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RNAi - Articles and news items
As RNA interference (RNAi) enters its teenage years from the first critical observations, it has now reached a multi-billion pound industry. There are few research areas that have expanded as quickly and spectacularly as the field of RNAi. The potential of RNAi initially sparked a functional genomics gold rush. Different uses of this technology in genomewide screens have identified genes involved in fundamental biological processes. There are now hundreds of research papers reporting genome-wide screens using cell culture to investigate the building blocks of the cell. However tempting it may be to speculate that this technology could be the new magic bullet to all our research needs, especially after some of the previous successes, some basic aspects of the RNAi technology and screening process still need to be addressed and improved upon. This review will investigate the strengths and weaknesses of our current technology, suggesting improvements and highlighting some of the novel growth areas in this field.
Our foundations of cell biology rely upon an understanding of cellular pathways, the components of which have been investigated over the last 40 years or so. Recent embellish – ment of the pathways has been carried out using models in cell culture with RNAi technology1. Many techniques have been used to reveal the functions of core pathway proteins, but few have sparked the imagination like the RNAi screen with the potential to systematically knock down the expression of every gene in the genome.
In 2001, small interfering RNA (siRNA) was discovered as the mediator of RNA interference (RNAi), a transient and specific repression mechanism of protein expression1. After the pharmaceutical industry became aware of the intrinsic versatility and potential of this molecule, a race to develop the first siRNA based drug began. However, the initial hype was followed by the realisation that due to the specific properties of this very fragile molecule, stability and delivery issues might limit its application to certain niche indications.
siRNAs have been rushed into the clinics before fully understanding their biological effects. As a result, some of the big pharmaceutical companies such as Roche or Pfizer, who were initially committed to siRNA drug development, have meanwhile scaled back their efforts or entirely stopped their siRNA programs.
An important property of siRNA to be controlled during the drug discovery process is its potential off-target effect, which limits its specificity. The key to developing a successful drug based on a well characterised siRNA molecule is its formulation, since the molecule is relatively big, heavily charged and susceptible to degradation in the body fluids, therefore, the delivery vehicle has to provide protection as well as enable cell penetration and release. An overview of the delivery-enabling excipients which have progressed into clinics can be found elsewhere2. Although so far no siRNA based therapeutic product has been commercialised, several clinical trials have been conducted or are currently on-going.
“We are very pleased to sponsor the joint research programs…”
microRNAs (miRNA) are a class of non-coding RNA that regulate the precise amounts of proteins expressed in a cell at a given time. These molecules were discovered in worms in 1993 and only known to exist in humans in the last decade. Despite the youth of the miRNA field, miRNA misexpression is known to occur in a range of human disease conditions and drugs based on modulating miRNA expression are now in development for treatment of cancer, cardiovascular, metabolic and inflammatory diseases. In the last six years, an increasing number of reports have also illuminated diverse roles of cellular miRNAs in viral infection and a miRNA-targeting therapy is currently in phase II clinical trials for treatment of the Hepatitis C virus. Here we review the literature related to miRNAs that regulate viral replication and highlight the factors that will influence the use of miRNA manipulation as a broader antiviral therapeutic strategy.
microRNAs (miRNA) are a class of small noncoding RNA that bind to messenger RNAs (mRNA) and regulate the amount of specific proteins that get expressed. These small RNAs are derived from longer primary transcripts that fold back on themselves to produce stem-loop structures which are recognised and processed by Drosha and co-factors in the nucleus followed by Dicer and co-factors in the cytoplasm, resulting in a ~ 22 nucleotide (nt) duplex RNA, for review see1,2. One strand of the duplex is preferentially incorporated into the RNA-induced silencing complex (RISC) where it then mediates binding to target mRNAs. These interactions lead to decreased protein getting produced from the transcript, due to RNA destabilisation and/or inhibited translation3 (Figure 1). miRNA-mRNA recognition generally requires perfect complementarity with only the first 6-8 nt of a miRNA, termed the ‘seed’ site4. Each miRNA therefore has the potential to interact with hundreds of target mRNAs3,4 and the majority of human protein-coding genes contain miRNA binding sites under selective pressure5. Therapeutic interest in miRNAs has been supported by studies in model organisms demonstrating key functions of individual miRNAs in cancer, cardiac disease, metabolic disease, neuronal and immune cell function6.
Genomics, Issue 5 2011 / 19 October 2011 / Marie Lundbæk, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology and Pål Sætrom, Department of Cancer Research and Molecular Medicine & Department of Computer and Information Science, Norwegian University of Science and Technology
RNA interference (RNAi) is now a standard tool in molecular biology. Short interfering RNAs (siRNAs) for knocking down your favourite human gene are only a couple of mouse-clicks away at your favourite reagent supplier’s website. Moreover, in contrast to initial attempts at siRNA design, these siRNAs usually give potent target gene knockdown. Nevertheless, siRNAs are not always a cure-all; therapeutic settings often require combinatorial treatments and may necessitate effects that are incompatible with standard siRNAs, such as targeted gene up-regulation. Here, we review the features of standard siRNAs before describing three unconventional but therapeutically relevant approaches to RNAi: multi-targeting siRNAs, immunostimulatory siRNAs, and transcription-modulating siRNAs.
Fire and Mello coined the term RNA interference when they discovered that long doublestranded RNAs cause sequence specific gene inhibition in worms1,2. The enzyme Dicer processes such long double-stranded RNAs into short double-stranded ~22 nt duplexes with 2 nt 3’ overhangs – the siRNAs. Argonaute 2 (Ago2) then incorporates one of the siRNA strands and uses the strand as a guide to bind and cleave single-stranded RNAs such as messenger RNAs (mRNAs).
Whitepapers / 1 August 2011 / Integrated DNA Technologies
Dr John Rossi is Chair and Professor of the Department of Molecular and Cellular Biology at the Beckman Research Institute of City of Hope (Duarte, CA). His research focuses on the biology and applications of eukaryotic small RNAs and, in particular, their therapeutic use in HIV/AIDS and cancer. One of IDT’s Scientific Writers recently had the opportunity to talk with Dr Rossi about the field of RNAi as therapeutics and his own path in science.
Recent advances in RNA interference (RNAi) technology and availability of RNAi libraries in various formats and genome coverage have impacted the direction and speed of drug target discovery and validation efforts. After the introduction of RNAi inducing reaagent libraries in various formats, systematic functional genome screens have been performed to query the functions of individual genes, pathways or an entire genome in many disease areas, including cancer, viral pathogenesis and others. As a consequence of these screens, novel mediators of cellular response to disease pathogenesis or treatment approaches have been identified leading to the discovery of novel drug targets, development of combinatorial treatment approaches and patient selection biomarkers.
Improved understanding of the molecular alterations in cancer cells has fuelled the development of more specific and directed cancer therapies. However, it has become clear that response rates can be low due to confounding genetic alterations that render these highly specific therapies ineffective. As a result, the costs of cancer treatment will increase enormously unless we are able to identify those patients that will benefit most from these directed therapies. In addition, it will be necessary to identify additional targets in these complex molecular networks that can be further exploited to increase overall response rates in the highly heterogenic populations of human tumours. In recent years, great expectations have been put forward for the use of functional genomic screening technologies to reach these goals.
In the past decade, the pharmaceutical industry has exploited the naturally occurring cellular RNAi pathway to enhance drug discovery research. The RNAi pathway, triggered by dsRNA, selectively, although not always specifically, degrades mRNA leading to substantial decreases in post-transcriptional gene expression1. Researchers have capitalised on this intrinsic pathway by synthesising RNAi reagents to modify the expression of any desired gene. RNAi libraries consisting of synthetic siRNAs or plasmid based shRNAs are amendable to largescale genome-wide screening campaigns to search for new therapeutic targets. Such loss of function screens can reveal novel targets and synthetic lethal interactions for cancer therapy2,3. These screens have also been used to identify novel host factors for diseases such as Hepatitis C4-7 and HIV8-14. Selective gene silencing can deconvolute molecular pathways implicated in disease onset and progression15.
Since the discovery of RNA interference (RNAi) in 19981 and the demonstration of RNAi in mammalian cells in 20012, research into the mechanisms and applications of this pathway has moved swiftly. RNAi is capable of mediating potent and specific silencing of genes and has therefore shown promise in the development of alternative anti-viral therapies with the potential to avoid disadvantages associated with conventional drug regimens. A number of synthetic and expressed constructs have been investigated against HIV with varying success. Despite rapid progress, important hurdles need to be surmounted before a safe, effective and widely applicable therapy can be implemented clinically. Here, we review different RNAi-based strategies against HIV and highlight future developments necessary for the realisation of an effective anti-HIV therapy.
Issue 6 2009 / 12 December 2009 /
The use of RNAi screening to identify potential drug targets has enjoyed great success in recent years as a robust method for linking genes to a disease process through a functional assessment of a gene in an experimental model1. True, RNAi screening is complicated by problems such as off-target effects and toxicities associated with various properties of RNAi molecules, but effort from many groups has produced a coherent set of guidelines that are useful for most RNAi screens2-4. As such, large scale RNAi screens can be performed by moderately-resourced laboratories. RNAi screens have identified genes involved in resistance to anticancer chemotherapeutics, viral and bacterial infection and specific signal transduction pathways. Now that the technical complications have been identified and largely mitigated, RNAi screening has joined other genomic technologies, such as transcriptional profiling, as a method for broadly surveying the human genome for roles in a given disease or a response to a treatment.
The field of oligonucleotide-based therapy experienced a revival with the discovery of RNA interference (RNAi) in 19981. RNAi is a conserved endogenous mechanism, which is triggered by double-stranded (ds) RNAs leading to target-specific inhibition of gene expression by promoting mRNA degradation or translational repression. There are two RNAi pathways that are guided either by small interfering RNAs (siRNAs), which are perfectly complementary to the mRNA or by microRNAs (miRNAs), which bind imperfectly to their target mRNA2. SiRNAs can also induce direct transcriptional gene silencing (TGS) in the nucleus, although the mechanisms underlying this are well understood in mammalian systems3,4.