Development Of RNA Interference Research Articles

Abstract LB-239: Genetic approaches for next generation of breast cancer tailored therapies

Abstract Cancer therapy has radically changed during the last decade. Novel therapies based on the specific molecular changes that drive tumorigenesis in every patient are emerging as low toxicity and more efficient alternatives to classical treatments. However, even these ideal tailored therapies fail to provide a long-term cure and can only delay the progression of the disease. An alternative promising approach is the use of genetic synthetic lethal interactions. These occur when two genetic alterations that are individually innocuous appear in the same cell causing growth inhibition. This concept can be exploited to identify genes that, when inhibited, exclusively reduce the viability of tumor cells that carry a preexisting genetic lesion. Although synthetic lethality has always been proposed as an attractive anticancer approach with proven success, identification of these genetic interactions has remained elusive mainly because of the lack of proper genetic tools. However, RNA interference (RNAi) technology has emerged as a very powerful approach to attenuate the expression of any chosen gene. Thus, we envision using RNAi to identify genes that, when attenuated, exclusively reduce the viability of tumor cells carrying specific genetic lesions without affecting normal ones. During the last years, we have pioneered the development of RNAi based genetic tools for studies in mammalian cells. Thus, we have generated a highly efficient RNAi library that allows us to block any gene of interest in the human genome. Additionally, we have developed a microarray-based analytical platform that, when combined with our RNAi library, greatly facilitates genome-wide RNAi studies. This technology represents a unique opportunity to identify synthetic lethal effects with major cancer alterations. In this project we have started to apply our state-of-the-art technology to uncover synthetic lethal interaction with the major breast cancer genes (ErbB2, c-Myc, Cyclin-D1 and RB). These functional studies have already revealed that Stat-3 is synthetic lethal with activation of ErbB2. Thus, attenuating the activation levels of Stat-3 using small molecules inhibitors or RNAi silencing differentially reduced the viability of cells transformed with an ErbB2 oncogene. Mechanistically, we have linked this effect to the activation of an autocrine loop in which ErbB2 transformed cells secrete interleukin-6 (IL6) and upregulate its bona fide receptor IL6R. This activates the canonical JAK/STAT3 pathway increasing their tolerance to oncogene-induced stress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-239. doi:10.1158/1538-7445.AM2011-LB-239

RNAi Screening for the Discovery of Novel Modulators of Human Disease

The development of RNA interference (RNAi)-mediated gene inhibition has changed the direction and speed of drug target discovery and validation. RNAi technology has already influenced strategies for the pharmacological treatment of many diseases including cancer, viral diseases, bacterial pathogens, inflammation, diseases of central nervous systems (CNS), and others. This technology provides a better understanding of the mechanisms which underlie disease pathogenesis and lead to the identification of novel factors that alter the disease phenotype. With the introduction of RNAi libraries in various formats, systematic loss-of-function screens can now be conducted to interrogate the biological functions of specific genes and pathways or an entire genome in various disease areas. The identification of novel mediators of cellular response to disease pathogenesis or treatment approaches may lead to the identification of novel drug targets, development of combinatorial treatment approaches, or pharmacodynamic and patient selection biomarkers and expand our understanding of disease pathogenesis. Here, we review the use of RNAi in various screening formats, and examine the types of targets pursued for oncology and other disease indications.

Cloning and expression pattern of chitin synthase (CHS) gene in epidermis of Ectropis obliqua Prout

Ectropis obliqua Prout is a major pest in tea fields. Chitin synthase (CHS) plays an important role in biosynthesis of chitin and growth of the pest. A cDNA sequence encoding the CHS and its expression pattern during development of E.obliqua was investigated. The CHS cDNA sequence was 5496 bp nucleotides, with an open reading frame of 4692 bp encoding a protein of 1563 amino acids. It belonged to CHS-A member of CHS gene family. Alternative splice was found in the CHS-A cDNA and the alternatively spliced fragments were 177 bp and shared 65% identity with each other at the nucleotide level. The CHS-A expression was the strongest in the third and fourth instar larvae, during which the growth rate of E. obliqua larvae was the rapidest. Catalysis model of CHS-A enzyme in E. obliqua was also hypothesized according to the specific motifs and topological structure prediction of the protein. This study provided an important information for further research on development of RNA interference (RNAi) technology to control E. obliqua . Keywords: Ectropis oblique, tea pest, chitin synthase, gene cloning, RNAi, biological control African Journal of Biotechnology Vol. 9(33), pp. 5297-5308, 16 August, 2010

Highly efficient siRNA delivery system into human and murine cells using single-wallcarbon nanotubes

Development of RNA interference (RNAi) technology utilizing short interfering RNAsequences (siRNA) has focused on creating methods for delivering siRNAs to cells and forenhancing siRNA stability in vitro and in vivo. Here, we describe a novel approach forsiRNA cellular delivery using siRNA coiling into carboxyl-functionalized single-wallcarbon nanotubes (SWCNTs). The CNT–siRNA delivery system successfullydemonstrates nonspecific toxicity and transfection efficiency greater than 95%. Thisapproach offers the potential for siRNA delivery into different types of cells, includinghard-to-transfect cells, such as neuronal cells and cardiomyocytes. We also tested theCNT–siRNA system in a non-metastatic human hepatocellular carcinoma cell line(SKHep1). In all types of cells used in this work the CNT–siRNA delivery systemshowed high efficiency and apparent no side effects for various in vitro applications.

Discordant influence of Blastomyces dermatitidis yeast-phase-specific gene BYS1 on morphogenesis and virulence.

Blastomyces dermatitidis is a thermally induced dimorphic fungus capable of causing lung and systemic infections in immunocompetent animal hosts. With the publication of genomic sequences from three different strains of B. dermatitidis and the development of RNA interference as a gene-silencing tool, it has become possible to easily ascertain the virulence and morphological effects of knocking down the expression of candidate genes of interest. BYS1 (Blastomyces yeast-phase-specific 1), first identified by Burg and Smith, is expressed at high levels in yeast cells and is undetectable in mold. The deduced protein sequence of BYS1 has a putative signal sequence at its N terminus, opening the possibility that the BYS1-encoded protein is associated with the yeast cell wall. Herein, strains of B. dermatitidis with silenced expression of BYS1 were engineered and tested for morphology and virulence. The silenced strains produced rough-surfaced cultures on agar medium and demonstrated a propensity to form pseudohyphal cells on prolonged culture in vitro and in vivo, as measured in the mouse lung. Tests using a mouse model of blastomycosis with either yeast or spore inocula showed that the bys1-silenced strains were as virulent as control strains. Thus, although silencing of BYS1 alters morphology at 37 degrees C, it does not appear to impair the pathogenicity of B. dermatitidis.

RNA interference as an anticancer therapy: a patent perspective

Background: The development of RNA interference (RNAi)-based gene silencing approaches has revolutionized biomedical research. These technologies have been applied for functional genomic studies in a variety of areas, including cancer research, by facilitating a better understanding of the mechanisms that underlie tumorigenicity and the identification of novel factors that either promote or inhibit oncogenic transformation. Objective: These approaches have laid the groundwork for the development of a novel class of genetic therapies. Preclinical results have exposed both the unique therapeutic opportunities and challenges that are encountered in adapting these technologies for clinical applications. These themes are reflected in the patent literature that has mirrored the rise in complexity and sophistication of RNAi-based therapeutic approaches. This review focuses on the identification of potential anticancer therapeutic targets and the development of clinically relevant delivery approaches. Conclusions: Thus far, the patent landscape in the RNAi field has been dominated by a handful of key patents that describe the original identification and characterization of inhibitory double-stranded RNA molecules. Only time will tell how these original patents will hold up in the face of the development of new approaches and reagents as RNAi-based therapeutics approach transition from the bench to the clinic.

Development of RNA interference (RNAi) as potential antiviral strategy against enterovirus 70

Enterovirus 70 (EV70) is recognized as the main causative agent of acute hemorrhagic conjunctivitis (AHC), a highly contagious viral infection of the eye. Currently, there is no available treatment for EV70 infections. In this study, we developed a potential intervention strategy using RNA interference (RNAi) against EV70 infection in an in vitro system. Two synthetic 19-mer siRNAs, si-3D1 and si-3D2, were designed to target the 3Dpol region of the EV70 genome. Significant dosage dependent inhibition of EV70 in rhabdomyosarcoma cell line, as shown by reduction of viral RNA and VP1 production, was observed. Both siRNAs prevented EV70 replication in RD cells when transfected into these cells 48 hr prior to virus infection. Introduction of these siRNAs into RD cells 1-3 hr after infection with EV70 reduced production of viral RNA by approximately 60%. Thus, RNAi is a promising strategy to prevent EV70 infections and may have therapeutic potential.

Technology Insight: therapeutic RNA interference—how far from the neurology clinic?

As an evolutionarily conserved cellular pathway to regulate endogenous gene expression, RNA interference (RNAi) has been implicated in diverse biological processes. Biologists now routinely exploit this cellular pathway to suppress virtually any target gene in a sequence-specific manner, including dominantly acting genes that cause incurable neurodegenerative disorders. The development of RNAi as potential therapy for such diseases has generated considerable interest, partly because of the success of early studies of therapeutic RNAi in rodent models for a range of neurodegenerative diseases. In this article, we review the progress of RNAi therapy to date, and assess the challenges ahead for the application of such therapy to neurodegenerative diseases. We discuss the various strategies that might be used to achieve this goal, outline the preclinical studies that have already been completed, and highlight the experimental questions that need to be answered before human clinical trials can begin.

Utilizing RNA interference to enhance cancer drug discovery

With the development of RNA interference (RNAi) libraries, systematic and cost-effective genome-wide loss-of-function screens can now be carried out with the aim of assessing the role of specific genes in neoplastic phenotypes, and the rapid identification of novel drug targets. Here, we discuss the existing applications of RNAi in cancer drug discovery and highlight areas in this process that may benefit from this technology in the future.

Gene knockdown by RNAi in the pea aphid Acyrthosiphon pisum

BackgroundRNA interference (RNAi) is a powerful method to inhibit gene expression in a sequence specific manner.ResultsHere, we described the development of RNAi by micro-injection of double-stranded RNA (dsRNA) in the pea aphid Acyrthosiphon pisum. Injection of dsRNA into whole aphid body induced the silencing of two marker genes with different expression patterns: the ubiquitously expressed Ap-crt genes encoding a calreticulin and the gut specific Ap-cath-L gene encoding a cathepsin-L. Time-course analysis of the silencing showed similar temporal patterns for both genes: inhibition started at 1 day after injection, reached its maximum at 5 days and stopped at 7 days. A comparable 40% decrease of gene expression was observed for Ap-crt and Ap-cath-L.ConclusionThe pea aphid is the first Hemipteran insect for which genome sequence will be available soon. The gene knockdown technique developed in this study will be an essential post-genomic tool for further investigations in aphidology.

SiRNA-containing liposomes modified with polyarginine effectively silence the targeted gene

Development of RNA interference (RNAi) technology utilizing the short interfering RNA sequences (siRNA) based ‘targeted’ therapeutics has focused on creating methods for delivering siRNAs to cells and for enhancing siRNA stability in vitro and in vivo. Here, we describe a novel approach for siRNA cellular delivery using siRNA encapsulated into liposomes additionally bearing arginine octamer (R8) molecules attached to their outer surface (R8-liposomes). The R8-liposomal human double minute gene 2 (HDM2)-siRNA demonstrated a significant stability against degradation in the blood serum (siRNA-loaded R8-liposomes remained intact even after 24-h incubation), and higher transfection efficiency into all three tested lung tumor cell lines. siRNA delivery successfully proceeds in the presence of plasma proteins, and R8-liposomes demonstrate low non-specific toxicity. The mechanism of action of R8-liposome-encapsulated siRNA is associated with the RNAi-mediated degradation of the target mRNA. siRNA in R8-liposomes effectively inhibited the targeted gene and significantly reduced the proliferation of cancer cells. The approach offers the potential for siRNA delivery for various in vitro and in vivo applications.

Chemical modification of siRNAs to improve serum stability without loss of efficacy

Development of RNA interference as a novel class of therapeutics requires improved pharmacokinetic properties of short interfering RNA (siRNA). To confer enhanced serum stability to Sur10058, a hyperfunctional siRNA which targets survivin mRNA, a systematic modification at the 2′-sugar position and phosphodiester linkage was introduced into Sur10058. End modification of three terminal nucleotides by 2′-OMe and phosphorothioate substitutions resulted in a modest increase in serum stability, with 3′ end modification being more effective. Alternating modification by 2′-OMe substitution significantly stabilized Sur10058, whereas phosphorothioate modification was only marginally effective. Through various combinations of 2′-OMe, 2′-F and phosphorothioate modifications that were directed mainly at pyrimidine nucleotides, we have identified several remarkably stable as well as efficient forms of Sur10058. Thus, our results provide an effective means to stabilize siRNA in human serum without compromising the knockdown efficiency. This advancement will prove useful for augmenting the in vivo potency of RNA interference.

Activation of the mammalian immune system by siRNAs

Inhibition of gene expression through RNA interference (RNAi) is emerging as a powerful experimental tool for gene function and target validation studies. The potential uses of this technology seem unlimited, extending to the prevention and therapy of human diseases. However, recent work demonstrating that there are unanticipated, different nonspecific effects associated with the use of small interfering RNAs in mammals has raised concerns about the safe use of RNAi in vivo. These nonspecific effects include activation of the immune system, potentially harming the individual. The application of screening assays for nonspecific activation of both innate and acquired immunity will be necessary for further development of RNAi as a therapeutic tool.

52. AAV-Delivered RNAi Improves Cellular and Motor Phenotypes in a Mouse Model for Huntington's Disease

Top of pageAbstract Huntington's Disease (HD) is a fatal, dominant neurodegenerative disorder caused by polyglutamine expansion (CAG codon, Q) in the protein huntingtin (htt). Hallmark HD characteristics include cognitive and behavioral disturbance, involuntary movements (chorea), neuronal inclusions, and striatal and cortical neurodegeneration. Therapies affecting events downstream of disease allele expression have been tested in HD animal models, but no effective treatment currently exists. Moreover, no therapy has been described that directly reduces mutant huntingtin gene expression, thereby targeting the fundamental, underlying pathological insult. RNA interference (RNAi) has emerged as a potential therapeutic tool for treating dominant diseases by directly reducing disease gene expression. We hypothesized that reduction of mutant htt by small hairpin RNA (shRNA) would improve HD-associated phenotypes in a transgenic mouse model for HD (HD-N171-82Q). We identified a U6 promoter-driven shRNA (shHD2.1) that caused a dose-dependent reduction of human htt transcript and protein in HEK 293 cells in contrast to a control hairpin targeting E. coli |[beta]|-galactosidase. AAV1s expressing shHD2.1 injected into HD-N171-82Q mice reduced human htt transgene levels by |[sim]|50% compared to those injected with AAV1.shLacZ. Additionally, the striatal and cerebellar inclusions evident in untreated or AAVshLacZ treated HD mice were significantly reduced following AAV1.shHD2.1 gene transfer. Finally, AAV1.shHD2.1, but not AAV1.shLacZ, led to a statistically significant correction of HD-associated motor abnormalities in the HD-N171-82Q mice as determined by rotarod performance and gait analysis. These exciting in vivo results show that shHD2.1 reduces htt mRNA, prevents protein aggregate formation, and improves motor and gait abnormalities in an HD mouse model. Our data provide support for the further development of RNAi for HD therapy.

RNA interference targeting cathepsin L and Z-like cysteine proteases of Onchocerca volvulus confirmed their essential function during L3 molting

We describe the successful use of RNA interference (RNAi) to investigate gene function in the human filarial parasite Onchocerca volvulus third-stage larvae (L3). We targeted two specific gene products, the O. volvulus cathepsin L (Ov-CPL) and cathepsin Z-like (Ov-CPZ) cysteine proteases, which were proposed to function during O. volvulus L3 molting. We show that fluorescent-labeled Cy3-dsRNA corresponding to cpl or cpz regions encoding the mature enzymes can enter the larvae. The molting rate of larvae treated overnight with 0.5 mg ml(-1) cpl was reduced by 92% and 86% in comparison to normal control worms. It appeared that although the larvae started the molting process the last stage of molting, ecdysis was inhibited. The effect was gene specific, as larvae that did not molt in the presence of cpl or cpz dsRNA expressed the other cysteine protease, CPZ and CPL, respectively. This was confirmed by immunoelectron microscopy using antibodies directed against each enzyme. Our present study validate conclusively that both enzymes are essential for the molting of O. volvulus L3 to fourth-stage larvae. We also confirmed that the activity of the enzymes is specific to the changes that occur during the molting process on days 1-3, when the separation between the cuticles is in progress. The development of RNAi in O. volvulus L3 could further help study many of the abundant L3 and molting L3 genes identified through the filarial genome project, many of which, although have no attributed function, were identified as vaccine candidates or potential drug targets.