl, and unmodified imidazole groups. These results suggest that PLH-Me controls the stability of siRNA polyion complexes by enhancing noncytotoxic siRNA delivery by optimizing the content balance of dimethyl, τ/π-methyl, and unmodified imidazole groups.

  6. Solid formulation of cell-penetrating peptide nanocomplexes with siRNA and their stability in simulated gastric conditions.

    Science.gov (United States)

    Ezzat, Kariem; Zaghloul, Eman M; El Andaloussi, Samir; Lehto, Taavi; El-Sayed, Ramy; Magdy, Tarek; Smith, C I Edvard; Langel, Ulo

    2012-08-20

    Cell-penetrating peptides (CPPs) are short cationic peptides that have been extensively studied as drug delivery vehicles for proteins, nucleic acids and nanoparticles. However, the formulation of CPP-based therapeutics into different pharmaceutical formulations and their stability in relevant biological environments have not been given the same attention. Here, we show that a newly developed CPP, PepFect 14 (PF14), forms non-covalent nanocomplexes with short interfering RNA (siRNA), which are able to elicit efficient RNA-interference (RNAi) response in different cell-lines. RNAi effect is obtained at low siRNA doses with a unique kinetic profile. Furthermore, the solid dispersion technique is utilized to formulate PF14/siRNA nanocomplexes into solid formulations that are as active as the freshly prepared nanocomplexes in solution. Importantly, the nanocomplexes are stable and active in mediating RNAi response after incubation with simulated gastric fluid (SGF) that is highly acidic. These results demonstrate the activity of PF14 in delivering and protecting siRNA in different pharmaceutical forms and biological environments.

  7. Continuous cell electroporation for efficient DNA and siRNA delivery based on laminar microfluidic chips.

    Science.gov (United States)

    Wei, Zewen; Li, Zhihong

    2014-01-01

    Electroporation is a high-efficiency and low-toxicity physical gene transfer method. Traditional electroporation is limited to only low volume cell samples. Here we present a continuous cell electroporation method based on commonly used microfluidic chip fabrication technology. Using easily fabricated PDMS microfluidic chip, syringe pumps, and pulse generator, we show efficient delivery of both DNA and siRNA into different cell lines. We describe the protocol of chip fabrication, apparatus setup, and cell electroporation assay. Typically, the fabrication of the devices takes 1 or 2 days and the continuous electroporation assay takes 1 h.

  8. MysiRNA: improving siRNA efficacy prediction using a machine-learning model combining multi-tools and whole stacking energy (ΔG).

    Science.gov (United States)

    Mysara, Mohamed; Elhefnawi, Mahmoud; Garibaldi, Jonathan M

    2012-06-01

    The investigation of small interfering RNA (siRNA) and its posttranscriptional gene-regulation has become an extremely important research topic, both for fundamental reasons and for potential longer-term therapeutic benefits. Several factors affect the functionality of siRNA including positional preferences, target accessibility and other thermodynamic features. State of the art tools aim to optimize the selection of target siRNAs by identifying those that may have high experimental inhibition. Such tools implement artificial neural network models as Biopredsi and ThermoComposition21, and linear regression models as DSIR, i-Score and Scales, among others. However, all these models have limitations in performance. In this work, a neural-network trained new siRNA scoring/efficacy prediction model was developed based on combining two existing scoring algorithms (ThermoComposition21 and i-Score), together with the whole stacking energy (ΔG), in a multi-layer artificial neural network. These three parameters were chosen after a comparative combinatorial study between five well known tools. Our developed model, 'MysiRNA' was trained on 2431 siRNA records and tested using three further datasets. MysiRNA was compared with 11 alternative existing scoring tools in an evaluation study to assess the predicted and experimental siRNA efficiency where it achieved the highest performance both in terms of correlation coefficient (R(2)=0.600) and receiver operating characteristics analysis (AUC=0.808), improving the prediction accuracy by up to 18% with respect to sensitivity and specificity of the best available tools. MysiRNA is a novel, freely accessible model capable of predicting siRNA inhibition efficiency with improved specificity and sensitivity. This multiclassifier approach could help improve the performance of prediction in several bioinformatics areas. MysiRNA model, part of MysiRNA-Designer package [1], is expected to play a key role in siRNA selection and evaluation.

  9. Blockage of Src by Specific siRNA as a Novel Therapeutic Strategy to Prevent Destructive Repair in Steroid-Associated Osteonecrosis in Rabbits.

    Science.gov (United States)

    Zheng, Li-zhen; Cao, Hui-juan; Chen, Shi-hui; Tang, Tao; Fu, Wei-min; Huang, Le; Chow, Dick Ho Kiu; Wang, Yi-xiang; Griffith, James Francis; He, Wei; Zhou, Hong; Zhao, De-wei; Zhang, Ge; Wang, Xin-luan; Qin, Ling

    2015-11-01

    Vascular hyperpermeability and highly upregulated bone resorption in the destructive repair progress of steroid-associated osteonecrosis (SAON) are associated with a high expression of VEGF and high Src activity (Src is encoded by the cellular sarcoma [c-src] gene). This study was designed to prove our hypothesis that blocking the VEGF-Src signaling pathway by specific Src siRNA is able to prevent destructive repair in a SAON rabbit model. Destructive repair in SAON was induced in rabbits. At 2, 4, and 6 weeks after SAON induction, VEGF, anti-VEGF, Src siRNA, Src siRNA+VEGF, control siRNA, and saline were introduced via intramedullary injection into proximal femora for each group, respectively. Vascularization and permeability were quantified by dynamic contrast-enhanced (DCE) MRI. At week 6 after SAON induction, proximal femurs were dissected for micro-computed tomography (μCT)-based trabecular architecture with finite element analysis (FEA), μCT-based angiography, and histological analysis. Histological evaluation revealed that VEGF enhanced destructive repair, whereas anti-VEGF prevented destructive repair and Src siRNA and Src siRNA+VEGF prevented destructive repair and enhanced reparative osteogenesis. Findings of angiography and histomorphometry were consistent with those determined by DCE MRI. Src siRNA inhibited VEGF-mediated vascular hyperpermeability but preserved VEGF-induced neovascularization. Bone resorption was enhanced in the VEGF group and inhibited in the anti-VEGF, Src siRNA, Src siRNA+VEGF groups as determined by both 3D μCT and 2D histomorphometry. FEA showed higher estimated failure load in the Src siRNA and Src siRNA+VEGF groups when compared to the vehicle control group. Blockage of VEGF-Src signaling pathway by specific Src siRNA was able to prevent steroid-associated destructive repair while improving reconstructive repair in SAON, which might become a novel therapeutic strategy.

  10. Effect of rumen-protected choline supplementation on liver and adipose gene expression during the transition period in dairy cattle

    NARCIS (Netherlands)

    Goselink, R.M.A.; Baal, van J.; Widjaja, H.C.A.; Dekker, R.A.; Zom, R.L.G.; Veth, M.J.; Vuuren, van A.M.

    2013-01-01

    We previously reported that supplementation of rumen-protected choline (RPC) reduces the hepatic triacylglycerol concentration in periparturient dairy cows during early lactation. Here, we investigated the effect of RPC on the transcript levels of lipid metabolism-related genes in liver and adipose

  11. Screening Target Specificity of siRNAs by Rapid Amplification of cDNA Ends (RACE) for Non-Sequenced Species

    OpenAIRE

    Sabirzhanov, Boris; Sabirzhanova, Inna B.; Keifer, Joyce

    2011-01-01

    RNA interference (RNAi) is the process of sequence-specific posttranslational gene silencing triggered by double-stranded RNAs (dsRNAs). RNAi is a widely used approach for studying gene function. However, studies have shown that using siRNA can lead to off-target effects when the siRNA contains sufficient sequence identity to non-target mRNA sequences. One of the important steps in designing dsRNA is verification that it has sequence identity to only the target mRNA. In this report, we propos...

  12. 6-Gingerol Protects against Nutritional Steatohepatitis by Regulating Key Genes Related to Inflammation and Lipid Metabolism

    Directory of Open Access Journals (Sweden)

    Thing-Fong Tzeng

    2015-02-01

    Full Text Available Non-alcoholic fatty liver disease, including non-alcoholic steatohepatitis (NASH, appears to be increasingly common worldwide. The aim of the study was to investigate the effects of 6-gingerol ((S-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl-3-decanone, a bioactive ingredient of plants belonging to the Zingiberaceae family, on experimental models of NASH. In HepG2 cells, 6-gingerol (100 μmol/L treatment inhibited free fatty acids mixture (0.33 mmol/L palmitate and 0.66 mmol/L oleate-induced triglyceride and inflammatory marker accumulations. Male C57BL/6 mice were fed with a methionine and choline-deficient (MCD diet to induce steatohepatitis. After four weeks of MCD diet feeding, the mice were dosed orally with 6-gingerol (25, 50 or 100 mg/kg/day once daily for another four weeks. 6-Gingerol (100 mg/kg/day attenuated liver steatosis and necro-inflammation in MCD diet-fed mice. The expressions of inflammatory cytokine genes, including those for monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-6, and nuclear transcription factor (NF-κB, which were increased in the livers of MCD diet-fed mice, were attenuated by 6-gingerol. 6-Gingerol possesses a repressive property on hepatic steatosis, which is associated with induction of peroxisome proliferator-activated receptor α. Our study demonstrated the protective role of 6-gingerol in ameliorating nutritional steatohepatitis. The effect was mediated through regulating key genes related to lipid metabolism and inflammation.

  13. siRNA, miRNA and HIV: promises and challenges

    Institute of Scientific and Technical Information of China (English)

    Man Lung YEUNG; Yamina BENNASSER; Shu Yun LE; Kuan Teh JEANG

    2005-01-01

    Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing.The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNAtargeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNAspecified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells' antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).

  14. Unraveling siRNA Unzipping Kinetics with Graphene

    CERN Document Server

    Mogurampelly, Santosh; Bhattacharyya, Dhananjay; Sood, A K; Maiti, Prabal K

    2012-01-01

    Using all atom molecular dynamics simulations, we report spontaneous unzipping and strong binding of small interfering RNA (siRNA) on graphene. Our dispersion corrected density functional theory based calculations suggest that nucleosides of RNA have stronger attractive interactions with graphene as compared to DNA residues. These stronger interactions force the double stranded siRNA to spontaneously unzip and bind to the graphene surface. Unzipping always nucleates at one end of the siRNA and propagates to the other end after few base-pairs get unzipped. While both the ends get unzipped, the middle part remains in double stranded form because of torsional constraint. Unzipping probability distributions fitted to single exponential function give unzipping time (t) of the order of few nanoseconds which decrease exponentially with temperature. From the temperature variation of unzipping time we estimate the energy barrier to unzipping.

  15. Lentivirus-expressed siRNA vectors against Alzheimer disease.

    Science.gov (United States)

    Peng, Kevin A; Masliah, Eliezer

    2010-01-01

    Amyloid precursor protein (APP) has been implicated in the pathogenesis of Alzheimer disease, and the accumulation of APP products ultimately leads to the familiar histopathological and clinical manifestations associated with this most common form of dementia. A protein that has been shown to promote APP accumulation is beta-secretase (beta-site APP cleaving enzyme 1, or BACE1), which is increased in the cerebrospinal fluid in those affected with Alzheimer disease. Through in vivo studies using APP transgenic mice, we demonstrated that decreasing the expression of BACE1 via lentiviral vector delivery of BACE1 siRNA has the potential for significantly reducing the cleavage of APP, accumulation of these products, and consequent neurodegeneration. As such, lentiviral-expressed siRNA against BACE1 is a therapeutic possibility in the treatment of Alzheimer disease. We detail the use of lentivirus-expressed siRNA as a method to ameliorate Alzheimer disease neuropathology in APP transgenic mice.

  16. Protease-triggered siRNA delivery vehicles.

    Science.gov (United States)

    Rozema, David B; Blokhin, Andrei V; Wakefield, Darren H; Benson, Jonathan D; Carlson, Jeffrey C; Klein, Jason J; Almeida, Lauren J; Nicholas, Anthony L; Hamilton, Holly L; Chu, Qili; Hegge, Julia O; Wong, So C; Trubetskoy, Vladimir S; Hagen, Collin M; Kitas, Eric; Wolff, Jon A; Lewis, David L

    2015-07-10

    The safe and efficacious delivery of membrane impermeable therapeutics requires cytoplasmic access without the toxicity of nonspecific cytoplasmic membrane lysis. We have developed a mechanism for control of cytoplasmic release which utilizes endogenous proteases as a trigger and results in functional delivery of small interfering RNA (siRNA). The delivery approach is based on reversible inhibition of membrane disruptive polymers with protease-sensitive substrates. Proteolytic hydrolysis upon endocytosis restores the membrane destabilizing activity of the polymers thereby allowing cytoplasmic access of the co-delivered siRNA. Protease-sensitive polymer masking reagents derived from polyethylene glycol (PEG), which inhibit membrane interactions, and N-acetylgalactosamine, which targets asialoglycoprotein receptors on hepatocytes, were synthesized and used to formulate masked polymer-siRNA delivery vehicles. The size, charge and stability of the vehicles enable functional delivery of siRNA after subcutaneous administration and, with modification of the targeting ligand, have the potential for extrahepatic targeting.

  17. Effects of a human plasma membrane-associated sialidase siRNA on prostate cancer invasion

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiaojie [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Taizhou Polytechnic College, Taizhou (China); Zhang, Ling; Shao, Yueting; Liang, Zuowen; Shao, Chen; Wang, Bo; Guo, Baofeng; Li, Na; Zhao, Xuejian [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Li, Yang, E-mail: lyang@jlu.edu.cn [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Xu, Deqi [Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD (United States)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Neu3 is as one of the sialidases and regulates cell surface functions. Black-Right-Pointing-Pointer A Neu3-specific siRNA inhibited prostrate cancer cell invasion and migration. Black-Right-Pointing-Pointer The Neu3-specific siRNA inhibited prostate cancer metastasis in mice. Black-Right-Pointing-Pointer Targeting Neu3 may have utility for gene-based therapy of human cancer metastasis. -- Abstract: Human plasma membrane-associated sialidase (Neu3) is one of several sialidases that hydrolyze sialic acids in the terminal position of the carbohydrate groups of glycolipids and glycoproteins. Neu3 is mainly localized in plasma membranes and plays crucial roles in the regulation of cell surface functions. In this study, we investigated the effects and molecular mechanisms of Neu3 on cell invasion and migration in vivo and in vitro. Initially, we found that the levels of Neu3 expression were higher in prostate cancer tissues and cell lines than in normal prostate tissues based on RT-PCR and Western blotting analyses. We then applied a Neu3 siRNA approach to block Neu3 signaling using PC-3M cells as model cells. Transwell invasion assays and wound assays showed significantly decreased invasion and migration potential in the Neu3 siRNA-transfected cells. RT-PCR and Western blotting analyses revealed that Neu3 knockdown decreased the expressions of the matrix metalloproteinases MMP-2 and MMP-9. In vivo, mice injected with PC-3M cell tumors were evaluated by SPECT/CT to determine the presence of bone metastases. Mice treated with attenuated Salmonella carrying the Neu3 siRNA developed fewer bone metastases than mice treated with attenuated Salmonella carrying a control Scramble siRNA, attenuated Salmonella alone or PBS. The results for bone metastasis detection by pathology were consistent with the data obtained by SPECT/CT. Tumor blocks were evaluated by histochemical, RT-PCR and Western blotting analyses. The results revealed

  18. Reversal of chemoresistance with small interference RNA (siRNA) in etoposide resistant acute myeloid leukemia cells (HL-60).

    Science.gov (United States)

    Kachalaki, Saeed; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi; Shanehbandi, Dariush; Mohammadinejad, Sina; Mansoori, Behzad

    2015-10-01

    Overexpression of ATP-binding cassette (ABC) drug transporters is a major barrier in the success of cancer chemotherapy. One way to overcome overexpression of ABC drug transporter-mediated chemoresistance in acute myeloid leukemia is to suppress ABC drug transporter genes expression by small interference RNA (siRNA). In this study was assessed the involvement of ABCB1 gene in the mechanisms of resistance to etoposide in AML cells. The etoposide-resistant HL-60 cells were generated by stepwise exposure increasing concentrations of etoposide. The etoposide-resistant HL-60 cells were transfected with siRNAs using Transfection Reagent. The ABCB1 mRNA expression were assessed by real-time quantitative PCR. The MDR1/P-gp levels were measured by Western blotting. The sensitivity of resistant HL-60 cells to etoposide after transfection was determined using MTT assay. Apoptosis of resistant HL-60 cells after transfection was detected by flow cytometer. It was found that siRNA effectively inhibited ABCB1 expression at both mRNA and protein levels. Knockdown of the ABCB1 gene correlated with increased sensitivity of the resistant HL-60 cells to etoposide and was observed to lower the cytotoxic index (IC50 etoposide value) after transfection. Our results indicate that product of the ABCB1 gene have effective role in resistance to etoposide in acute myeloid leukemia cells. Copyright © 2015. Published by Elsevier Masson SAS.

  19. Systemic delivery of siRNA by aminated poly(α)glutamate for the treatment of solid tumors.

    Science.gov (United States)

    Polyak, Dina; Krivitsky, Adva; Scomparin, Anna; Eliyahu, Shay; Kalinski, Hagar; Avkin-Nachum, Sharon; Satchi-Fainaro, Ronit

    2016-06-26

    Small interfering RNA (siRNA) can silence the expression of a targeted gene in a process known as RNA interference (RNAi). As a consequence, RNAi has immense potential as a novel therapeutic approach in cancer targeted therapy. However, successful application of siRNA for therapeutic purposes is challenging due to its rapid renal clearance, degradation by RNases in the bloodstream, poor cellular penetration, immunogenicity and aggregation in the blood. In addition, the few oligonucleotide-based nanomedicines that reached clinical trials either go to the liver following systemic administration or are applied topically. Treatment of solid tumors requires selective distribution of siRNA to the target tissue, hence there is an unmet medical need for an efficacious and safe nano-sized delivery system for their clinical use. To overcome these hurdles, we have designed, synthesized and physico-chemically characterized a novel nanocarrier based on aminated poly(α)glutamate (PGAamine). This cathepsin B-biodegradable polymer interacts electrostatically with the siRNA to form a nano-sized polyplex stable in plasma. Treatment with PGAamine-Rac1 siRNA polyplex (siRac1-polyplex) caused specific gene silencing by 80% in HeLa and SKOV-3 human ovarian adenocarcinoma cells as opposed to PGAamine-control non-targeting siRNA polyplex (siCtrl-polyplex) leading to inhibition of cell migration and wound healing abilities. A stepwise dose escalation was performed in order to determine the in vivo maximum tolerated dose (MTD). This was followed by intraperitoneal administration of siRac1-polyplex to mCherry-labeled ovarian adenocarcinoma-bearing mice leading to preferred tumor accumulation of siRac1 (8-fold) which resulted in 38% Rac1 knockdown. Furthermore, the polyplex was administered intravenously to lung carcinoma-bearing mice in which it caused 33% Rac1 knockdown. These promising results led to efficacy studies administering systemic treatment with an anticancer siRNA, siPlk1

  20. siRNA release from pri-miRNA scaffolds is controlled by the sequence and structure of RNA.

    Science.gov (United States)

    Galka-Marciniak, Paulina; Olejniczak, Marta; Starega-Roslan, Julia; Szczesniak, Michal W; Makalowska, Izabela; Krzyzosiak, Wlodzimierz J

    2016-04-01

    shmiRs are pri-miRNA-based RNA interference triggers from which exogenous siRNAs are expressed in cells to silence target genes. These reagents are very promising tools in RNAi in vivo applications due to their good activity profile and lower toxicity than observed for other vector-based reagents such as shRNAs. In this study, using high-resolution northern blotting and small RNA sequencing, we investigated the precision with which RNases Drosha and Dicer process shmiRs. The fidelity of siRNA release from the commonly used pri-miRNA shuttles was found to depend on both the siRNA insert and the pri-miR scaffold. Then, we searched for specific factors that may affect the precision of siRNA release and found that both the structural features of shmiR hairpins and the nucleotide sequence at Drosha and Dicer processing sites contribute to cleavage site selection and cleavage precision. An analysis of multiple shRNA intermediates generated from several reagents revealed the complexity of shmiR processing by Drosha and demonstrated that Dicer selects substrates for further processing. Aside from providing new basic knowledge regarding the specificity of nucleases involved in miRNA biogenesis, our results facilitate the rational design of more efficient genetic reagents for RNAi technology.

  1. Onset Time and Durability of Huntingtin Suppression in Rhesus Putamen After Direct Infusion of Antihuntingtin siRNA.

    Science.gov (United States)

    Grondin, Richard; Ge, Pei; Chen, Qingmin; Sutherland, Jessica E; Zhang, Zhiming; Gash, Don M; Stiles, David K; Stewart, Gregory R; Sah, Dinah W Y; Kaemmerer, William F

    2015-06-30

    One possible treatment for Huntington's disease involves direct infusion of a small, interfering RNA (siRNA) designed to reduce huntingtin expression into brain tissue from a chronically implanted programmable pump. Here, we studied the suppression of huntingtin mRNA achievable with short infusion times, and investigated how long suppression may persist after infusion ceases. Rhesus monkeys received 3 days of infusion of Magnevist into the putamen to confirm catheter patency and fluid distribution. After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin. After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later. Results indicate that the onset of huntingtin mRNA suppression in the rhesus putamen occurs rapidly, achieving a plateau throughout the putamen within 4 days. Conversely, loss of huntingtin suppression progresses slowly, persisting an estimated 27-39 days in the putamen and surrounding white matter. These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

  2. Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer.

    Science.gov (United States)

    Kim, Sun Hwa; Jeong, Ji Hoon; Lee, Soo Hyeon; Kim, Sung Wan; Park, Tae Gwan

    2008-07-14

    For efficient cancer therapy, small interfering RNA (siRNA) should be stably and efficiently delivered into the target tissue and readily taken up by cancer cells. To address these needs, a polyelectrolyte complex (PEC) micelle-based siRNA delivery system was developed for anti-angiogenic gene therapy. The interaction between poly(ethylene glycol) (PEG)-conjugated vascular endothelial growth factor siRNA (VEGF siRNA-PEG) and polyethylenimine (PEI) led to the spontaneous formation of nanoscale polyelectrolyte complex micelles (VEGF siRNA-PEG/PEI PEC micelles), having a characteristic siRNA/PEI PEC inner core with a surrounding PEG shell layer. Intravenous as well as intratumoral administration of the PEC micelles significantly inhibited VEGF expression at the tumor tissue and suppressed tumor growth in an animal tumor model without showing any detectable inflammatory responses in mice. Upon examination of the PEC micelle distribution and in vivo optical imaging following intravenously injection, enhanced accumulation of the PEC micelles was also observed in the tumor region. This study demonstrates the feasibility of using PEC micelles as a potential carrier for therapeutic siRNAs in local and systemic treatment of cancer.

  3. Inhibition of high-mobility group box 1 expression by siRNA in rat hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Wen-Song Ge; Jian-Xin Wu; Jian-Gao Fan; Yao-Jun Wang; Ying-Wei Chen

    2011-01-01

    AIM: To explore the role of high-mobility group box 1 (HMGB1) protein during liver fibrogenesis and investigate the functional effects of HMGB1 gene silencing in hepatic stellate cells (HSCs) using siRNA. METHODS: Hepatic fibrosis in rats was induced through serial subcutaneous injections of dimethylnitrosamine, and expression of HMGB1 was detected by immunohistochemistry. HMGB1 siRNAs were developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000. HMGB1 expression was evaluated by real-time polymerase chain reaction (PCR) and Western blotting analysis. Expression of α-smooth muscle actin (α-SMA) and collagen types Ⅰ and Ⅲ was evaluated by real-time PCR. Cell proliferation and the cell cycle were determined using the methyl thiazolyl tetrazolium method. Finally, collagen content in HSC supernatant was evaluated by an enzyme-linked immunosorbent assay. RESULTS: The results showed that HMGB1 was upregulated during liver fibrosis and that its expression was closely correlated with the deposition of collagen. siRNA molecules were successfully transfected into HSCs and induced inhibition of HMGB1 expression in a time-dependent manner. Moreover, HMGB1 siRNA treatment inhibited synthesis of α-SMA and collagen types Ⅰ and Ⅲ in transfected HSCs. CONCLUSION: This study suggests a significant functional role for HMGB1 in the development of liver fibrosis. It also demonstrates that downregulation of HMGB1 expression might be a potential strategy to treat liver fibrosis.

  4. Onset Time and Durability of Huntingtin Suppression in Rhesus Putamen After Direct Infusion of Antihuntingtin siRNA

    Directory of Open Access Journals (Sweden)

    Richard Grondin

    2015-01-01

    Full Text Available One possible treatment for Huntington's disease involves direct infusion of a small, interfering RNA (siRNA designed to reduce huntingtin expression into brain tissue from a chronically implanted programmable pump. Here, we studied the suppression of huntingtin mRNA achievable with short infusion times, and investigated how long suppression may persist after infusion ceases. Rhesus monkeys received 3 days of infusion of Magnevist into the putamen to confirm catheter patency and fluid distribution. After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin. After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later. Results indicate that the onset of huntingtin mRNA suppression in the rhesus putamen occurs rapidly, achieving a plateau throughout the putamen within 4 days. Conversely, loss of huntingtin suppression progresses slowly, persisting an estimated 27–39 days in the putamen and surrounding white matter. These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

  5. Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene.

    Directory of Open Access Journals (Sweden)

    Nidhi Thakur

    Full Text Available BACKGROUND: Expression of double strand RNA (dsRNA designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi, thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci upon oral feeding. The v-ATPase subunit A (v-ATPaseA coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation. METHODOLOGY/PRINCIPAL FINDINGS: Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect. CONCLUSIONS/SIGNIFICANCE: Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops.

  6. Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene.

    Science.gov (United States)

    Thakur, Nidhi; Upadhyay, Santosh Kumar; Verma, Praveen C; Chandrashekar, Krishnappa; Tuli, Rakesh; Singh, Pradhyumna K

    2014-01-01

    Expression of double strand RNA (dsRNA) designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi), thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci) upon oral feeding. The v-ATPase subunit A (v-ATPaseA) coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation. Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect. Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops.

  7. Risperidone and NAP protect cognition and normalize gene expression in a schizophrenia mouse model.

    Science.gov (United States)

    Vaisburd, Sinaya; Shemer, Zeev; Yeheskel, Adva; Giladi, Eliezer; Gozes, Illana

    2015-11-10

    Mutated disrupted in schizophrenia 1 (DISC1), a microtubule regulating protein, leads to schizophrenia and other psychiatric illnesses. It is hypothesized that microtubule stabilization may provide neuroprotection in schizophrenia. The NAP (NAPVSIPQ) sequence of activity-dependent neuroprotective protein (ADNP) contains the SxIP motif, microtubule end binding (EB) protein target, which is critical for microtubule dynamics leading to synaptic plasticity and neuroprotection. Bioinformatics prediction for FDA approved drugs mimicking SxIP-like motif which displace NAP-EB binding identified Risperidone. Risperidone or NAP effectively ameliorated object recognition deficits in the mutated DISC1 mouse model. NAP but not Risperidone, reduced anxiety in the mutated mice. Doxycycline, which blocked the expression of the mutated DISC1, did not reverse the phenotype. Transcripts of Forkhead-BOX P2 (Foxp2), a gene regulating DISC1 and associated with human ability to acquire a spoken language, were increased in the hippocampus of the DISC1 mutated mice and were significantly lowered after treatment with NAP, Risperidone, or the combination of both. Thus, the combination of NAP and standard of care Risperidone in humans may protect against language disturbances associated with negative and cognitive impairments in schizophrenia.

  8. Evaluation of carrier-mediated siRNA delivery

    DEFF Research Database (Denmark)

    Colombo, Stefano; Nielsen, Hanne Mørck; Foged, Camilla

    2013-01-01

    RNA delivered by use of carriers remains an analytical challenge. The purpose of the present study was to optimize and validate an analytical protocol based on stem-loop reverse transcription quantitative polymerase chain reaction (RT qPCR) to quantitatively monitor the carrier-mediated intracellular si......RNA delivery. An in vitro cell culture model system expressing enhanced green fluorescent protein (EGFP) was used to develop the assay, which was based on the intracellular quantification of a full-length double-stranded Dicer substrate siRNA by stem-loop RT qPCR. The result is a well-documented protocol...

  9. Oxime ether lipids containing hydroxylated head groups are more superior siRNA delivery agents than their nonhydroxylated counterparts

    Science.gov (United States)

    Gupta, Kshitij; Mattingly, Stephanie J; Knipp, Ralph J; Afonin, Kirill A; Viard, Mathias; Bergman, Joseph T; Stepler, Marissa; Nantz, Michael H; Puri, Anu; Shapiro, Bruce A

    2015-01-01

    Aim: To evaluate the structure–activity relationship of oxime ether lipids (OELs) containing modifications in the hydrophobic domains (chain length, degree of unsaturation) and hydrophilic head groups (polar domain hydroxyl groups) toward complex formation with siRNA molecules and siRNA delivery efficiency of resulting complexes to a human breast cancer cell line (MDA-MB-231). Materials & methods: Ability of lipoplex formation between oxime ether lipids with nucleic acids were examined using biophysical techniques. The potential of OELs to deliver nucleic acids and silence green fluorescent protein (GFP) gene was analyzed using MDA-MB-231 and MDA-MB-231/GFP cells, respectively. Results & conclusion: Introduction of hydroxyl groups to the polar domain of the OELs and unsaturation into the hydrophobic domain favor higher transfection and gene silencing in a cell culture system. PMID:26107486

  10. Exploring in vitro roles of siRNA in cardiovascular disease

    Institute of Scientific and Technical Information of China (English)

    Yu TANG; Yu-zhi GE; James Q YIN

    2007-01-01

    RNA interference (RNAi) is an adaptive defense mechanism through which double-stranded RNAs silence cognate genes in a sequence-specific manner. It has been employed widely as a powerful tool in functional genomics studies, target valida-tion and therapeutic product development. Similarly, the application of small-interfering RNA (siRNA) to the silencing of the disease-causing genes involved in cardiovascular diseases has made great progress. In this overview, we attempt to provide a brief outline of the current understanding of the mechanism of RNAi and its potential application to the cardiovascular system, with particular empha-sis on its ability to identify the pathophysiological function of genes related to several important cardiovascular disorders. The prospects of RNAi-based therapeutics, as well as the advantages and potential problems, are also discussed.

  11. HER-2/neu·siRNA表达载体的构建及对人乳腺癌细胞株的影响%Vector construction and silencing effects of siRNA to HER-2/neu gene of human breast cancer cell line SK-BR-3

    Institute of Scientific and Technical Information of China (English)

    万光霞; 林辉; 刘浔阳

    2010-01-01

    目的:探讨siRNA 对人乳腺癌细胞株SK-BR-3 的HER-2/neu基因表达的影响.方法:以HER-2/neu mRNA 序列为模板设计合成2对siRNA 序列,构建pGenesil-1-HER-2/neu siRNA 重组质粒,转化感受态的大肠杆菌,质粒酶切、测序鉴定.转染SK-BR-3 细胞48 h后,提取RNA 进行RT-PCR,采用方差分析(ANOVA),分析RNA 干扰效应.结果:成功构建了pGenesil-1-HER-2/neu siRNA 重组质粒,成功转染SK-BR-3 细胞.重组质粒抑制HER-2/neu基因的表达接近54.45 %( P =0.000).结论: HER-2/neu siRNA 重组质粒明显下调HER-2/neu 基因在乳腺癌细胞中的表达.

  12. Telomerase inhibition by siRNA causes senescence and apoptosis in Barrett's adenocarcinoma cells: mechanism and therapeutic potential

    Directory of Open Access Journals (Sweden)

    Batchu Ramesh B

    2005-07-01

    Full Text Available Abstract Background In cancer cells, telomerase induction helps maintain telomere length and thereby bypasses senescence and provides enhanced replicative potential. Chemical inhibitors of telomerase have been shown to reactivate telomere shortening and cause replicative senescence and apoptotic cell death of tumor cells while having little or no effect on normal diploid cells. Results We designed siRNAs against two different regions of telomerase gene and evaluated their effect on telomere length, proliferative potential, and gene expression in Barrett's adenocarcinoma SEG-1 cells. The mixture of siRNAs in nanomolar concentrations caused a loss of telomerase activity that appeared as early as day 1 and was essentially complete at day 3. Inhibition of telomerase activity was associated with marked reduction in median telomere length and complete loss of detectable telomeres in more than 50% of the treated cells. Telomere loss caused senescence in 40% and apoptosis in 86% of the treated cells. These responses appeared to be associated with activation of DNA sensor HR23B and subsequent activation of p53 homolog p73 and p63 and E2F1. Changes in these gene regulators were probably the source of observed up-regulation of cell cycle inhibitors, p16 and GADD45. Elevated transcript levels of FasL, Fas and caspase 8 that activate death receptors and CARD 9 that interacts with Bcl10 and NFKB to enhance mitochondrial translocation and activation of caspase 9 were also observed. Conclusion These studies show that telomerase siRNAs can cause effective suppression of telomerase and telomere shortening leading to both cell cycle arrest and apoptosis via mechanisms that include up-regulation of several genes involved in cell cycle arrest and apoptosis. Telomerase siRNAs may therefore be strong candidates for highly selective therapy for chemoprevention and treatment of Barrett's adenocarcinoma.

  13. Influence of the Internalization Pathway on the Efficacy of siRNA Delivery by Cationic Fluorescent Nanodiamonds in the Ewing Sarcoma Cell Model

    Science.gov (United States)

    Alhaddad, Anna; Durieu, Catherine; Dantelle, Géraldine; Le Cam, Eric; Malvy, Claude; Treussart, François; Bertrand, Jean-Rémi

    2012-01-01

    Small interfering RNAs (siRNAs) are powerful tools commonly used for the specific inhibition of gene expression. However, vectorization is required to facilitate cell penetration and to prevent siRNA degradation by nucleases. We have shown that diamond nanocrystals coated with cationic polymer can be used to carry siRNAs into Ewing sarcoma cells, in which they remain traceable over long periods, due to their intrinsic stable fluorescence. We tested two cationic polymers, polyallylamine and polyethylenimine. The release of siRNA, accompanied by Ewing sarcoma EWS-Fli1 oncogene silencing, was observed only with polyethylenimine. We investigated cell penetration and found that the underlying mechanisms accounted for these differences in behavior. Using drugs selectively inhibiting particular pathways and a combination of fluorescence and electronic microscopy, we showed that siRNA gene silencing occurred only if the siRNA:cationic nanodiamond complex followed the macropinocytosis route. These results have potential implications for the design of efficient drug-delivery vectors. PMID:23284935

  14. Systemic delivery of siRNA by hyaluronan-functionalized calcium phosphate nanoparticles for tumor-targeted therapy

    Science.gov (United States)

    Qiu, Chong; Wei, Wei; Sun, Jing; Zhang, Hai-Tao; Ding, Jing-Song; Wang, Jian-Cheng; Zhang, Qiang

    2016-06-01

    In this study, hyaluronan (HA)-functionalized calcium phosphate nanoparticles (CaP-AHA/siRNA NPs) were developed for an injectable and targetable delivery of siRNA, which were prepared by coating the alendronate-hyaluronan graft polymer (AHA) around the surface of calcium phosphate-siRNA co-precipitates. The prepared CaP-AHA/siRNA NPs had a uniform spherical core-shell morphology with an approximate size of 170 nm and zeta potential of -12 mV. The coating of hydrophilic HA improved the physical stability of nanoparticles over one month due to the strong interactions between phosphonate and calcium. In vitro experiments demonstrated that the negatively charged CaP-AHA/siRNA NPs could effectively deliver EGFR-targeted siRNA into A549 cells through CD44-mediated endocytosis and significantly down-regulate the level of EGFR expression. Also, the internalized CaP-AHA/siRNA NPs exhibited a pH-responsive release of siRNA, indicating that the acidification of lysosomes probably facilitated the disassembling of nanoparticles and the resultant ions sharply increased the inner osmotic pressure and thus expedited the release of siRNA from late lysosomes into the cytoplasm. Furthermore, in vivo tumor therapy demonstrated that high accumulation of CaP-AHA/siEGFR NPs in tumor led to a significant tumor growth inhibition with a specific EGFR gene silencing effect after intravenous administration in nude mice xenografted with A549 tumor, along with a negligible body weight loss. These results suggested that the CaP-AHA/siRNA NPs could be an effective and safe systemic siRNA delivery system for a RNAi-based tumor targeted therapy strategy.In this study, hyaluronan (HA)-functionalized calcium phosphate nanoparticles (CaP-AHA/siRNA NPs) were developed for an injectable and targetable delivery of siRNA, which were prepared by coating the alendronate-hyaluronan graft polymer (AHA) around the surface of calcium phosphate-siRNA co-precipitates. The prepared CaP-AHA/siRNA NPs had a uniform

  15. [Genomic noncoding sequences and the size of eukaryotic cell nucleus as important factors of gene protection from chemical mutagens].

    Science.gov (United States)

    Minkevich, I G; Patrushev, L I

    2007-01-01

    An improved quantitative model describing a protective function of eukaryotic genomic noncoding sequences was developed. In this new model, two factors affecting gene protection from chemical mutagens are considered: (1) the ratio of the total lengths of coding and noncoding genomic sequences and (2) the volume of the cell nucleus. An increase in the noncoding DNA in the genome reduces the number of mutagen-damaged nucleotides in the coding region, whereas an increase in the volume of the nucleus decreases the flow of mutagens per unit of nuclear volume that attacks its surface.

  16. siRNAs from an X-linked satellite repeat promote X-chromosome recognition in Drosophila melanogaster.

    Science.gov (United States)

    Menon, Debashish U; Coarfa, Cristian; Xiao, Weimin; Gunaratne, Preethi H; Meller, Victoria H

    2014-11-18

    Highly differentiated sex chromosomes create a lethal imbalance in gene expression in one sex. To accommodate hemizygosity of the X chromosome in male fruit flies, expression of X-linked genes increases twofold. This is achieved by the male- specific lethal (MSL) complex, which modifies chromatin to increase expression. Mutations that disrupt the X localization of this complex decrease the expression of X-linked genes and reduce male survival. The mechanism that restricts the MSL complex to X chromatin is not understood. We recently reported that the siRNA pathway contributes to localization of the MSL complex, raising questions about the source of the siRNAs involved. The X-linked 1.688 g/cm(3) satellite related repeats (1.688(X) repeats) are restricted to the X chromosome and produce small RNA, making them an attractive candidate. We tested RNA from these repeats for a role in dosage compensation and found that ectopic expression of single-stranded RNAs from 1.688(X) repeats enhanced the male lethality of mutants with defective X recognition. In contrast, expression of double-stranded hairpin RNA from a 1.688(X) repeat generated abundant siRNA and dramatically increased male survival. Consistent with improved survival, X localization of the MSL complex was largely restored in these males. The striking distribution of 1.688(X) repeats, which are nearly exclusive to the X chromosome, suggests that these are cis-acting elements contributing to identification of X chromatin.

  17. Stearylated octaarginine and artificial virus-like particles for transfection of siRNA into primary rat neurons

    Science.gov (United States)

    Tönges, Lars; Lingor, Paul; Egle, Roman; Dietz, Gunnar P.H.; Fahr, Alfred; Bähr, Mathias

    2006-01-01

    RNA interference (RNAi) provides a powerful experimental tool for sequence-specific gene silencing, allowing efficient analysis of gene function in a multitude of cell types. However, application of RNAi in primary mammalian neurons has been limited by low-transfection efficiency and considerable toxicity of conventional transfection methods. In this study, we evaluated a peptide-mediated and a polymer/lipid-based cellular delivery method for siRNA into rat primary neurons and compared the results with a commonly used liposomal transfection reagent. Stearylated octaarginine (Stearyl-R8) was used as polypeptide and artificial virus-like particles (AVPs) were used as a combined liposomal-polymeric vector, since both reagents have been previously shown to successfully transfect DNA into cell lines. Stearyl-R8 and AVPs both promoted siRNA transfection into primary hippocampal neurons via the endosomal pathway. SiRNA-mediated gene silencing could be effectively induced in primary neuron cultures. In comparison with the commonly used cationic liposome transfection agent, both novel reagents were less detrimental to cell metabolic activity. We conclude that these novel transfection methods yield performances comparable to cationic liposome-mediated transfection for siRNA, while being less cytotoxic in primary neurons. Stearyl-R8 and AVPs may therefore represent novel and more cost-efficient alternatives to conventional siRNA-transfection reagents. PMID:16699166

  18. siRNA delivery with lipid-based systems

    DEFF Research Database (Denmark)

    Foged, Camilla

    2012-01-01

    in vivo, toxicity and non-specific stimulation of the immune system. To optimally design and tailor the lipidic systems for siRNA delivery, better insight is needed into the mechanisms of cell delivery. More specifically, further clarification is need regarding the nature of cell surface interactions...

  19. Poly(amidoamine) dendrimer nanocarriers and their aerosol formulations for siRNA delivery to the lung epithelium.

    Science.gov (United States)

    Conti, Denise S; Brewer, Daniel; Grashik, Jordan; Avasarala, Sumant; da Rocha, Sandro R P

    2014-06-02

    Small interfering RNA (siRNA)-based therapies have great promise in the treatment of a number of prevalent pulmonary disorders including lung cancer, asthma and cystic fibrosis. However, progress in this area has been hindered due to the lack of carriers that can efficiently deliver siRNA to lung epithelial cells, and also due to challenges in developing oral inhalation (OI) formulations for the regional administration of siRNA and their carriers to the lungs. In this work we report the ability of generation four, amine-terminated poly(amidoamine) (PAMAM) dendrimer (G4NH2)-siRNA complexes (dendriplexes) to silence the enhanced green fluorescent protein (eGFP) gene on A549 lung alveolar epithelial cells stably expressing eGFP. We also report the formulation of the dendriplexes and their aerosol characteristics in propellant-based portable OI devices. The size and gene silencing ability of the dendriplexes was seen not to be a strong function of the N/P ratio. Silencing efficiencies of up to 40% are reported. Stable dispersions of the dendriplexes encapsulated in mannitol and also in a biodegradable and water-soluble co-oligomer were prepared in hydrofluoroalkane (HFA)-based pressurized metered-dose inhalers (pMDIs). Their aerosol characteristics were very favorable, and conducive to deep lung deposition, with respirable fractions of up to 77%. Importantly, siRNA formulated as dendriplexes in pMDIs was shown to keep its integrity after the particle preparation processes, and also after long-term exposures to HFA. The relevance of this study stems from the fact that this is the first work to report the formulation of inhalable siRNA with aerosol properties suitable to deep lung deposition using pMDIs devices that are the least expensive and most widely used portable inhalers. This study is relevant because, also for the first time, it shows that siRNA-G4NH2 dendriplexes can efficiently target lung alveolar epithelial A549 cells and silence genes even after siRNA has

  20. Connexin43 Mediated Delivery of ADAMTS5 Targeting siRNAs from Mesenchymal Stem Cells to Synovial Fibroblasts.

    Directory of Open Access Journals (Sweden)

    Shuo Liu

    Full Text Available Osteoarthritis is a joint-destructive disease that has no effective cure. Human mesenchymal stem cells (hMSCs could offer therapeutic benefit in the treatment of arthritic diseases by suppressing inflammation and permitting tissue regeneration, but first these cells must overcome the catabolic environment of the diseased joint. Likewise, gene therapy also offers therapeutic promise given its ability to directly modulate key catabolic factors that mediate joint deterioration, although it too has limitations. In the current study, we explore an approach that combines hMSCs and gene therapy. Specifically, we test the use of hMSC as a vehicle to deliver ADAMTS5 (an aggrecanase with a key role in osteoarthritis-targeting siRNAs to SW982 synovial fibroblast-like cells via connexin43 containing gap junctions. Accordingly, we transduced hMSCs with ADAMTS5-targeting shRNA or non-targeted shRNA, and co-cultured them with synovial fibroblasts to allow delivery of siRNAs from hMSC to synovial fibroblasts. We found that co-culture of hMSCs-shRNA-ADAMTS5 and synovial fibroblasts reduced ADAMTS5 expression relative to co-culture of hMSCs-shRNA-control and synovial fibroblasts. Furthermore, ADAMTS5 was specifically reduced in the synovial fibroblasts populations as determined by fluorescence-activated cell sorting, suggesting transfer of the siRNA between cells. To test if Cx43-containing gap junctions are involved in the transfer of siRNA, we co-cultured hMSCs-shRNA-ADAMTS5 cells with synovial fibroblasts in which connexin43 was knocked down. Under these conditions, ADAMTS5 levels were not inhibited by co-culture, indicating that connexin43 mediates the delivery of siRNA from hMSCs to synovial fibroblasts. In total, our findings demonstrate that hMSCs can function as donor cells to host and deliver siRNAs to synovial fibroblasts via connexin43 gap junction in vitro. These data may have implications in the combination of hMSCs and gene therapy to treat diseases

  1. Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes.

    Science.gov (United States)

    Yang, Ji Hye; Shin, Bo Yeon; Han, Jae Yun; Kim, Mi Gwang; Wi, Ji Eun; Kim, Young Woo; Cho, Il Je; Kim, Sang Chan; Shin, Sang Mi; Ki, Sung Hwan

    2014-01-15

    Isorhamentin is a 3'-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes.

  2. Beclin 1 and UVRAG confer protection from radiation-induced DNA damage and maintain centrosome stability in colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Jae Myung Park

    Full Text Available Beclin 1 interacts with UV-irradiation-resistance-associated gene (UVRAG to form core complexes that induce autophagy. While cells with defective autophagy are prone to genomic instability that contributes to tumorigenesis, it is unknown whether Beclin1 or UVRAG can regulate the DNA damage/repair response to cancer treatment in established tumor cells. We found that siRNA knockdown of Beclin 1 or UVRAG can increase radiation-induced DNA double strand breaks (DSBs, shown by pATM and γH2Ax, and promote colorectal cancer cell death. Furthermore, knockdown of Beclin 1, UVRAG or ATG5 increased the percentage of irradiated cells with nuclear foci expressing 53BP1, a marker of nonhomologous end joining but not RAD51 (homologous recombination, compared to control siRNA. Beclin 1 siRNA was shown to attenuate UVRAG expression. Cells with a UVRAG deletion mutant defective in Beclin 1 binding showed increased radiation-induced DSBs and cell death compared to cells with ectopic wild-type UVRAG. Knockdown of Beclin 1 or UVRAG, but not ATG5, resulted in a significant increase in centrosome number (γ-tubulin staining in irradiated cells compared to control siRNA. Taken together, these data indicate that Beclin 1 and UVRAG confer protection against radiation-induced DNA DSBs and may maintain centrosome stability in established tumor cells.

  3. The siRNA targeted to mdr1b and mdr1a mRNAs in vivo sensitizes murine lymphosarcoma to chemotherapy

    OpenAIRE

    Vlassov Valentin V; Nikolin Valery P; Kaledin Vasily I; Popova Nelly A; Mironova Nadezda L; Patutina Olga A; Zenkova Marina A

    2010-01-01

    Abstract Background One of the main obstacles for successful cancer polychemotherapy is multiple drug resistance phenotype (MDR) acquired by tumor cells. Currently, RNA interference represents a perspective strategy to overcome MDR via silencing the genes involved in development of this deleterious phenotype (genes of ABC transporters, antiapoptotic genes, etc.). Methods In this study, we used the siRNAs targeted to mdr1b, mdr1a, and bcl-2 mRNAs to reverse the MDR of tumors and increase tumor...

  4. Construction of eukaryotic expression vectors of siRNA against TNF-α gene and their silencing effects on TNF-α and TGF-β1 in A549 cells%TNF-α基因siRNA真核表达载体的构建及其对A549细胞TNF-α和TGF-β1表达的影响

    Institute of Scientific and Technical Information of China (English)

    张立英; 刘绍霞; 赵国强; 张国俊

    2012-01-01

    目的:构建靶向肿瘤坏死因子α(TNF-α)基因的小干扰RNA(siRNA)真核表达载体,观察其对人肺腺癌A549细胞中TNF-α和转化生长因子-β1(TGF-β1)表达的影响.方法:依据GenBank中人TNF-α(NM_000594)序列和siRNA靶序列设计原则,设计并合成一对针对TNF-α的特异性序列(siTNF-α)和一对无关序列(siCon).退火合成DNA双链,再分别重组入pRNAT-U6.1载体,经过PCR和DNA测序鉴定.将构建好的重组载体pRNAT-U6.1-siTNF-α、pRNAT-U6.1-siCon及空载体pRNAT-U6.1用脂质体介导转染A549细胞株,分别采用Real time PCR和Western blot方法检测细胞中TNF-α和TGF-β1 mRNA和蛋白的表达水平.结果:PCR和DNA测序鉴定证实载体构建成功;转染pRNAT-U6.1-siTNF-α的A549细胞中TNF-α和TGF-β1 mRNA和蛋白表达水平均低于其他3组(FmRNA=478.663,4.081;F蛋白=123.420,6.312,P均<0.05).结论:成功构建了靶向TNF-α基因的siRNA真核表达载体,该载体能够有效沉默A549细胞中TNF-α的表达,TGF-β1基因表达亦受到抑制.%Aim: To clone the eukaryotic expression vectors of small interfering RNA ( siRNA ) against TNF-a gene and to evaluate their silencing effects on TNF-a and TGF-β1 in A549 cells. Methods: According to TNF-a gene ( NM_ 000594 ) sequence of GenBank, using RNAi Designer software to design the siRNA specific target sequence ( siTNF-α ) and the unrelated control sequence ( siCon ) for TNF-α. The annealing products were recombined into pRNAT-U6. 1, PCR and DNA sequencing confirmed the insertion sequence. The recombinant plasmids ( pRNAT-U6. l-siTNF-a,pRNAT-U6. 1-siCon ) were transfected to the A549 cells with lipofectamine,and the expressions of TNF-α, TGF-β1 mRNA and protein in cells detected by Real time PCR and Western blot methods. Results: PCR and DNA sequencing confirmed the vectors were consistent;the expressions of TNF-α, TGF-β1 mRNA and protein in the cells transfected pRNAT-U6. 1-siTNF-a were markedly lower than the other three

  5. Therapeutic silence of pleiotrophin by targeted delivery of siRNA and its effect on the inhibition of tumor growth and metastasis.

    Science.gov (United States)

    Zha, Lisha; He, Lichun; Xie, Weidong; Cheng, Jin; Li, Tong; Mohsen, Mona O; Lei, Fan; Storni, Federico; Bachmann, Martin; Chen, Hongquan; Zhang, Yaou

    2017-01-01

    Pleiotrophin (PTN) is a secreted cytokine that is expressed in various cancer cell lines and human tumor such as colon cancer, lung cancer, gastric cancer and melanoma. It plays significant roles in angiogenesis, metastasis, differentiation and cell growth. The expression of PTN in the adult is limited to the hippocampus in an activity-dependent manner, making it a very attractive target for cancer therapy. RNA interference (RNAi) offers great potential as a new powerful therapeutic strategy based on its highly specific and efficient silencing of a target gene. However, efficient delivery of small interfering RNA (siRNA) in vivo remains a significant hurdle for its successful therapeutic application. In this study, we first identified, on a cell-based experiment, applying a 1:1 mixture of two PTN specific siRNA engenders a higher silencing efficiency on both mRNA and protein level than using any of them discretely at the same dose. As a consequence, slower melanoma cells growth was also observed for using two specific siRNA combinatorially. To establish a robust way for siRNA delivery in vivo and further investigate how silence of PTN affects tumor growth, we tested three different methods to deliver siRNA in vivo: first non-targeted in-vivo delivery of siRNA via jetPEI; second lung targeted delivery of siRNA via microbubble coated jetPEI; third tumor cell targeted delivery of siRNA via transferrin-polyethylenimine (Tf-PEI). As a result, we found that all three in-vivo siRNAs delivery methods led to an evident inhibition of melanoma growth in non-immune deficiency C57BL/6 mice without a measureable change of ALT and AST activities. Both targeted delivery methods showed more significant curative effect than jetPEI. The lung targeted delivery by microbubble coated jetPEI revealed a comparable therapeutic effect with Tf-PEI, indicating its potential application for target delivery of siRNA in vivo.

  6. Therapeutic silence of pleiotrophin by targeted delivery of siRNA and its effect on the inhibition of tumor growth and metastasis

    Science.gov (United States)

    Xie, Weidong; Cheng, Jin; Li, Tong; Mohsen, Mona O.; Lei, Fan; Storni, Federico; Bachmann, Martin; Chen, Hongquan; Zhang, Yaou

    2017-01-01

    Pleiotrophin (PTN) is a secreted cytokine that is expressed in various cancer cell lines and human tumor such as colon cancer, lung cancer, gastric cancer and melanoma. It plays significant roles in angiogenesis, metastasis, differentiation and cell growth. The expression of PTN in the adult is limited to the hippocampus in an activity-dependent manner, making it a very attractive target for cancer therapy. RNA interference (RNAi) offers great potential as a new powerful therapeutic strategy based on its highly specific and efficient silencing of a target gene. However, efficient delivery of small interfering RNA (siRNA) in vivo remains a significant hurdle for its successful therapeutic application. In this study, we first identified, on a cell-based experiment, applying a 1:1 mixture of two PTN specific siRNA engenders a higher silencing efficiency on both mRNA and protein level than using any of them discretely at the same dose. As a consequence, slower melanoma cells growth was also observed for using two specific siRNA combinatorially. To establish a robust way for siRNA delivery in vivo and further investigate how silence of PTN affects tumor growth, we tested three different methods to deliver siRNA in vivo: first non-targeted in-vivo delivery of siRNA via jetPEI; second lung targeted delivery of siRNA via microbubble coated jetPEI; third tumor cell targeted delivery of siRNA via transferrin-polyethylenimine (Tf-PEI). As a result, we found that all three in-vivo siRNAs delivery methods led to an evident inhibition of melanoma growth in non-immune deficiency C57BL/6 mice without a measureable change of ALT and AST activities. Both targeted delivery methods showed more significant curative effect than jetPEI. The lung targeted delivery by microbubble coated jetPEI revealed a comparable therapeutic effect with Tf-PEI, indicating its potential application for target delivery of siRNA in vivo. PMID:28562667

  7. Synthetic siRNAs effectively target cystein protease 12 and α-actinin transcripts in Trichomonas vaginalis.

    Science.gov (United States)

    Ravaee, Roya; Ebadi, Parimah; Hatam, Gholamreza; Vafafar, Arghavan; Ghahramani Seno, Mohammad Mahdi

    2015-10-01

    The flagellated protozoan Trichomonas vaginalis (T. vaginalis) causes trichomoniasis, a reproductive tract infection, in humans. Trichomoniasis is the most common non-viral sexually transmitted disease worldwide. In addition to direct consequences such as infertility and abortion, there are indications that trichomoniasis favours development of prostate cancer and it has also been associated with increased risk of spreading human immunodeficiency virus and papillomavirus infections. Reports from around the world show that the rate of drug resistance in T. vaginalis is increasing, and therefore new therapeutic approaches have to be developed. Studying molecular biology of T. vaginalis will be quite helpful in identifying new drugable targets. RNAi is a powerful technique which allows biologist to specifically target gene products (i.e. mRNA) helping them in unravelling gene functions and biology of systems. However, due to lack of some parts of the required intrinsic RNAi machinery, the RNAi system is not functional in all orders of life. Here, by using synthetic siRNAs targeting two genes, i.e. α-actinin and cystein protease 12 (cp12), we demonstrate T. vaginalis cells are amenable to RNAi experiments conducted by extrinsic siRNAs. Electroporation of siRNAs targeting α-actinin or cp12 into T. vaginalis cells resulted in, respectively, 48-67% and 33-72% downregulation of the cognate transcripts compared to the T. vaginalis cells received siRNAs targeting GL2 luciferase as a control. This finding is helpful in that it demonstrates the potential of using extrinsically induced RNAi in studies on molecular biology of T. vaginalis such as those aiming at identifying new drug targets.

  8. Identification and Molecular Characterization of a Gene Encoding a Protective Leishmania amazonensis Trp-Asp (WD) Protein

    OpenAIRE

    2004-01-01

    Several Leishmania proteins have been identified and characterized in pursuit of understanding pathogenesis and protection in cutaneous leishmaniasis. In the present study, we utilized sera from infected BALB/c mice to screen a Leishmania amazonensis amastigote cDNA expression library and obtained the full-length gene that encodes a novel Trp-Asp (WD) protein designated LAWD (for Leishmania antigenic WD protein). The WD family of proteins mediates protein-protein interactions and coordinates ...

  9. Early life DNA vaccination with the H gene of Canine distemper virus induces robust protection against distemper

    DEFF Research Database (Denmark)

    Jensen, Trine Hammer; Nielsen, Line; Aasted, Bent;

    2009-01-01

    Young mink kits (n = 8)were vaccinated withDNA plasmids encoding the viral haemagglutinin protein (H) of a vaccine strain of Canine distemper virus (CDV). Virus neutralising (VN) antibodieswere induced after 2 immunisations and after the third immunisation all kits had high VN antibody titres...... demonstrate that early life DNA vaccination with the H gene of a CDV vaccine strain induced robust protective immunity against a recent wild type CDV....

  10. Immunostimulatory motifs enhance antiviral siRNAs targeting highly pathogenic avian influenza H5N1.

    Directory of Open Access Journals (Sweden)

    Cameron R Stewart

    Full Text Available Highly pathogenic avian influenza (HPAI H5N1 virus is endemic in many regions around the world and remains a significant pandemic threat. To date H5N1 has claimed almost 300 human lives worldwide, with a mortality rate of 60% and has caused the death or culling of hundreds of millions of poultry since its initial outbreak in 1997. We have designed multi-functional RNA interference (RNAi-based therapeutics targeting H5N1 that degrade viral mRNA via the RNAi pathway while at the same time augmenting the host antiviral response by inducing host type I interferon (IFN production. Moreover, we have identified two factors critical for maximising the immunostimulatory properties of short interfering (siRNAs in chicken cells (i mode of synthesis and (ii nucleoside sequence to augment the response to virus. The 5-bp nucleoside sequence 5'-UGUGU-3' is a key determinant in inducing high levels of expression of IFN-α, -β, -λ and interleukin 1-β in chicken cells. Positioning of this 5'-UGUGU-3' motif at the 5'-end of the sense strand of siRNAs, but not the 3'-end, resulted in a rapid and enhanced induction of type I IFN. An anti-H5N1 avian influenza siRNA directed against the PB1 gene (PB1-2257 tagged with 5'-UGUGU-3' induced type I IFN earlier and to a greater extent compared to a non-tagged PB1-2257. Tested against H5N1 in vitro, the tagged PB1-2257 was more effective than non-tagged PB1-2257. These data demonstrate the ability of an immunostimulatory motif to improve the performance of an RNAi-based antiviral, a finding that may influence the design of future RNAi-based anti-influenza therapeutics.

  11. Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Ji Hye; Shin, Bo Yeon; Han, Jae Yun; Kim, Mi Gwang; Wi, Ji Eun [College of Pharmacy, Chosun University, Gwangju, 501-759 (Korea, Republic of); Kim, Young Woo; Cho, Il Je; Kim, Sang Chan [Medical Research Center for Globalization of Herbal Formulation, College of Korean Medicine, Daegu Haany University, Gyeongsan 712-715 (Korea, Republic of); Shin, Sang Mi [College of Pharmacy, Chosun University, Gwangju, 501-759 (Korea, Republic of); Ki, Sung Hwan, E-mail: shki@chosun.ac.kr [College of Pharmacy, Chosun University, Gwangju, 501-759 (Korea, Republic of)

    2014-01-15

    Isorhamentin is a 3′-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes. - Highlights: • We investigated the effect of isorhamnetin on Nrf2 activation. • Isorhamnetin increased Nrf2

  12. Molecular characteristics and efficacy of 16D10 siRNAs in inhibiting root-knot nematode infection in transgenic grape hairy roots.

    Directory of Open Access Journals (Sweden)

    Yingzhen Yang

    Full Text Available Root-knot nematodes (RKNs infect many annual and perennial crops and are the most devastating soil-born pests in vineyards. To develop a biotech-based solution for controlling RKNs in grapes, we evaluated the efficacy of plant-derived RNA interference (RNAi silencing of a conserved RKN effector gene, 16D10, for nematode resistance in transgenic grape hairy roots. Two hairpin-based silencing constructs, containing a stem sequence of 42 bp (pART27-42 or 271 bp (pART27-271 of the 16D10 gene, were transformed into grape hairy roots and compared for their small interfering RNA (siRNA production and efficacy on suppression of nematode infection. Transgenic hairy root lines carrying either of the two RNAi constructs showed less susceptibility to nematode infection compared with control. Small RNA libraries from four pART27-42 and two pART27-271 hairy root lines were sequenced using an Illumina sequencing technology. The pART27-42 lines produced hundred times more 16D10-specific siRNAs than the pART27-271 lines. On average the 16D10 siRNA population had higher GC content than the 16D10 stem sequences in the RNAi constructs, supporting previous observation that plant dicer-like enzymes prefer GC-rich sequences as substrates for siRNA production. The stems of the 16D10 RNAi constructs were not equally processed into siRNAs. Several hot spots for siRNA production were found in similar positions of the hairpin stems in pART27-42 and pART27-271. Interestingly, stem sequences at the loop terminus produced more siRNAs than those at the stem base. Furthermore, the relative abundance of guide and passenger single-stranded RNAs from putative siRNA duplexes was largely correlated with their 5' end thermodynamic strength. This study demonstrated the feasibility of using a plant-derived RNAi approach for generation of novel nematode resistance in grapes and revealed several interesting molecular characteristics of transgene siRNAs important for optimizing plant RNAi

  13. Lipid nanoparticle siRNA treatment of Ebola-virus-Makona-infected nonhuman primates.

    Science.gov (United States)

    Thi, Emily P; Mire, Chad E; Lee, Amy C H; Geisbert, Joan B; Zhou, Joy Z; Agans, Krystle N; Snead, Nicholas M; Deer, Daniel J; Barnard, Trisha R; Fenton, Karla A; MacLachlan, Ian; Geisbert, Thomas W

    2015-05-21

    The current outbreak of Ebola virus in West Africa is unprecedented, causing more cases and fatalities than all previous outbreaks combined, and has yet to be controlled. Several post-exposure interventions have been employed under compassionate use to treat patients repatriated to Europe and the United States. However, the in vivo efficacy of these interventions against the new outbreak strain of Ebola virus is unknown. Here we show that lipid-nanoparticle-encapsulated short interfering RNAs (siRNAs) rapidly adapted to target the Makona outbreak strain of Ebola virus are able to protect 100% of rhesus monkeys against lethal challenge when treatment was initiated at 3 days after exposure while animals were viraemic and clinically ill. Although all infected animals showed evidence of advanced disease including abnormal haematology, blood chemistry and coagulopathy, siRNA-treated animals had milder clinical features and fully recovered, while the untreated control animals succumbed to the disease. These results represent the first, to our knowledge, successful demonstration of therapeutic anti-Ebola virus efficacy against the new outbreak strain in nonhuman primates and highlight the rapid development of lipid-nanoparticle-delivered siRNA as a countermeasure against this highly lethal human disease.

  14. Supramolecular Self-Assembly of Histidine-Capped-Dialkoxy-Anthracene: A Visible Light Triggered Platform for facile siRNA Delivery

    KAUST Repository

    Patil, Sachin

    2016-06-29

    Supramolecular self-assembly of histidine-capped-dialkoxy-anthracene (HDA) results in the formation of light responsive nanostructures.Single-crystal X-ray diffraction analysis of HDA shows two types of hydrogen bonding. The first hydrogen bond is established between the imidazole moieties while the second involves the oxygen atom of one amide group and the hydrogen atom of a second amide group. When protonated in acidic aqueous media, HDA successfully complexes siRNA yielding spherical nanostructures. This biocompatible platform controllably delivers siRNA with high efficacy upon visible light irradiation leading up to 90% of gene silencing in live cells.

  15. In vivo screening of modified siRNAs for non-specific antiviral effect in a small fish model: number and localization in the strands are important

    DEFF Research Database (Denmark)

    Schyth, Brian Dall; Bramsen, Jesper Bertram; Pakula, Malgorzata Maria;

    2012-01-01

    but often only examining the expression of specific immunologically relevant genes in selected cell populations typically blood cells from treated animals or humans. Assays using a relevant physiological state in biological models as read-out are not common. Here we use a fish model where the innate......, increase the antiviral effect of siRNAs. The applied fish model represents a potent tool for conducting fast but statistically and scientifically relevant evaluations of chemically optimized siRNAs with respect to non-specific antiviral effects in vivo....

  16. The Small Interfering RNA Pathway Is Not Essential for Wolbachia-Mediated Antiviral Protection in Drosophila melanogaster

    OpenAIRE

    Lauren M Hedges; Yamada, Ryuichi; O'Neill, Scott L; Karyn N. Johnson

    2012-01-01

    Wolbachia pipientis delays RNA virus-induced mortality in Drosophila spp. We investigated whether Wolbachia-mediated protection was dependent on the small interfering RNA (siRNA) pathway, a key antiviral defense. Compared to Wolbachia-free flies, virus-induced mortality was delayed in Wolbachia-infected flies with loss-of-function of siRNA pathway components, indicating that Wolbachia-mediated protection functions in the absence of the canonical siRNA pathway.

  17. Mechanism of Induction of Apoptosis by siRNA Targeting hTERT in HeLa Cells

    Institute of Scientific and Technical Information of China (English)

    WANG Jian; REN Chang-shan

    2008-01-01

    Objective:To investigate the molecular mechanism of induction of apoptosis by siRNA targeting human telomerase reverse transcriptase(hTERT)in HeLa Cells.Methods:HeLa cells were transfected with siRNAs by liposome method.RT-PCR was used to examine mRNA levels of hTERT in HeLa cells.Microarray assay was adopted to explore the transcriptional profiling of apoptosis associated genes.The protein levels of hTERT,TRAIL,Bcl-2,and cytoplasm Cyt C were detected by Western Blotting.The apoptosis rate was determined by flow cytometry using PI staining.Relative activity of Caspase-3 and Caspase-8 was measured by colorimetric assay.Results:The siRNA targeting hTERT suppressed the expression of hTERT gene significantly.Forty-eight hours after transfection,the expression level of TRAIL was increased,the expression level of Bcl-2 was decreased,the releasing of Cyt C was enhanced,the activation of Caspase-3 was increased and the apoptosis rate was increased.Conclusion:hTERT-siRNA induces apoptosis of HeLa cells via activating mitochondrial signal transduction pathway.

  18. Protection of stem cell-derived lymphocytes in a primate AIDS gene therapy model after in vivo selection.

    Directory of Open Access Journals (Sweden)

    Grant D Trobridge

    Full Text Available BACKGROUND: There is currently no effective AIDS vaccine, emphasizing the importance of developing alternative therapies. Recently, a patient was successfully transplanted with allogeneic, naturally resistant CCR5-negative (CCR5Delta32 cells, setting the stage for transplantation of naturally resistant, or genetically modified stem cells as a viable therapy for AIDS. Hematopoietic stem cell (HSC gene therapy using vectors that express various anti-HIV transgenes has also been attempted in clinical trials, but inefficient gene transfer in these studies has severely limited the potential of this approach. Here we evaluated HSC gene transfer of an anti-HIV vector in the pigtailed macaque (Macaca nemestrina model, which closely models human transplantation. METHODS AND FINDINGS: We used lentiviral vectors that inhibited both HIV-1 and simian immunodeficiency virus (SIV/HIV-1 (SHIV chimera virus infection, and also expressed a P140K mutant methylguanine methyltransferase (MGMT transgene to select gene-modified cells by adding chemotherapy drugs. Following transplantation and MGMT-mediated selection we demonstrated transgene expression in over 7% of stem-cell derived lymphocytes. The high marking levels allowed us to demonstrate protection from SHIV in lymphocytes derived from gene-modified macaque long-term repopulating cells that expressed an HIV-1 fusion inhibitor. We observed a statistically significant 4-fold increase of gene-modified cells after challenge of lymphocytes from one macaque that received stem cells transduced with an anti-HIV vector (p<0.02, Student's t-test, but not in lymphocytes from a macaque that received a control vector. We also established a competitive repopulation assay in a second macaque for preclinical testing of promising anti-HIV vectors. The vectors we used were HIV-based and thus efficiently transduce human cells, and the transgenes we used target HIV-1 genes that are also in SHIV, so our findings can be rapidly

  19. Effect of siRNAs on HSV-1 Plaque Formation and Relative Expression Levels of RR mRNA

    Institute of Scientific and Technical Information of China (English)

    Zhe Ren; Pei-zhuo Zhang; Shen Li; Qiao-li Wang; Yang-fei Xiang; Yun-xia Cui; Yi-fei Wang; Ren-bin Qi; Da-xiang Lu; Shu-min Zhang

    2011-01-01

    RNA interference(RNAi)is a process by which introduced small interfering RNA(siRNA)can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1(HSV-1)RR is composed of two distinct homodimeric subunits encoded by UL39 and UL40, respectively. In this study, we applied siRNAs targeting the UL39 and UL40 genes of HSV-1. We showed that synthetic siRNA silenced effectively and specifically UL39 and UL40 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.

  20. Delivery of multiple siRNAs using lipid-coated PLGA nanoparticles for treatment of prostate cancer.

    Science.gov (United States)

    Hasan, Warefta; Chu, Kevin; Gullapalli, Anuradha; Dunn, Stuart S; Enlow, Elizabeth M; Luft, J Christopher; Tian, Shaomin; Napier, Mary E; Pohlhaus, Patrick D; Rolland, Jason P; DeSimone, Joseph M

    2012-01-11

    Nanotechnology can provide a critical advantage in developing strategies for cancer management and treatment by helping to improve the safety and efficacy of novel therapeutic delivery vehicles. This paper reports the fabrication of poly(lactic acid-co-glycolic acid)/siRNA nanoparticles coated with lipids for use as prostate cancer therapeutics made via a unique soft lithography particle molding process called Particle Replication In Nonwetting Templates (PRINT). The PRINT process enables high encapsulation efficiency of siRNA into neutral and monodisperse PLGA particles (32-46% encapsulation efficiency). Lipid-coated PLGA/siRNA PRINT particles were used to deliver therapeutic siRNA in vitro to knockdown genes relevant to prostate cancer.

  1. ROBUST HYPERPARAMETER ESTIMATION PROTECTS AGAINST HYPERVARIABLE GENES AND IMPROVES POWER TO DETECT DIFFERENTIAL EXPRESSION

    Science.gov (United States)

    Phipson, Belinda; Lee, Stanley; Majewski, Ian J.; Alexander, Warren S.; Smyth, Gordon K.

    2017-01-01

    One of the most common analysis tasks in genomic research is to identify genes that are differentially expressed (DE) between experimental conditions. Empirical Bayes (EB) statistical tests using moderated genewise variances have been very effective for this purpose, especially when the number of biological replicate samples is small. The EB procedures can however be heavily influenced by a small number of genes with very large or very small variances. This article improves the differential expression tests by robustifying the hyperparameter estimation procedure. The robust procedure has the effect of decreasing the informativeness of the prior distribution for outlier genes while increasing its informativeness for other genes. This effect has the double benefit of reducing the chance that hypervariable genes will be spuriously identified as DE while increasing statistical power for the main body of genes. The robust EB algorithm is fast and numerically stable. The procedure allows exact small-sample null distributions for the test statistics and reduces exactly to the original EB procedure when no outlier genes are present. Simulations show that the robustified tests have similar performance to the original tests in the absence of outlier genes but have greater power and robustness when outliers are present. The article includes case studies for which the robust method correctly identifies and downweights genes associated with hidden covariates and detects more genes likely to be scientifically relevant to the experimental conditions. The new procedure is implemented in the limma software package freely available from the Bioconductor repository.

  2. Development of allele-specific therapeutic siRNA in Meesmann epithelial corneal dystrophy.

    Directory of Open Access Journals (Sweden)

    Haihui Liao

    Full Text Available BACKGROUND: Meesmann epithelial corneal dystrophy (MECD is an inherited eye disorder caused by dominant-negative mutations in either keratins K3 or K12, leading to mechanical fragility of the anterior corneal epithelium, the outermost covering of the eye. Typically, patients suffer from lifelong irritation of the eye and/or photophobia but rarely lose visual acuity; however, some individuals are severely affected, with corneal scarring requiring transplant surgery. At present no treatment exists which addresses the underlying pathology of corneal dystrophy. The aim of this study was to design and assess the efficacy and potency of an allele-specific siRNA approach as a future treatment for MECD. METHODS AND FINDINGS: We studied a family with a consistently severe phenotype where all affected persons were shown to carry heterozygous missense mutation Leu132Pro in the KRT12 gene. Using a cell-culture assay of keratin filament formation, mutation Leu132Pro was shown to be significantly more disruptive than the most common mutation, Arg135Thr, which is associated with typical, mild MECD. A siRNA sequence walk identified a number of potent inhibitors for the mutant allele, which had no appreciable effect on wild-type K12. The most specific and potent inhibitors were shown to completely block mutant K12 protein expression with negligible effect on wild-type K12 or other closely related keratins. Cells transfected with wild-type K12-EGFP construct show a predominantly normal keratin filament formation with only 5% aggregate formation, while transfection with mutant K12-EGFP construct resulted in a significantly higher percentage of keratin aggregates (41.75%; p<0.001 with 95% confidence limits. The lead siRNA inhibitor significantly rescued the ability to form keratin filaments (74.75% of the cells contained normal keratin filaments; p<0.001 with 95% confidence limits. CONCLUSIONS: This study demonstrates that it is feasible to design highly potent siRNA

  3. Subtle changes to polymer structure and degradation mechanism enable highly effective nanoparticles for siRNA and DNA delivery to human brain cancer

    Science.gov (United States)

    Tzeng, Stephany Y.

    2013-01-01

    Polymeric materials can be used to deliver nucleic acids such as DNA plasmids and siRNA, but often have low efficacy in human cells. To improve gene delivery, we synthesized an array of over 70 hydrolytically degradable and bioreducible poly(beta-amino ester)s and evaluated properties of over 200 nanoparticle formulations fabricated from these biomaterials. We determined the effect of different polymer structures on the delivery of nucleic acids of different structures and sizes, including siRNA, linear DNA, and circular DNAs (1.8–26 kb). Significantly, leading hydrolytically degradable polymeric nanoparticles delivered DNA to 90±2% of primary human glioblastoma cells with Polymer molecular weight was a driving factor of transfection efficacy for some polymer structures (correlation of r2=0.63) but had no influence on transfection for other structures (r2=0.01). Polymers with a reducible cystamine functional group dramatically improved siRNA delivery by facilitating quick release while generally decreasing DNA delivery compared with non-reducible counterparts (p<0.01). Other material properties facilitated DNA delivery compared to siRNA delivery or increased delivery of both DNA and siRNA. PMID:23184674

  4. Targeted tumor-penetrating siRNA nanocomplexes for credentialing the ovarian cancer oncogene ID4.

    Science.gov (United States)

    Ren, Yin; Cheung, Hiu Wing; von Maltzhan, Geoffrey; Agrawal, Amit; Cowley, Glenn S; Weir, Barbara A; Boehm, Jesse S; Tamayo, Pablo; Karst, Alison M; Liu, Joyce F; Hirsch, Michelle S; Mesirov, Jill P; Drapkin, Ronny; Root, David E; Lo, Justin; Fogal, Valentina; Ruoslahti, Erkki; Hahn, William C; Bhatia, Sangeeta N

    2012-08-15

    The comprehensive characterization of a large number of cancer genomes will eventually lead to a compendium of genetic alterations in specific cancers. Unfortunately, the number and complexity of identified alterations complicate endeavors to identify biologically relevant mutations critical for tumor maintenance because many of these targets are not amenable to manipulation by small molecules or antibodies. RNA interference provides a direct way to study putative cancer targets; however, specific delivery of therapeutics to the tumor parenchyma remains an intractable problem. We describe a platform for the discovery and initial validation of cancer targets, composed of a systematic effort to identify amplified and essential genes in human cancer cell lines and tumors partnered with a novel modular delivery technology. We developed a tumor-penetrating nanocomplex (TPN) that comprised small interfering RNA (siRNA) complexed with a tandem tumor-penetrating and membrane-translocating peptide, which enabled the specific delivery of siRNA deep into the tumor parenchyma. We used TPN in vivo to evaluate inhibitor of DNA binding 4 (ID4) as a novel oncogene. Treatment of ovarian tumor-bearing mice with ID4-specific TPN suppressed growth of established tumors and significantly improved survival. These observations not only credential ID4 as an oncogene in 32% of high-grade ovarian cancers but also provide a framework for the identification, validation, and understanding of potential therapeutic cancer targets.

  5. Dual targeted therapy with p53 siRNA and Epigallocatechingallate in a triple negative breast cancer cell model.

    Directory of Open Access Journals (Sweden)

    Cornelia Braicu

    Full Text Available Triple-negative breast cancer (TNBC is a highly aggressive phenotype that is resistant to standard therapy. Thus, the development of alternative therapeutic strategies for TNBC is essential. The purpose of our in vitro study was to evaluate the impact of p53 gene silencing in conjunction with the administration of a natural compound, epigallocatechingallate (EGCG. RT2Profiler PCR Array technology was used to evaluate the impact of dual treatment on the main genes involved in apoptosis in the Hs578T cell culture model of TNBC. Gene expression analysis revealed 28 genes were significantly altered (16 upregulated and 12 downregulated in response to combined p53 siRNA and EGCG treatment. Further analysis revealed that p53 siRNA and EGCG dual therapy leads to the activation of pro-apoptotic genes and the inhibition of pro-survival genes, autophagy, and cell network formation. These results indicate that this dual therapy targets both the apoptotic and angiogenic pathways, which may improve treatment effectiveness for tumors resistant to conventional treatment.

  6. Genome-wide identification of microRNA and siRNA responsive to endophytic beneficial diazotrophic bacteria in maize.

    Science.gov (United States)

    Thiebaut, Flávia; Rojas, Cristian A; Grativol, Clícia; Motta, Mariana Romeiro; Vieira, Tauan; Regulski, Michael; Martienssen, Robert A; Farinelli, Laurent; Hemerly, Adriana S; Ferreira, Paulo C G

    2014-09-06

    Small RNA (sRNA) has been described as a regulator of gene expression. In order to understand the role of maize sRNA (Zea mays-hybrid UENF 506-8) during association with endophytic nitrogen-fixing bacteria, we analyzed the sRNA regulated by its association with two diazotrophic bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. Deep sequencing analysis was done with RNA extracted from plants inoculated with H. seropedicae, allowing the identification of miRNA and siRNA. A total of 25 conserved miRNA families and 15 novel miRNAs were identified. A dynamic regulation in response to inoculation was also observed. A hypothetical model involving copper-miRNA is proposed, emphasizing the fact that the up-regulation of miR397, miR398, miR408 and miR528, which is followed by inhibition of their targets, can facilitate association with diazotrophic bacteria. Similar expression patterns were observed in samples inoculated with A. brasilense. Moreover, novel miRNA and siRNA were classified in the Transposable Elements (TE) database, and an enrichment of siRNA aligned with TE was observed in the inoculated samples. In addition, an increase in 24-nt siRNA mapping to genes was observed, which was correlated with an increase in methylation of the coding regions and a subsequent reduction in transcription. Our results show that maize has RNA-based silencing mechanisms that can trigger specific responses when plants interact with beneficial endophytic diazotrophic bacteria. Our findings suggest important roles for sRNA regulation in maize, and probably in other plants, during association with diazotrophic bacteria, emphasizing the up-regulation of Cu-miRNA.

  7. Reversal of multidrug resistance by small interfering RNA (siRNA) in doxorubicin-resistant MCF-7 breast cancer cells.

    Science.gov (United States)

    Dönmez, Yaprak; Gündüz, Ufuk

    2011-03-01

    Resistance to anticancer drugs is a serious obstacle to cancer chemotherapy. A common form of multidrug resistance (MDR) is caused by the overexpression of transmembrane transporter proteins P-glycoprotein (P-gp) and multidrug resistance-associated protein-1 (MRP1), encoded by MDR1 and MRP1 genes, respectively. These proteins lead to reduced intracellular drug concentration and decreased cytotoxicity by means of their ability to pump the drugs out of the cells. Breast cancer tumor resistance is mainly associated with overexpression of P-gp/MDR1. Although some chemical MDR modulators aim to overcome MDR by interfering functioning of P-gp, their toxicities limit their usage in clinics. Consequently, RNA interference mediated sequence specific inhibition of the expression of P-gp/MDR1 mRNA may be an efficient tool to reverse MDR phenotype and increase the success of chemotherapy. Aim of this study was resensitizing doxorubicin-resistant breast cancer cells to anticancer agent doxorubicin by selective downregulation of P-gp/MDR1 mRNA. The effect of the selected MDR1 siRNA, and MRP1 expression after MDR1 silencing was determined by qPCR analysis. Intracellular drug accumulation and localization was investigated by confocal laser scanning microscopy after treatment with MDR1 siRNA. XTT cell proliferation assay was performed to determine the effect of MDR1 silencing on doxorubicin sensitivity. The results demonstrated that approximately 90% gene silencing occurred by the selected siRNA targeting MDR1 mRNA. However, the level of MRP1 mRNA did not change after MDR1 downregulation. Silencing of P-gp encoding MDR1 gene resulted in almost complete restoration of the intracellular doxorubicin accumulation and relocalization of the drug in the nuclei. Introduction of siRNA resulted in about 70% resensitization to doxorubicin. Selected siRNA duplex was shown to effectively inhibit MDR1 gene expression, restore doxorubicin accumulation and localization, and enhance

  8. Synaptic protection in the brain of WldS mice occurs independently of age but is sensitive to gene-dose.

    Directory of Open Access Journals (Sweden)

    Ann K Wright

    Full Text Available BACKGROUND: Disruption of synaptic connectivity is a significant early event in many neurodegenerative conditions affecting the aging CNS, including Alzheimer's disease and Parkinson's disease. Therapeutic approaches that protect synapses from degeneration in the aging brain offer the potential to slow or halt the progression of such conditions. A range of animal models expressing the slow Wallerian Degeneration (Wld(S gene show robust neuroprotection of synapses and axons from a wide variety of traumatic and genetic neurodegenerative stimuli in both the central and peripheral nervous systems, raising that possibility that Wld(S may be useful as a neuroprotective agent in diseases with synaptic pathology. However, previous studies of neuromuscular junctions revealed significant negative effects of increasing age and positive effects of gene-dose on Wld(S-mediated synaptic protection in the peripheral nervous system, raising doubts as to whether Wld(S is capable of directly conferring synapse protection in the aging brain. METHODOLOGY/PRINCIPAL FINDINGS: We examined the influence of age and gene-dose on synaptic protection in the brain of mice expressing the Wld(S gene using an established cortical lesion model to induce synaptic degeneration in the striatum. Synaptic protection was found to be sensitive to Wld(S gene-dose, with heterozygous Wld(S mice showing approximately half the level of protection observed in homozygous Wld(S mice. Increasing age had no influence on levels of synaptic protection. In contrast to previous findings in the periphery, synapses in the brain of old Wld(S mice were just as strongly protected as those in young mice. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that Wld(S-mediated synaptic protection in the CNS occurs independently of age, but is sensitive to gene dose. This suggests that the Wld(S gene, and in particular its downstream endogenous effector pathways, may be potentially useful therapeutic agents for

  9. In vivo siRNA delivery system for targeting to the liver by poly-l-glutamic acid-coated lipoplex

    Directory of Open Access Journals (Sweden)

    Yoshiyuki Hattori

    2014-01-01

    Full Text Available In this study, we developed anionic polymer-coated liposome/siRNA complexes (lipoplexes with chondroitin sulfate C (CS, poly-l-glutamic acid (PGA and poly-aspartic acid (PAA for siRNA delivery by intravenous injection, and evaluated the biodistribution and gene silencing effect in mice. The sizes of CS-, PGA- and PAA-coated lipoplexes were about 200 nm and their ζ-potentials were negative. CS-, PGA- and PAA-coated lipoplexes did not induce agglutination after mixing with erythrocytes. In terms of biodistribution, siRNAs after intravenous administration of cationic lipoplexes were largely observed in the lungs, but those of CS-, PGA- and PAA-coated lipoplexes were in both the liver and the kidneys, indicating that siRNA might be partially released from the anionic polymer-coated lipoplexes in the blood circulation and accumulate in the kidney, although the lipoplexes can prevent the agglutination with blood components. To increase the association between siRNA and cationic liposome, we used cholesterol-modified siRNA (siRNA-Chol for preparation of the lipoplexes. When CS-, PGA- and PAA-coated lipoplexes of siRNA-Chol were injected into mice, siRNA-Chol was mainly observed in the liver, not in the kidneys. In terms of the suppression of gene expression in vivo, apolipoprotein B (ApoB mRNA in the liver was significantly reduced 48 h after single intravenous injection of PGA-coated lipoplex of ApoB siRNA-Chol (2.5 mg siRNA/kg, but not cationic, CS- and PAA-coated lipoplexes. In terms of toxicity after intravenous injection, CS-, PGA- and PAA-coated lipoplexes did not increase GOT and GPT concentrations in blood. From these findings, PGA coatings for cationic lipoplex of siRNA-Chol might produce a systemic vector of siRNA to the liver.

  10. The Armc10/SVH gene: genome context, regulation of mitochondrial dynamics and protection against Aβ-induced mitochondrial fragmentation

    Science.gov (United States)

    Serrat, R; Mirra, S; Figueiro-Silva, J; Navas-Pérez, E; Quevedo, M; López-Doménech, G; Podlesniy, P; Ulloa, F; Garcia-Fernàndez, J; Trullas, R; Soriano, E

    2014-01-01

    Mitochondrial function and dynamics are essential for neurotransmission, neural function and neuronal viability. Recently, we showed that the eutherian-specific Armcx gene cluster (Armcx1–6 genes), located in the X chromosome, encodes for a new family of proteins that localise to mitochondria, regulating mitochondrial trafficking. The Armcx gene cluster evolved by retrotransposition of the Armc10 gene mRNA, which is present in all vertebrates and is considered to be the ancestor gene. Here we investigate the genomic organisation, mitochondrial functions and putative neuroprotective role of the Armc10 ancestor gene. The genomic context of the Armc10 locus shows considerable syntenic conservation among vertebrates, and sequence comparisons and CHIP-data suggest the presence of at least three conserved enhancers. We also show that the Armc10 protein localises to mitochondria and that it is highly expressed in the brain. Furthermore, we show that Armc10 levels regulate mitochondrial trafficking in neurons, but not mitochondrial aggregation, by controlling the number of moving mitochondria. We further demonstrate that the Armc10 protein interacts with the KIF5/Miro1-2/Trak2 trafficking complex. Finally, we show that overexpression of Armc10 in neurons prevents Aβ-induced mitochondrial fission and neuronal death. Our data suggest both conserved and differential roles of the Armc10/Armcx gene family in regulating mitochondrial dynamics in neurons, and underscore a protective effect of the Armc10 gene against Aβ-induced toxicity. Overall, our findings support a further degree of regulation of mitochondrial dynamics in the brain of more evolved mammals. PMID:24722288

  11. Analyses of a Glycine max Degradome Library Identify microRNA Targets and MicroRNAs that Trigger Secondary SiRNA Biogenesis

    Institute of Scientific and Technical Information of China (English)

    Zheng Hu; Qiyan Jiang; Zhiyong Ni; Rui Chen; Shuo Xu; Hui Zhang

    2013-01-01

    Plant microRNAs (miRNAs) regulate gene expression mainly by guiding cleavage of target mRNAs.In this study,a degradome library constructed from different soybean (Glycine max (L.) Merr.) tissues was deep-sequenced.428 potential targets of small interfering RNAs and 25 novel miRNA families were identified.A total of 211 potential miRNA targets,including 174 conserved miRNA targets and 37 soybeanspecific miRNA targets,were identified.Among them,121 targets were first discovered in soybean.The signature distribution of soybean primary miRNAs (pri-miRNAs) showed that most pri-miRNAs had the characteristic pattern of Dicer processing.The biogenesis of TAS3 small interfering RNAs (siRNAs) was conserved in soybean,and nine Auxin Response Factors were identified as TAS3 siRNA targets.Twentythree miRNA targets produced secondary small interfering RNAs (siRNAs) in soybean.These targets were guided by five miRNAs:gma-miR393,gma-miR1508,gma-miR1510,gma-miR1514,and novel-11.Multiple targets of these secondary siRNAs were detected.These 23 miRNA targets may be the putative novel TAS genes in soybean.Global identification of miRNA targets and potential novel TAS genes will contribute to research on the functions of miRNAs in soybean.

  12. Analyses of a Glycine max degradome library identify microRNA targets and microRNAs that trigger secondary siRNA biogenesis.

    Science.gov (United States)

    Hu, Zheng; Jiang, Qiyan; Ni, Zhiyong; Chen, Rui; Xu, Shuo; Zhang, Hui

    2013-02-01

    Plant microRNAs (miRNAs) regulate gene expression mainly by guiding cleavage of target mRNAs. In this study, a degradome library constructed from different soybean (Glycine max (L.) Merr.) tissues was deep-sequenced. 428 potential targets of small interfering RNAs and 25 novel miRNA families were identified. A total of 211 potential miRNA targets, including 174 conserved miRNA targets and 37 soybean-specific miRNA targets, were identified. Among them, 121 targets were first discovered in soybean. The signature distribution of soybean primary miRNAs (pri-miRNAs) showed that most pri-miRNAs had the characteristic pattern of Dicer processing. The biogenesis of TAS3 small interfering RNAs (siRNAs) was conserved in soybean, and nine Auxin Response Factors were identified as TAS3 siRNA targets. Twenty-three miRNA targets produced secondary small interfering RNAs (siRNAs) in soybean. These targets were guided by five miRNAs: gma-miR393, gma-miR1508, gma-miR1510, gma-miR1514, and novel-11. Multiple targets of these secondary siRNAs were detected. These 23 miRNA targets may be the putative novel TAS genes in soybean. Global identification of miRNA targets and potential novel TAS genes will contribute to research on the functions of miRNAs in soybean.

  13. The siRNA targeted to mdr1b and mdr1a mRNAs in vivo sensitizes murine lymphosarcoma to chemotherapy

    Directory of Open Access Journals (Sweden)

    Vlassov Valentin V

    2010-05-01

    Full Text Available Abstract Background One of the main obstacles for successful cancer polychemotherapy is multiple drug resistance phenotype (MDR acquired by tumor cells. Currently, RNA interference represents a perspective strategy to overcome MDR via silencing the genes involved in development of this deleterious phenotype (genes of ABC transporters, antiapoptotic genes, etc.. Methods In this study, we used the siRNAs targeted to mdr1b, mdr1a, and bcl-2 mRNAs to reverse the MDR of tumors and increase tumor sensitivity to chemotherapeutics. The therapy consisting in ex vivo or in vivo application of mdr1b/1a siRNA followed by cyclophosphamide administration was studied in the mice bearing RLS40 lymphosarcoma, displaying high resistance to a wide range of cytostatics. Results Our data show that a single application of mdr1b/1a siRNA followed by treatment with conventionally used cytostatics results in more than threefold decrease in tumor size as compared with the control animals receiving only cytostatics. Conclusions In perspective, mdr1b/1a siRNA may become a well-reasoned adjuvant tool in the therapy of MDR malignancies.

  14. Anti-gp120 minibody gene transfer to female genital epithelial cells protects against HIV-1 virus challenge in vitro.

    Directory of Open Access Journals (Sweden)

    Ussama M Abdel-Motal

    Full Text Available BACKGROUND: Although cervico-vaginal epithelial cells of the female lower genital tract provide the initial defense system against HIV-1 infection, the protection is sometimes incomplete. Thus, enhancing anti-HIV-1 humoral immunity at the mucosal cell surface by local expression of anti-HIV-1 broadly neutralizing antibodies (BnAb that block HIV-1 entry would provide an important new intervention that could slow the spread of HIV/AIDS. METHODS AND FINDINGS: This study tested the hypothesis that adeno-associated virus (AAV-BnAb gene transfer to cervico-vaginal epithelial cells will lead to protection against HIV-1. Accordingly, a recombinant AAV vector that encodes human b12 anti-HIV gp120 BnAb as a single-chain variable fragment Fc fusion (scFvFc, or "minibody" was constructed. The secreted b12 minibody was shown to be biologically functional in binding to virus envelope protein, neutralizing HIV-1 and importantly, blocking transfer and infectivity of HIV-1(bal in an organotypic human vaginal epithelial cell (VEC model. Furthermore, cervico-vaginal epithelial stem cells were found to be efficiently transduced by the optimal AAV serotype mediated expression of GFP. CONCLUSION: This study provides the foundation for a novel microbicide strategy to protect against sexual transmission of HIV-1 by AAV transfer of broadly neutralizing antibody genes to cervico-vaginal epithelial stem cells that could replenish b12 BnAb secreting cells through multiple menstrual cycles.

  15. Local administration of siRNA through Microneedle: Optimization, Bio-distribution, Tumor Suppression and Toxicity

    Science.gov (United States)

    Tang, Tao; Deng, Yan; Chen, Jiao; Zhao, Yi; Yue, Ruifeng; Choy, Kwong Wai; Wang, Chi Chiu; Du, Quan; Xu, Yan; Han, Linxiao; Chung, Tony Kwok Hung

    2016-07-01

    Although RNA interference may become a novel therapeutic approach for cancer treatment, target-site accumulation of siRNA to achieve therapeutic dosage will be a major problem. Microneedle represents a better way to deliver siRNAs and we have evaluated for the first time the capability of a silicon microneedle array for delivery of Gapdh siRNA to the skin in vivo and the results showed that the microneedle arrays could effectively deliver siRNA to relevant regions of the skin noninvasively. For the further study in this field, we evaluated the efficacy of the injectable microneedle device for local delivery of siRNA to the mouse xenograft. The results presented here indicate that local administration of siRNA through injectable microneedle could effectively deliver siRNA into the tumor region, and inhibit tumor progression without major adverse effects.

  16. Long-term skeletal muscle protection after gene transfer in a mouse model of LGMD-2D.

    Science.gov (United States)

    Pacak, Christina A; Walter, Glenn A; Gaidosh, Gabe; Bryant, Nathan; Lewis, Melissa A; Germain, Sean; Mah, Cathryn S; Campbell, Kevin P; Byrne, Barry J

    2007-10-01

    Limb girdle muscular dystrophy (LGMD) describes a group of inherited diseases resulting from mutations in genes encoding proteins involved in maintaining skeletal muscle membrane stability. LGMD type-2D is caused by mutations in alpha-sarcoglycan (sgca). Here we describe muscle-specific gene delivery of the human sgca gene into dystrophic muscle using an adeno-associated virus 1 (AAV1) capsid and creatine kinase promoter. Delivery of this construct to adult sgca(-/-) mice resulted in localization of the sarcoglycan complex to the sarcolemma and a reduction in muscle fiber damage. Sgca expression prevented disease progression as observed in vivo by T(2)-weighted magnetic resonance imaging (MRI) and confirmed in vitro by decreased Evan's blue dye accumulation. The ability of recombinant AAV-mediated gene delivery to restore normal muscle mechanical properties in sgca(-/-) mice was verified by in vitro force mechanics on isolated extensor digitorum longus (EDL) muscles, with a decrease in passive resistance to stretch as compared with untreated controls. In summary, AAV/AAV-sgca gene transfer provides long-term muscle protection from LGMD and can be non-invasively evaluated using magnetic resonance imaging.

  17. Cancer-targeted BikDD gene therapy elicits protective antitumor immunity against lung cancer.

    Science.gov (United States)

    Sher, Yuh-Pyng; Liu, Shih-Jen; Chang, Chun-Mien; Lien, Shu-Pei; Chen, Chien-Hua; Han, Zhenbo; Li, Long-Yuan; Chen, Jin-Shing; Wu, Cheng-Wen; Hung, Mien-Chie

    2011-04-01

    Targeted cancer-specific gene therapy is a promising strategy for treating metastatic lung cancer, which is a leading cause of lung cancer-related deaths. Previously, we developed a cancer-targeted gene therapy expression system with high tumor specificity and strong activity that selectively induced lung cancer cell killing without affecting normal cells in immunocompromised mice. Here, we found this cancer-targeted gene therapy, SV-BikDD, composed of the survivin promoter in the VP16-GAL4-WPRE integrated systemic amplifier system to drive the apoptotic gene BikDD, not only caused cytotoxic effects in cancer cells but also elicited a cancer-specific cytotoxic T lymphocyte response to synergistically increase the therapeutic effect and further develop an effective systemic antitumoral immunity against rechallenges of tumorigenic dose of parental tumor cells inoculated at distant sites in immunocompetent mice. In addition, this cancer-targeted gene therapy does not elicit an immune response against normal tissues, but CMV-BikDD treatment does. The therapeutic vector could also induce proinflammatory cytokines to activate innate immunity and provide some benefits in antitumor gene therapy. Thus, this study provides a promising strategy with benefit of antitumoral immune response worthy of further development in clinical trials for treating lung cancer via cancer-targeted gene therapy.

  18. Protective effects of L-selenomethionine on space radiation induced changes in gene expression.

    Science.gov (United States)

    Stewart, J; Ko, Y-H; Kennedy, A R

    2007-06-01

    Ionizing radiation can produce adverse biological effects in astronauts during space travel. Of particular concern are the types of radiation from highly energetic, heavy, charged particles known as HZE particles. The aims of our studies are to characterize HZE particle radiation induced biological effects and evaluate the effects of L-selenomethionine (SeM) on these adverse biological effects. In this study, microarray technology was used to measure HZE radiation induced changes in gene expression, as well as to evaluate modulation of these changes by SeM. Human thyroid epithelial cells (HTori-3) were irradiated (1 GeV/n iron ions) in the presence or in the absence of 5 microM SeM. At 6 h post-irradiation, all cells were harvested for RNA isolation. Gene Chip U133Av2 from Affymetrix was used for the analysis of gene expression, and ANOVA and EASE were used for a determination of the genes and biological processes whose differential expression is statistically significant. Results of this microarray study indicate that exposure to small doses of radiation from HZE particles, 10 and 20 cGy from iron ions, induces statistically significant differential expression of 196 and 610 genes, respectively. In the presence of SeM, differential expression of 77 out of 196 genes (exposure to 10 cGy) and 336 out of 610 genes (exposure to 20 cGy) is abolished. In the presence or in the absence of SeM, radiation from HZE particles induces differential expression of genes whose products have roles in the induction of G1/S arrest during the mitotic cell cycle, as well as heat shock proteins. Some of the genes, whose expressions were affected by radiation from HZE particles and were unchanged in irradiated cells treated with SeM, have been shown to have altered expression levels in cancer cells. The conclusions of this report are that radiation from HZE particles can induce differential expression of many genes, some of which are known to play roles in the same processes that have

  19. The Mx1 gene protects mice against the pandemic 1918 and highly lethal human H5N1 influenza viruses.

    Science.gov (United States)

    Tumpey, Terrence M; Szretter, Kristy J; Van Hoeven, Neal; Katz, Jacqueline M; Kochs, Georg; Haller, Otto; García-Sastre, Adolfo; Staeheli, Peter

    2007-10-01

    Mice carrying a wild-type Mx1 gene (Mx1+/+) differ from standard laboratory mice (Mx1-/-) in being highly resistant to infection with common laboratory strains of influenza A virus. We report that Mx1 also protects mice against the pandemic human 1918 influenza virus and a highly lethal human H5N1 strain from Vietnam. Resistance to H5N1 of Mx1+/+ but not Mx1-/- mice was enhanced if the animals were treated with a single dose of exogenous alpha interferon before infection. Thus, the interferon-induced resistance factor Mx1 represents a key component of the murine innate immune system that mediates protection against epidemic and pandemic influenza viruses.

  20. Identification of Novel Fibrosis Modifiers by In Vivo siRNA Silencing

    Directory of Open Access Journals (Sweden)

    Elisabeth H. Vollmann

    2017-06-01

    Full Text Available Fibrotic diseases contribute to 45% of deaths in the industrialized world, and therefore a better understanding of the pathophysiological mechanisms underlying tissue fibrosis is sorely needed. We aimed to identify novel modifiers of tissue fibrosis expressed by myofibroblasts and their progenitors in their disease microenvironment through RNA silencing in vivo. We leveraged novel biology, targeting genes upregulated during liver and kidney fibrosis in this cell lineage, and employed small interfering RNA (siRNA-formulated lipid nanoparticles technology to silence these genes in carbon-tetrachloride-induced liver fibrosis in mice. We identified five genes, Egr2, Atp1a2, Fkbp10, Fstl1, and Has2, which modified fibrogenesis based on their silencing, resulting in reduced Col1a1 mRNA levels and collagen accumulation in the liver. These genes fell into different groups based on the effects of their silencing on a transcriptional mini-array and histological outcomes. Silencing of Egr2 had the broadest effects in vivo and also reduced fibrogenic gene expression in a human fibroblast cell line. Prior to our study, Egr2, Atp1a2, and Fkbp10 had not been functionally validated in fibrosis in vivo. Thus, our results provide a major advance over the existing knowledge of fibrogenic pathways. Our study is the first example of a targeted siRNA assay to identify novel fibrosis modifiers in vivo.

  1. Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA.

    Science.gov (United States)

    Zang, Xinlong; Ding, Huaiwei; Zhao, Xiufeng; Li, Xiaowei; Du, Zhouqi; Hu, Haiyang; Qiao, Mingxi; Chen, Dawei; Deng, Yuihui; Zhao, Xiuli

    2016-01-01

    Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Here, a pH-sensitive cholesterol-Schiff base-polyethylene glycol (Chol-SIB-PEG)-modified cationic liposome-siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph), was developed as an efficient nonviral siRNA delivery system. Chol-SIB-PEG was successfully synthesized and confirmed with FTIR and (1)H-NMR. An Eph-PEG-SIB-Chol-modified liposome-siRNA complex (EPSLR) was prepared and characterized by size, zeta potential, gel retardation, and encapsulation efficiency. Electrophoresis results showed that EPSLR was resistant to heparin replacement and protected siRNA from fetal bovine serum digestion. EPSLR exhibited only minor cytotoxicity in MCF-7/ADR cells. The results of flow cytometry and confocal laser scanning microscopy suggested that EPSLR enhanced siRNA transfection in MCF-7/ADR cells. Intracellular distribution experiment revealed that EPSLR could escape from the endo-lysosomal organelle and release siRNA into cytoplasm at 4 hours posttransfection. Western blot experiment demonstrated that EPSLR was able to significantly reduce the levels of MDR1 protein in MCF-7/ADR cells. The in vivo study of DIR-labeled complexes in mice bearing MCF-7/ADR tumor indicated that EPSLR could reach the tumor site rather than other organs more effectively. All these results demonstrate that EPSLR has much potential for effective siRNA delivery and may facilitate its therapeutic application.

  2. Lactobacillus zeae protects Caenorhabditis elegans from enterotoxigenic Escherichia coli-caused death by inhibiting enterotoxin gene expression of the pathogen.

    Directory of Open Access Journals (Sweden)

    Mengzhou Zhou

    Full Text Available BACKGROUND: The nematode Caenorhabditis elegans has become increasingly used for screening antimicrobials and probiotics for pathogen control. It also provides a useful tool for studying microbe-host interactions. This study has established a C. elegans life-span assay to preselect probiotic bacteria for controlling K88(+ enterotoxigenic Escherichia coli (ETEC, a pathogen causing pig diarrhea, and has determined a potential mechanism underlying the protection provided by Lactobacillus. METHODOLOGY/PRINCIPAL FINDINGS: Life-span of C. elegans was used to measure the response of worms to ETEC infection and protection provided by lactic acid-producing bacteria (LAB. Among 13 LAB isolates that varied in their ability to protect C. elegans from death induced by ETEC strain JG280, Lactobacillus zeae LB1 offered the highest level of protection (86%. The treatment with Lactobacillus did not reduce ETEC JG280 colonization in the nematode intestine. Feeding E. coli strain JFF4 (K88(+ but lacking enterotoxin genes of estA, estB, and elt did not cause death of worms. There was a significant increase in gene expression of estA, estB, and elt during ETEC JG280 infection, which was remarkably inhibited by isolate LB1. The clone with either estA or estB expressed in E. coli DH5α was as effective as ETEC JG280 in killing the nematode. However, the elt clone killed only approximately 40% of worms. The killing by the clones could also be prevented by isolate LB1. The same isolate only partially inhibited the gene expression of enterotoxins in both ETEC JG280 and E. coli DH5α in-vitro. CONCLUSIONS/SIGNIFICANCE: The established life-span assay can be used for studies of probiotics to control ETEC (for effective selection and mechanistic studies. Heat-stable enterotoxins appeared to be the main factors responsible for the death of C. elegans. Inhibition of ETEC enterotoxin production, rather than interference of its intestinal colonization, appears to be the

  3. The Mx1 Gene Protects Mice against the Pandemic 1918 and Highly Lethal Human H5N1 Influenza Viruses▿

    OpenAIRE

    2007-01-01

    Mice carrying a wild-type Mx1 gene (Mx1+/+) differ from standard laboratory mice (Mx1−/−) in being highly resistant to infection with common laboratory strains of influenza A virus. We report that Mx1 also protects mice against the pandemic human 1918 influenza virus and a highly lethal human H5N1 strain from Vietnam. Resistance to H5N1 of Mx1+/+ but not Mx1−/− mice was enhanced if the animals were treated with a single dose of exogenous alpha interferon before infection. Thus, the interferon...

  4. Changes in Gene Expression in the Hippocampus Following Exposure to 56Fe Particles and Protection by Berry Diets

    Science.gov (United States)

    Shukitt-Hale, Barbara; Lau, Francis; Carey, Amanda; Carrihill-Knoll, Kirsty; Rabin, Bernard; Joseph, James

    DNA damage caused by radiation to new cells. Supplementation with the berry diets enhanced neuronal communication and cell signaling by altering gene regulation of some of the protective stress signals. Therefore, these data suggest that 56 Fe particle irradiation causes deficits in gene expression in rats which are ameliorated by berry fruit diets.

  5. Cell-Internalization SELEX: Method for Identifying Cell-Internalizing RNA Aptamers for Delivering siRNAs to Target Cells

    Science.gov (United States)

    Thiel, William H.; Thiel, Kristina W.; Flenker, Katie S.; Bair, Tom; Dupuy, Adam J.; McNamara, James O.; Miller, Francis J.; Giangrande, Paloma H.

    2015-01-01

    After a decade of work to address cellular uptake, the principal obstacle to RNAi-based therapeutics, there is now well-deserved, renewed optimism about RNAi-based drugs. Phase I and II studies have shown safe, strong, and durable-gene knockdown (80–90 %, lasting for a month after a single injection) and/or clinical benefit in treating several liver pathologies. Although promising, these studies have also highlighted the need for robust delivery techniques to develop RNAi therapeutics for treating other organ systems and diseases. Conjugation of siRNAs to cell-specific, synthetic RNA ligands (aptamers) is being proposed as a viable solution to this problem. While encouraging, the extended use of RNA aptamers as a delivery tool for siRNAs awaits the identification of RNA aptamer sequences capable of targeting and entering the cytoplasm of many different cell types. We describe a cell-based selection process for the rapid identification and characterization of RNA aptamers suited for delivering siRNA drugs into the cytoplasm of target cells. This process, termed “cell-internalization SELEX (Systematic Evolution of Ligands by Exponential Enrichment),” entails the combination of multiple sophisticated technologies, including cell culture-based SELEX procedures, next-generation sequencing (NGS), and novel bioinformatics tools. PMID:25319652

  6. EGF receptor targeted lipo-oligocation polyplexes for antitumoral siRNA and miRNA delivery

    Science.gov (United States)

    Müller, Katharina; Klein, Philipp M.; Heissig, Philipp; Roidl, Andreas; Wagner, Ernst

    2016-11-01

    Antitumoral siRNA and miRNA delivery was demonstrated by epidermal growth factor receptor (EGFR) targeted oligoaminoamide polyplexes. For this purpose, the T-shaped lipo-oligomer 454 was used to complex RNA into a core polyplex, which was subsequently functionalized with the targeting peptide ligand GE11 via a polyethylene glycol (PEG) linker. To this end, free cysteines on the surface of 454 polyplex were coupled with a maleimide-PEG-GE11 reagent (Mal-GE11). Resulting particles with sizes of 120-150 nm showed receptor-mediated uptake into EGFR-positive T24 bladder cancer cells, MDA-MB 231 breast cancer cells and Huh7 liver cancer cells. Furthermore, these formulations led to ligand-dependent gene silencing. RNA interference (RNAi) triggered antitumoral effects were observed for two different therapeutic RNAs, a miRNA-200c mimic or EG5 siRNA. Using polyplexes modified with a ratio of 0.8 molar equivalents of Mal-GE11, treatment of T24 or MDA-MB 231 cancer cells with miR-200c led to the expected decreased proliferation and migration, changes in cell cycle and enhanced sensitivity towards doxorubicin. Delivery of EG5 siRNA into Huh7 cells resulted in antitumoral activity with G2/M arrest, triggered by loss of mitotic spindle separation and formation of mono-astral spindles. These findings demonstrate the potential of GE11 ligand-containing RNAi polyplexes for cancer treatment.

  7. Protection against Vibrio alginolyticus in crimson snapper Lutjanus erythropterus immunized with a DNA vaccine containing the ompW gene.

    Science.gov (United States)

    Cai, Shuang-Hu; Lu, Yi-Shan; Jian, Ji-Chang; Wang, Bei; Huang, Yu-Cong; Tang, Ju-Fen; Ding, Yu; Wu, Zao-He

    2013-09-24

    The outer membrane proteins of Vibrio alginolyticus play an important role in the virulence of the bacterium and are potential candidates for vaccine development. In the present study, the ompW gene was cloned, expressed and purified. A DNA vaccine was constructed by inserting the ompW gene into a pcDNA plasmid. Crimson snapper Lutjanus erythropterus (Bloch) were injected intramuscularly with the recombinant plasmid pcDNA-ompW. The expression of the DNA vaccine was detected in gill, head kidney, heart, liver, spleen and injection site muscle of crimson snapper by RT-PCR 7 and 28 d post-vaccination. The ELISA results demonstrated that the DNA vaccine produced an observable antibody response in all sera of the vaccinated fish. In addition, crimson snapper immunized with the DNA vaccine showed a relative percentage survival (RPS) of 92.53%, indicating effective protection against V. alginolyticus infection.

  8. Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain

    Directory of Open Access Journals (Sweden)

    Julia F Alterman

    2015-01-01

    Full Text Available Applications of RNA interference for neuroscience research have been limited by a lack of simple and efficient methods to deliver oligonucleotides to primary neurons in culture and to the brain. Here, we show that primary neurons rapidly internalize hydrophobically modified siRNAs (hsiRNAs added directly to the culture medium without lipid formulation. We identify functional hsiRNAs targeting the mRNA of huntingtin, the mutation of which is responsible for Huntington's disease, and show that direct uptake in neurons induces potent and specific silencing in vitro. Moreover, a single injection of unformulated hsiRNA into mouse brain silences Htt mRNA with minimal neuronal toxicity. Thus, hsiRNAs embody a class of therapeutic oligonucleotides that enable simple and straightforward functional studies of genes involved in neuronal biology and neurodegenerative disorders in a native biological context.

  9. Polyphosphonium polymers for siRNA delivery: An efficient and nontoxic alternative to polyammonium carriers

    KAUST Repository

    Ornelas-Megiatto, Cátia

    2012-02-01

    A water-soluble polyphosphonium polymer was synthesized and directly compared with its ammonium analog in terms of siRNA delivery. The triethylphosphonium polymer shows transfection efficiency up to 65% with 100% cell viability, whereas the best result obtained for the ammonium analog reaches only 25% transfection with 85% cell viability. Moreover, the nature of the alkyl substituents on the phosphonium cations is shown to have an important influence on the transfection efficiency and toxicity of the polyplexes. The present results show that the use of positively charged phosphonium groups is a worthy choice to achieve a good balance between toxicity and transfection efficiency in gene delivery systems. © 2012 American Chemical Society.

  10. SiRNAs in vivo imaging: methodology of fluorine-18 radiolabelling and application for the optimization of the siRNAs biodistribution and pharmaceutical properties; Imagerie in vivo des ARN interferentiels: methodologie de marquage au fluor-18 et application pour l'optimisation par imagerie de leur biodistribution et de leurs proprietes pharmacologiques

    Energy Technology Data Exchange (ETDEWEB)

    Viel, Th

    2008-01-15

    As RNA interference is a natural process which enables eukaryote cells to regulate the gene expressions, to control transposons, and to struggle against some viruses, two imagery techniques have been used in this research, i.e. optical imagery and Positron Emission Tomography (PET) imagery, to study the various modifications of the small interferential RNAs (siRNA). Different chemically modified siRNAs have been prepared and their in vitro activity, their in vivo metabolism (by HPLC analysis), their bio-distribution and their pharmacokinetic properties (by PET imagery) after marking them with fluorine-18. Their in vivo activity has been assessed by optical imagery.

  11. A sulfated galactans supplemented diet enhances the expression of immune genes and protects against Vibrio parahaemolyticus infection in shrimp.

    Science.gov (United States)

    Rudtanatip, Tawut; Boonsri, Nantavadee; Asuvapongpatana, Somluk; Withyachumnarnkul, Boonsirm; Wongprasert, Kanokpan

    2017-06-01

    A sulfated galactans (SG) supplemented diet was evaluated for the potential to stimulate immune activity in shrimp Penaeus vannamei (P. vannamei). Shrimp given the SG supplemented diet (0.5, 1 and 2% w/w) for 7 days showed enhanced expression of the downstream signaling mediator of lipopolysaccharide and β-1,3-glucan binding protein (LGBP) and immune related genes including p-NF-κB, IMD, IKKβ and IKKε, antimicrobial peptide PEN-4, proPO-I and II. Following immersion with Vibrio parahaemolyticus (V. parahaemolyticus) for 14 days, the shrimp given the SG supplemented diet (1 and 2% w/w) showed a decrease in bacterial colonies and bacterial toxin gene expression, compared to shrimp given a normal diet, and they reached 50% mortality at day 14. However, shrimp given the normal diet and challenged with the bacteria reached 100% mortality at day 6. SG-fed shrimp increased expression of immune genes related to LGBP signaling at day 1 after the bacterial immersion compared to control (no immersion), which later decreased to control levels. Shrimp on the normal diet also increased expression of immune related genes at day 1 after immersion which however decreased below control levels by day 3. Taken together, the results indicate the efficacy of the SG supplemented diet to enhance the immune activity in shrimp which could offer protection from V. parahaemolyticus infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production.

    Science.gov (United States)

    Brueggeman, Andrew J; Kuehler, Daniel; Weeks, Donald P

    2014-09-01

    Genes conferring resistance to the herbicides glyphosate, oxyfluorfen and norflurazon were developed and tested for use as dominant selectable markers in genetic transformation of Chlamydomonas reinhardtii and as potential tools for the protection of commercial-scale algal production facilities against contamination by organisms sensitive to these broad-spectrum herbicides. A synthetic glyphosate acetyltransferase (GAT) gene, when fitted with a strong Chlamydomonas promoter, conferred a 2.7×-fold increase in tolerance to the EPSPS inhibitor, glyphosate, in transgenic cells compared with progenitor WT cells. A mutant Chlamydomonas protoporphyrinogen oxidase (protox, PPO) gene previously shown to produce an enzyme insensitive to PPO-inhibiting herbicides, when genetically engineered, generated transgenic cells able to tolerate up to 136× higher levels of the PPO inhibitor, oxyfluorfen, than nontransformed cells. Genetic modification of the Chlamydomonas phytoene desaturase (PDS) gene-based gene sequences found in various norflurazon-resistant organisms allowed production of transgenic cells tolerant to 40× higher levels of norflurazon than nontransgenic cells. The high efficiency of all three herbicide resistance genes in producing transgenic cells demonstrated their suitability as dominant selectable markers for genetic transformation of Chlamydomonas and, potentially, other eukaryotic algae. However, the requirement for high concentrations of glyphosate and its associated negative effects on cell growth rates preclude its consideration for use in large-scale production facilities. In contrast, only low doses of norflurazon and oxyfluorfen (~1.5 μm and ~0.1 μm, respectively) are required for inhibition of cell growth, suggesting that these two herbicides may prove effective in large-scale algal production facilities in suppressing growth of organisms sensitive to these herbicides.

  13. Inhibition of c-REL using siRNA increased apoptosis and decreased proliferation in pre-B ALL blasts: Therapeutic implications.

    Science.gov (United States)

    Mohammadi, Seyedeh Momeneh; Mohammadnejad, Daryosh; Hosseinpour Feizi, Abbas Ali; Movassaghpour, Ali Akbar; Montazersaheb, Soheila; Nozad Charoudeh, Hojjatollah

    2017-10-01

    The c-Rel transcription factor is a unique member of the NF-kB family that has a role in apoptosis, proliferation and cell survival. Overexpression of c-Rel is detected in many human B cell tumors, including B-cell leukemia and several cancers. The study aimed to investigate the effects of c-Rel siRNA on the proliferation and apoptosis of relapsed pre-B acute leukemia cells. The c-Rel siRNA was transfected into Leukemia cells using an Amaxa cell line Nucleofector kit L (Lonza). Quantitative real-time RT-PCR (qRT-PCR) and western blot were done to measure the expression levels of mRNA and protein, respectively. The flow cytometry was used to analyze the effect of c-Rel siRNA on the apoptosis and proliferation of Leukemia cells. Observed c-Rel expression in the 5 pre-B Acute lymphoblastic leukemia (ALL) patients were higher than the normal cells. The c-Rel siRNA transfection significantly blocked the expression of c-Rel mRNA in a time-dependent manner, leading to a strong growth inhibition and enhanced apoptosis (Pc-Rel plays a fundamental role in the survival. Therefore, c-Rel can be considered as an attractive target for gene therapy in ALL patients. Also siRNA-mediated silencing of this gene may be a novel strategy in ALL treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Nucleolin-targeting liposomes guided by aptamer AS1411 for the delivery of siRNA for the treatment of malignant melanomas.

    Science.gov (United States)

    Li, Liyu; Hou, Jianjun; Liu, Xinjie; Guo, Yujia; Wu, Yun; Zhang, Lihe; Yang, Zhenjun

    2014-04-01

    BRAF gene mutation is found in more than 60% of malignant melanomas, which are difficult to treat. In this study, a new tumor-targeting liposome was developed to deliver anti-BRAF siRNA (siBraf) for the treatment of melanomas. Nucleolin is overexpressed on the surface of cancer cells. AS1411, an aptamer showing specific binding to nucleolin, was conjugated to PEGylated cationic liposome as the targeting probe ASLP (AS1411-PEG-liposome). The ASLP/siRNA complex was formed through electrostatic interaction between ASLP and siRNA. The binding of AS1411 to the surface of PEGylated liposomes was confirmed by gel electrophoresis and capillary electrophoresis. Real-time PCR and Western blot analysis showed that ASLP/siBraf exhibited strong silencing activity of BRAF gene. The much higher accumulation of the siRNA in tumor cells comparing with normal cells indicated that ASLP displayed excellent tumor-targeting capability. Notably, ASLP/siBraf showed significant silencing activity in A375 tumor xenograft mice and inhibited the melanoma growth. These results suggested that the new nucleolin-targeted siRNA delivery system by AS1411 may have the potential for the treatment of melanoma.

  15. Combinations of siRNAs against La Autoantigen with NS5B or hVAP-A Have Additive Effect on Inhibition of HCV Replication

    Science.gov (United States)

    Mandal, Anirban; Ganta, Krishna Kumar

    2016-01-01

    Hepatitis C virus is major cause of chronic liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Presently available direct-acting antiviral drugs have improved success rate; however, high cost limits their utilization, especially in developing countries like India. In the present study, we evaluated anti-HCV potential of several siRNAs targeted against the HCV RNA-dependent RNA polymerase NS5B and cellular factors, La autoantigen, PSMA7, and human VAMP-associated protein to intercept different steps of viral life cycle. The target genes were downregulated individually as well as in combinations and their impact on viral replication was evaluated. Individual downregulation of La autoantigen, PSMA7, hVAP-A, and NS5B resulted in inhibition of HCV replication by about 67.2%, 50.7%, 39%, and 52%, respectively. However, antiviral effect was more pronounced when multiple genes were downregulated simultaneously. Combinations of siRNAs against La autoantigen with NS5B or hVAP-A resulted in greater inhibition in HCV replication. Our findings indicate that siRNA is a potential therapeutic tool for inhibiting HCV replication and simultaneously targeting multiple viral steps with the combination of siRNAs is more effective than silencing a single target. PMID:27446609

  16. Silencing of the AV2 gene by antisense RNA protects transgenic plants against a bipartite begomovirus

    Directory of Open Access Journals (Sweden)

    Zafar Yusuf

    2007-01-01

    Full Text Available Abstract Whitefly-transmitted geminiviruses (genus Begomovirus are phytopathogens that cause heavy losses to crops worldwide. Efforts to engineer resistance against these viruses are focused mainly on silencing of complementary-sense virus genes involved in virus replication. Here we have targeted a virion-sense gene (AV2 to develop resistance against Tomato leaf curl New Delhi virus, a bipartite begomovirus prevalent throughout the Indian subcontinent. We show that tobacco plants transformed with an antisense construct targeting this gene are resistant to the virus. Following challenged with the virus, transgenic plants remained symptomless, although viral DNA could be detected in some plants by PCR. This is the first report of transgenic resistance against a bipartite begomovirus obtained by targeting a virion-sense gene. The relatively conserved nature of the gene suggests that the technology may be useful to develop broad-spectrum resistance which is required because of the fact that plants are often infected with multiple begomoviruses in the field.

  17. SiRNA Inhibits Replication of Langat Virus, a Member of the Tick-Borne Encephalitis Virus Complex in Organotypic Rat Brain Slices

    Science.gov (United States)

    Maffioli, Carola; Grandgirard, Denis; Leib, Stephen L.; Engler, Olivier

    2012-01-01

    Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000–3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3′ untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro. PMID:22984545

  18. The (PrS/HGF-pDNA) multilayer films for gene-eluting stent coating: Gene-protecting, anticoagulation, antibacterial properties, and in vivo antirestenosis evaluation.

    Science.gov (United States)

    Chang, Hao; Ren, Ke-feng; Zhang, He; Wang, Jin-lei; Wang, Bai-liang; Ji, Jian

    2015-02-01

    Vascular gene-eluting stents (GES) is a promising strategy for treatment of cardiovascular disease. Very recently, we have proved that the (protamine sulfate/plasmid DNA encoding hepatocyte growth factor) (PrS/HGF-pDNA) multilayer can serve as a powerful tool for enhancing competitiveness of endothelial cell over smooth muscle cell, which opens perspectives for the regulation of intercellular competitiveness in the field of interventional therapy. However, before the gene multilayer films could be used in vascular stents for real clinical application, the preservation of gene bioactivity during the industrial sterilization and the hemocompatibility of film should be taken into account. Actually, both are long been ignored issues in the field of gene coating for GES. In this study, we demonstrate that the (PrS/HGF-pDNA) multilayer film exhibits the good gene-protecting abilities, which is confirmed by using the industrial sterilizations (gamma irradiation and ethylene oxide) and a routine storage condition (dry state at 4°C for 30 days). Furthermore, hemocompatible measurements (such as platelet adhesion and whole blood coagulation) and antibacterial assays (bacteria adhesion and growth inhibition) indicate the good anticoagulation and antibacterial properties of the (PrS/HGF-pDNA) multilayer film. The in vivo preliminary data of angiography and histological analysis suggest that the (PrS/HGF-pDNA) multilayer coated stent can reduce the in-stent restenosis. This work reveals that the (PrS/HGF-pDNA) multilayer film could be a promising candidate as coating for GES, which is of great potential in future clinic application.

  19. Intravenous Delivery of pDNA and siRNA into Muscle with Bubble Liposomes and Ultrasound

    Science.gov (United States)

    Negishi, Yoichi; Sekine, Shohko; Endo, Yoko; Nishijima, Nobuaki; Suzuki, Ryo; Maruyama, Kazuo; Aramaki, Yukihiko

    2010-03-01

    Skeletal muscle is an attractive target tissue for numerous gene therapy strategies. Gene delivery into muscle has been extensively studied. Of the strategies, intravascular delivery of naked pDNA is desirable. Muscle has a high density of capillaries that are in close contact with myofibers. Previously, we developed polyethylene glycol (PEG)-modified liposomes entrapping echo-contrast gas, known as ultrasound (US) imaging gas. We called the liposomes "Bubble liposomes" (BLs). It has been reported that BLs improve the tissue permeability by cavitation on US exposure. Here, we modified the naked pDNA or siRNA transfer method into hind limb muscle through blood vessels using BLs and US. The intravenous delivery of pDNA into muscle can be markedly enhanced when the pDNA is delivered in combination with BLs and US. In addition, the expression of pDNA is high in the US-focused site. Moreover, efficient gene delivery can be achieved by the intravenous delivery of pDNA into muscle with BLs and US. Expression is also down-regulated by delivering siRNA with BLs and US. Thus, this US-mediated BL technique involving veins may be an effective method for gene therapy.

  20. Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants

    KAUST Repository

    Khraiwesh, Basel

    2012-02-01

    Small, non-coding RNAs are a distinct class of regulatory RNAs in plants and animals that control a variety of biological processes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved through a series of pathways. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs control the expression of cognate target genes by binding to reverse complementary sequences, resulting in cleavage or translational inhibition of the target RNAs. siRNAs have a similar structure, function, and biogenesis as miRNAs but are derived from long double-stranded RNAs and can often direct DNA methylation at target sequences. Besides their roles in growth and development and maintenance of genome integrity, small RNAs are also important components in plant stress responses. One way in which plants respond to environmental stress is by modifying their gene expression through the activity of small RNAs. Thus, understanding how small RNAs regulate gene expression will enable researchers to explore the role of small RNAs in biotic and abiotic stress responses. This review focuses on the regulatory roles of plant small RNAs in the adaptive response to stresses. This article is part of a Special Issue entitled: Plant gene regulation in response to abiotic stress. © 2011 Elsevier B.V.

  1. Glycine betaine protects tomato (Solanum lycopersicum) plants at low temperature by inducing fatty acid desaturase7 and lipoxygenase gene expression.

    Science.gov (United States)

    Karabudak, T; Bor, M; Özdemir, F; Türkan, İ

    2014-03-01

    Cold stress is among the environmental stressors limiting productivity, yield and quality of agricultural plants. Tolerance to cold stress is associated with the increased unsaturated fatty acids ratio in the plant membranes which are also known to be substrates of octadecanoid pathway for jasmonate and other oxylipins biosynthesis. Accumulation of osmoprotectant, glycine betaine (GB) is wel