RNA interference by small hairpin RNAs synthesised under control of the human 7S K RNA promoter

2004 ◽  
Vol 385 (9) ◽  
pp. 791-794 ◽  
Author(s):  
Dorota Koper-Emde ◽  
Lutz Herrmann ◽  
Björn Sandrock ◽  
Bernd-Joachim Benecke
Keyword(s):  
Rna Interference ◽  
Gene Promoter ◽  
Target Sequence ◽  
Small Nuclear Rna ◽  
Small Interfering Rnas ◽  
Rna Duplexes ◽  
Inhibit Gene Expression ◽  
Short Hairpin Rnas ◽  
Rna Promoter

AbstractSmall interfering RNAs (siRNAs) represent RNA duplexes of 21 nucleotides in length that inhibit gene expression. We have used the human gene-external 7S K RNA promoter for synthesis of short hairpin RNAs (shRNAs) which efficiently target human lamin mRNA via RNA interference (RNAi). Here we demonstrate that orientation of the target sequence within the shRNA construct is important for interference. Furthermore, effective interference also depends on the length and/or structure of the shRNA. Evidence is presented that the human 7S K promoter is more activein vivothan other gene-external promoters, such as the human U6 small nuclear RNA (snRNA) gene promoter.

siRNA, miRNA, and shRNA: in vivo Applications

2008 ◽  
Vol 87 (11) ◽  
pp. 992-1003 ◽  
Author(s):  
P.N. Pushparaj ◽  
J.J. Aarthi ◽  
J. Manikandan ◽  
S.D. Kumar
Keyword(s):  
Metabolic Diseases ◽  
Small Interfering Rnas ◽  
Base Pairs ◽  
Rna Molecules ◽  
Rna Activation ◽  
Research Findings ◽  
Dental Diseases ◽  
Short Hairpin Rnas ◽  
Clinical Potential

RNA interference (RNAi), an accurate and potent gene-silencing method, was first experimentally documented in 1998 in Caenorhabditis elegans by Fire et al., who subsequently were awarded the 2006 Nobel Prize in Physiology/Medicine. Subsequent RNAi studies have demonstrated the clinical potential of synthetic small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) in dental diseases, eye diseases, cancer, metabolic diseases, neurodegenerative disorders, and other illnesses. siRNAs are generally from 21 to 25 base-pairs (bp) in length and have sequence-homology-driven gene-knockdown capability. RNAi offers researchers an effortless tool for investigating biological systems by selectively silencing genes. Key technical aspects—such as optimization of selectivity, stability, in vivo delivery, efficacy, and safety—need to be investigated before RNAi can become a successful therapeutic strategy. Nevertheless, this area shows a huge potential for the pharmaceutical industry around the globe. Interestingly, recent studies have shown that the small RNA molecules, either indigenously produced as microRNAs (miRNAs) or exogenously administered synthetic dsRNAs, could effectively activate a particular gene in a sequence-specific manner instead of silencing it. This novel, but still uncharacterized, phenomenon has been termed ‘RNA activation’ (RNAa). In this review, we analyze these research findings and discussed the in vivo applications of siRNAs, miRNAs, and shRNAs.

scholarly journals Human Immunodeficiency Virus Type 1 Escapes from RNA Interference-Mediated Inhibition

2004 ◽  
Vol 78 (5) ◽  
pp. 2601-2605 ◽  
Author(s):  
Atze T. Das ◽  
Thijn R. Brummelkamp ◽  
Ellen M. Westerhout ◽  
Monique Vink ◽  
Mandy Madiredjo ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Interference ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Antiviral Drug ◽  
Target Sequence ◽  
Small Interfering Rnas ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Short-term assays have suggested that RNA interference (RNAi) may be a powerful new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) infection. However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays. We stably introduced vectors expressing small interfering RNAs (siRNAs) directed against the HIV-1 genome into human T cells by retroviral transduction. We report here that an siRNA directed against the viral Nef gene (siRNA-Nef) confers resistance to HIV-1 replication. This block in replication is not absolute, and HIV-1 escape variants that were no longer inhibited by siRNA-Nef appeared after several weeks of culture. These RNAi-resistant viruses contained nucleotide substitutions or deletions in the Nef gene that modified or deleted the siRNA-Nef target sequence. These results demonstrate that efficient inhibition of HIV-1 replication through RNAi is possible in stably transduced cells. Therefore, RNAi could become a realistic gene therapy approach with which to overcome the devastating effect of HIV-1 on the immune system. However, as is known for antiviral drug therapy against HIV-1, antiviral approaches involving RNAi should be used in a combined fashion to prevent the emergence of resistant viruses.

scholarly journals Bottlebrush-architectured poly(ethylene glycol) as an efficient vector for RNA interference in vivo

2019 ◽  
Vol 5 (2) ◽  
pp. eaav9322 ◽  
Author(s):  
Dali Wang ◽  
Jiaqi Lin ◽  
Fei Jia ◽  
Xuyu Tan ◽  
Yuyan Wang ◽  
...  
Keyword(s):  
Rna Interference ◽  
Ethylene Glycol ◽  
Target Genes ◽  
Area Under The Curve ◽  
Fold Increase ◽  
Small Interfering Rnas ◽  
Poly Ethylene Glycol ◽  
Linear Poly ◽  
Poly Ethylene

Nonhepatic delivery of small interfering RNAs (siRNAs) remains a challenge for development of RNA interference–based therapeutics. We report a noncationic vector wherein linear poly(ethylene glycol) (PEG), a polymer generally considered as inert and safe biologically but ineffective as a vector, is transformed into a bottlebrush architecture. This topology provides covalently embedded siRNA with augmented nuclease stability and cellular uptake. Consisting almost entirely of PEG and siRNA, the conjugates exhibit a ~25-fold increase in blood elimination half-life and a ~19-fold increase in the area under the curve compared with unmodified siRNA. The improved pharmacokinetics results in greater tumor uptake and diminished liver capture. Despite the structural simplicity these conjugates efficiently knock down target genes in vivo without apparent toxic and immunogenic reactions. Given the benign biological nature of PEG and its widespread precedence in biopharmaceuticals, we anticipate the brush polymer–based technology to have a significant impact on siRNA therapeutics.

scholarly journals Synthetic Pre-miRNA-Based shRNA as Potent RNAi Triggers

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Kazuya Terasawa ◽  
Kazuharu Shimizu ◽  
Gozoh Tsujimoto
Keyword(s):  
Biological Activity ◽  
Rna Interference ◽  
Gene Function ◽  
Small Interfering Rnas ◽  
Therapeutic Applications ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Interferon Responses ◽  
Rnai Activity ◽  
Target Effects

RNA interference (RNAi) is a powerful tool for studying gene function owing to the ease with which it can selectively silence genes of interest, and it has also attracted attention because of its potential for therapeutic applications. Chemically synthesized small interfering RNAs (siRNAs) and DNA vector-based short hairpin RNAs (shRNAs) are now widely used as RNAi triggers. In contrast to expressed shRNAs, the use of synthetic shRNAs is limited. Here we designed shRNAs modeled on a precursor microRNA (pre-miRNA) and evaluated their biological activity. We demonstrated that chemically synthetic pre-miRNA-based shRNAs have more potent RNAi activity than their corresponding siRNAs and found that their antisense strands are more efficiently incorporated into the RNA-induced silencing complex. Although greater off-target effects and interferon responses were induced by shRNAs than by their corresponding siRNAs, these effects could be overcome by simply using a lower concentration or by optimizing and chemically modifying shRNAs similar to synthetic siRNAs. These are challenges for the future.

Exploring Promises of siRNA in Cancer Therapeutics

2020 ◽  
Vol 16 (1) ◽  
pp. 29-35
Author(s):  
Mahima Kaushik ◽  
Rddhima Raghunand ◽  
Shobhit Maheshwari
Keyword(s):  
Clinical Trials ◽  
Rna Interference ◽  
Cancer Therapy ◽  
Biomedical Applications ◽  
Cancer Therapeutics ◽  
Small Interfering Rnas ◽  
Polymeric Carriers ◽  
Drug Treatments ◽  
Anticancer Treatment

Since the discovery of the RNA interference (RNAi) in 2006, several attempts have been made to use it for designing and developing drug treatments for a variety of diseases, including cancer. In this mini-review, we focus on the potential of small interfering RNAs (siRNA) in anticancer treatment. We first describe the significant barriers that exist on the path to clinical application of siRNA drugs. Then the current delivery approaches of siRNAs using lipids, polymers, and, in particular, polymeric carriers that overcome the aforementioned obstacles have been reviewed. Also, few siRNA mediated drugs currently in clinical trials for cancer therapy, and a collated list of siRNA databases having a qualitative and/ or quantitative summary of the data in each database have been briefly mentioned. This mini review aims to facilitate our understanding about the siRNA, their delivery systems and the possible barriers in their in vivo usage for biomedical applications.

Abstract 3810: Antiviral RNA Interference Mediated by a Recombinant Cardiotropic AAV9 Vector Improves Cardiac Function in Coxsackievirus B3 Cardiomyopathy

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Wolfgang Poller ◽  
Isaac Sipo ◽  
Dirk Westermann ◽  
Jens Kurreck ◽  
Roland Vetter ◽  
...  
Keyword(s):  
Rna Interference ◽  
Coxsackievirus B3 ◽  
Left Ventricular ◽  
Lv Function ◽  
Target Cells ◽  
Small Interfering Rnas ◽  
Rat Cardiomyocytes ◽  
Terminal Heart Failure ◽  
Novel Therapeutic

RNA interference (RNAi) has potential to be a novel therapeutic strategy in diverse areas of medicine. We report here on targeted RNAi for the treatment of a viral cardiomyopathy which is a major cause of sudden cardiac death or terminal heart failure in children and young adults. RNAi therapy employs small regulatory RNAs to achieve its effect but in vivo use of synthetic small interfering RNAs is limited by instability in plasma and low transfer into target cells. We instead evaluated an RNAi strategy using short hairpin RNA (shRdRp) directed at the RNA polymerase (RdRP) of Coxsackievirus B3 (CoxB3) in HeLa cells, primary rat cardiomyocytes (PNCMs), and CoxB3-infected mice in vivo. A conventional AAV2 vector expressing shRdRp protected HeLa against virus-induced death, but this vector type was unable to transduce PNCMs. In contrast, an analogous pseudotyped AAV2.6 vector was protective also in PNCMs and reduced virus replication by >3 log10 steps. Finally, we evaluated intravenous treatment of mice with an AAV2.9-shRdRp vector since AAV9 carries the most cardiotropic AAV capsid currently known for in vivo use. Mice with CoxB3 cardiomyopathy had disturbed left ventricular (LV) function with impaired parameters of contractility (dP/dtmax 3006±287 vs. 7482±487 mmHg/s, p<0.01) and diastolic relaxation (dP/dtmin -2224±195 vs. -6456±356 mmHg/s, p<0.01 and Tau 16.2±1.1 vs. 10.7±0.6 ms, p<0.01) as compared to control mice. AAV2.9-shRdRp treatment significantly attenuated the cardiac dysfunction compared to control vector-treated mice on day 10 after CoxB3 infection: dP/dtmax 3865±354 vs. 3006±287 mmHg/s (p<0.05) and dP/dtmin -3245±231 vs. −2224±195, mmHg/s (p<0.05), and Tau 11.9±0.5 vs. 16.2±1.1 ms (p<0.01). The data show, for the first time, that intravenously injected AAV9 has the potential to target RNAi to the heart and suggest AAV9-shRNA vectors as a novel therapeutic approach for cardiac disorders.

scholarly journals Non-Viral Delivery and Therapeutic Application of Small Interfering RNAs

Acta Naturae ◽  
2013 ◽  
Vol 5 (3) ◽  
pp. 35-53 ◽  
Author(s):  
N. A. Nikitenko ◽  
V. S. Prassolov
Keyword(s):  
Rna Interference ◽  
Gene Expression Regulation ◽  
Sirna Delivery ◽  
Target Cells ◽  
Powerful Method ◽  
Small Interfering Rnas ◽  
Therapeutic Application ◽  
Rnai Technology ◽  
In Vivo Degradation

RNA interference (RNAi) is a powerful method used for gene expression regulation. The increasing knowledge about the molecular mechanism of this phenomenon creates new avenues for the application of the RNAi technology in the treatment of various human diseases. However, delivery of RNA interference mediators, small interfering RNAs (siRNAs), to target cells is a major hurdle. Effective and safe pharmacological use of siRNAs requires carriers that can deliver siRNA to its target site and the development of methods for protection of these fragile molecules from in vivo degradation. This review summarizes various strategies for siRNA delivery, including chemical modification and non-viral approaches, such as the polymer-based, peptide-based, lipid-based techniques, and inorganic nanosystems. The advantages, disadvantages, and prospects for the therapeutic application of these methods are also examined in this paper.

scholarly journals Novel dimeric Nur77 signaling mechanism in endocrine and lymphoid cells.

1997 ◽  
Vol 17 (10) ◽  
pp. 5946-5951 ◽  
Author(s):  
A Philips ◽  
S Lesage ◽  
R Gingras ◽  
M H Maira ◽  
Y Gauthier ◽  
...  
Keyword(s):  
T Cell ◽  
Gene Promoter ◽  
Cell Receptor ◽  
Lymphoid Cells ◽  
Target Sequence ◽  
Orphan Nuclear Receptors ◽  
Response Element ◽  
Signaling Mechanism ◽  
Induced Apoptosis

Within the nuclear receptor family, Nur77 (also known as NGFI-B) distinguishes itself by its ability to bind a target sequence (the NBRE) as a monomer and by its role in T-cell receptor (TCR)-induced apoptosis in T cells. We now report on a novel mechanism of Nur77 action that is mediated by homodimers. These dimers bind a Nur77 response element (NurRE), which has been identified as a target of CRH-induced Nur77 in the pro-opiomelanocortin (POMC) gene promoter. Both halves of the palindromic NurRE are required for responsiveness to physiological signals, like CRH in pituitary-derived AtT-20 cells. Similarly, in T-cell hybridomas, TCR activation induced NurRE but not NBRE reporters. The in vivo signaling function of Nur77 thus appears to be mediated by dimers acting on a palindromic response element of unusual spacing between its half-sites. This mechanism may represent the biologically relevant paradigm of action for this subfamily of orphan nuclear receptors.

scholarly journals RNA interference and the use of small interfering RNA to study gene function in mammalian systems

2004 ◽  
Vol 33 (3) ◽  
pp. 545-557 ◽  
Author(s):  
I Bantounas ◽  
L A Phylactou ◽  
J B Uney
Keyword(s):  
Rna Interference ◽  
Gene Function ◽  
Viral Vector ◽  
Sirna Delivery ◽  
Interferon Response ◽  
Rnai Pathway ◽  
Small Interfering Rnas ◽  
Rnai Technology

In the past 2 years, extraordinary developments in RNA interference (RNAi)-based methodologies have seen small interfering RNAs (siRNA) become the method of choice for researchers wishing to target specific genes for silencing. In this review, an historic overview of the biochemistry of the RNAi pathway is described together with the latest advances in the RNAi field. Particular emphasis is given to strategies by which siRNAs are used to study mammalian gene function. In this regard, the use of plasmid-based and viral vector-based systems to mediate long-term RNAi in vitro and in vivo are described. However, recent work has shown that non-specific silencing effects and activation of the interferon response may occur following the use of some siRNA and delivery vector combinations. Future goals must therefore be to understand the mechanisms by which siRNA delivery leads to unwanted gene silencing effects in cells and, in this way, RNAi technology can reach its tremendous potential as a scientific tool and ultimately be used for therapeutic purposes.

scholarly journals HER3-targeted protein chimera forms endosomolytic capsomeres and self-assembles into stealth nucleocapsids for systemic tumor homing of RNA interference in vivo

2019 ◽  
Vol 47 (21) ◽  
pp. 11020-11043 ◽  
Author(s):  
Felix Alonso-Valenteen ◽  
Sayuri Pacheco ◽  
Dustin Srinivas ◽  
Altan Rentsendorj ◽  
David Chu ◽  
...  
Keyword(s):  
Rna Interference ◽  
Sirna Delivery ◽  
Cell Uptake ◽  
Small Interfering Rnas ◽  
Penton Base ◽  
Molecular Approaches ◽  
Tumor Homing ◽  
Dynamics Simulations

AbstractRNA interference represents a potent intervention for cancer treatment but requires a robust delivery agent for transporting gene-modulating molecules, such as small interfering RNAs (siRNAs). Although numerous molecular approaches for siRNA delivery are adequate in vitro, delivery to therapeutic targets in vivo is limited by payload integrity, cell targeting, efficient cell uptake, and membrane penetration. We constructed nonviral biomaterials to transport small nucleic acids to cell targets, including tumor cells, on the basis of the self-assembling and cell-penetrating activities of the adenovirus capsid penton base. Our recombinant penton base chimera contains polypeptide domains designed for noncovalent assembly with anionic molecules and tumor homing. Here, structural modeling, molecular dynamics simulations, and functional assays suggest that it forms pentameric units resembling viral capsomeres that assemble into larger capsid-like structures when combined with siRNA cargo. Pentamerization forms a barrel lined with charged residues mediating pH-responsive dissociation and exposing masked domains, providing insight on the endosomolytic mechanism. The therapeutic impact was examined on tumors expressing high levels of HER3/ErbB3 that are resistant to clinical inhibitors. Our findings suggest that our construct may utilize ligand mimicry to avoid host attack and target the siRNA to HER3+ tumors by forming multivalent capsid-like structures.

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