Applied RNAi: From Fundamental Research to Therapeutic Applications | Book
Caister Academic Press
and Marc S. Weinberg1,2 1Antiviral Gene Therapy Research Unit, School of Pathology, University of the Witwatersrand, WITS 2050, South Africa; 2Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N Torrey Pines Rd., La Jolla, 92037, USA.
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Since the discovery of RNA interference (RNAi) in 1998, research on the topic has advanced at an impressive pace. Small RNAs, and in particular micro RNAs (miRNAs), play a fundamental role in gene regulation through the activation of RNAi. Detailed insights into the mechanisms of RNAi have led to an improved understanding of gene regulation in normal and disease states, and thereby enabled the exploitation of RNAi for a variety of applications.
In this book an international panel of RNAi experts critically reviews the most interesting advances in basic applied RNAi research, highlighting the applications in RNAi-based therapies and discussing the technical hurdles that remain. Topics covering the fundamental biological aspects of applied RNAi research include: the role of miRNAs in trinucleotide repeat disorders; miRNAs and HIV pathogenesis; miRNAs for epigenetic gene silencing; the role of miRNAs in virus-related cancers; non-canonical miRNA biogenesis. In the area of RNAi-based therapy, topics include: harnessing RNAi for the treatment of viral infections; optimising the design of exogenous RNAi activators; blocking miRNA function with synthetic agents; somatic cell reprogramming; high-content miRNA-based screening tools and the use of miRNA target sites for control of transgene expression.
Essential reading for everyone involved in RNAi research, drug discovery and delivery, biomedical engineering and biomaterials.
Overview of Biogenesis and Applications of MicroRNA
Patrick Arbuthnot and Marc S. Weinberg
Intensive research on the RNA interference (RNAi) pathway has provided detailed understanding of the intricacies of gene silencing by small RNAs. Insights into the regulation of micro RNA (miRNA) biogenesis have revealed a complex interplay of controlling mechanisms. Efficacies of miRNAs are potentially influenced by modulation at all steps of their biogenesis: transcription of primary miRNAs, nuclear processing and export, cytoplasmic Dicer cleavage of precursor miRNAs (pre-miRNAs) and loading of mature miRNAs onto the RNA Induced Silencing Complex (RISC). In addition competing endogenous sequences, especially circular RNA sponges, may function to sequester miRNAs and attenuate their effects on mRNA targets. Disruption of miRNA function is implicated in disease processes such as cancer and modulating natural miRNA function has potential for treating diseases. Remarkably, miRNAs may be secreted from cells and detection of particular miRNA profiles in serum has diagnostic utility. Synthetic and expressed RNAi activators are being applied to therapeutic inhibition of pathology-causing genes. Also incorporating miRNA targets into untranslated sequences has provided a useful mechanism of regulating therapeutic transgene expression. RNAi research has matured and remains an exciting field of investigation. Improved understanding has now provided the means for powerful application.
Non-Canonical MicroRNA Biogenesis and Function
Thomas C. Roberts and Matthew J.A. Wood
MicroRNAs (miRNAs) are small, single-stranded non-coding RNAs that act primarily as post-transcriptional negative regulators of gene expression and are implicated in a wide range of cellular processes and pathological conditions. As such, miRNAs are being investigated both as disease biomarkers and as novel therapeutic targets for the treatment of a plethora of disease conditions including cancers, viral infections and genetic disorders. Here we consider non-canonical processing and functions of miRNAs. Alternative sources of miRNAs are described including Dicer-negative miRNAs, mirtrons, endo-shRNAs as well as miRNAs derived from snoRNAs and tRNAs. Non-miRNA small RNA biogenesis pathways are also briefly discussed including piRNAs, endo-siRNAs and tiRNAs. In addition, we discuss the causes and consequences of heterogeneity in processing and post-transcriptional editing of miRNAs. Lastly, we consider non-canonical miRNA functions including miRNAs that activate translation or regulate gene expression at the epigenetic level, interactions between miRNAs and long non-coding RNAs (lncRNAs), regulation of miRNA activity by endogenous miRNA sponges, and the role of miRNAs in the extracellular environment.
Non-coding RNAs and the Epigenetic Control of Gene Expression
Kevin V. Morris
A paradigm shift is occurring in molecular biology with the realization that much of the genome is transcribed as non-coding RNA and that these non-coding transcripts are biologically relevant. Many examples have now been provided whereby long non-coding RNAs that are antisense to their protein-coding counterpart are actually involved in epigenetically regulating the protein coding genes expression. Importantly, small non-coding RNAs can be utilized to either take advantage of these endogenous non-coding pathways of gene regulation and silence gene expression or to target regulatory non-coding RNAs and de-repress particular protein-coding genes, essentially turning on a genes expression. Knowledge of this emerging RNA based epigenetic regulatory network and our ability to control gene expression has deep implications in the development of entirely new areas of therapeutics.
From Mice to Men: Towards the Clinical Translation of miRNA Technologies for Somatic Cell Reprogramming
Elena Senís and Dirk Grimm
The 2006/2007 discovery that somatic cells can be reprogrammed to induced pluripotent stem cells through the introduction of a defined class of transcription factors has revolutionized the field of regenerative medicine. More recently, the additional finding that the RNAi machinery and in particular miRNAs are fundamentally involved in pluripotency has sparked a flurry of efforts to develop new technologies for miRNA-regulated reprogramming of human cells. Here, we summarize and critically review the latest literature describing the three most prominent advances in this field, with a specific focus on the underlying methodologies and their clinical relevance. First, we compare the different experimental approaches - forward versus reverse screening - to identify endogenous miRNAs that are inherently involved in the induction and/or maintenance of pluripotency. Second, we comprehensively discuss the principal strategies for deliberate dysregulation of these miRNAs in order to reprogram somatic cells that have been tested thus far, from viral vector-mediated miRNA expression along with classical pluripotency factors, to exclusive reprogramming through delivery of “naked” miRNAs. Third and last, we highlight novel innovative concepts that exploit the inherent alterations in miRNA expression profiles between pluripotent and differentiated cells, with the aim to mark and track these distinct subpopulations, or to purify one cell type from contaminations with the other. Finally, we conclude with our outlook into the possible future of human cell reprogramming and with suggestions for improvements to existing miRNA technologies that may further foster their clinical translation.
Systems Biology Tools to Understand the Role of Host miRNAs in Infection: A Closer Look at HIV
Jerolen Naidoo, Robyn Brackin, Rethabile Khutlang, Anca Savulescu and Musa M. Mhlanga
The discovery of mammalian microRNAs (miRNAs) has greatly enhanced our appreciation for the complexity associated with the regulation of the mammalian transcriptional landscape. Endogenous miRNA pathways mediate the targeted and subtle variations in gene expression required to drive complex biological processes that must be coordinated both spatially and temporally within cells and tissues. It is not surprising then that the dysregulation of miRNA function has been implicated in various models of disease and pathogenesis. Increasing interest in miRNA function has facilitated the transfer of many existing technologies to miRNA-based formats. Expression-based tools like RNAseq and qPCR microarray technologies, as well as the use of synthetic molecules to inhibit or enhance miRNA functions have been employed to identify and characterize distinct miRNA expression profiles in various models of infection. In this chapter we take a closer look at the application of some of the existing tools for miRNA-based analyses with a focus on host-pathogen interactions. Aspects pertinent to high-content miRNA-based screens are also discussed using an HIV screening workflow as a backdrop to address the important considerations associated with miRNA-based studies.
Synthetic microRNA Blocking Agents
Peter Järver, Adrian G. Torres and Michael J. Gait
This chapter describes how synthetic miRNA blocking agents may be used to inhibit miRNA action in cells and in vivo. Particular emphasis is given to chemically synthesized oligonucleotide anti-miRs, types of synthetic analogues, mechanisms of action, and methods used to validate their activities. Also included are brief descriptions of miRNA sponges that are also used to inhibit miRNA activity, as well as other techniques, including use of mRNA target protectors and synthetic miRNA mimics.
Exploiting microRNAs to Regulate Transgene Expression
Virginie Pichard, Dejana Ivacik and Nicolas Ferry
RNA interference (RNAi) is as highly-conserved gene regulatory mechanism which is triggered by double-stranded RNA. The discovery of this naturally functioning gene silencing mechanism has contributed to a deeper understanding of the function and regulation of eukaryotic genes. In mammalian cells, RNAi functions in regulating gene expression via small, non-coding RNA molecules, known as microRNAs (miRNAs). Over the past decade, numerous studies have emphasized the role of miRNAs as powerful transcriptome regulators highlighting the potential usefulness of these small RNA effecters to regulate transgene expression. Precise tissue-specific control of transgene expression is a prerequisite for many investigational and therapeutic applications involving gene transfer. Consequently, miRNAs have been exploited by gene therapists to improve transcriptional regulation of gene transfer vectors and accomplish tight spatial and temporal regulation of transgene expression in different therapeutic contexts. In this chapter, we will emphasize the versatility of miRNAs to improve the efficacy of many gene therapy applications, with a particular focus on their potential for improving specificity and safety of gene therapy as well as their role in averting immune-mediated clearance of gene-modified cells.
Use of Artificial microRNAs for Gene Silencing
Betty Mowa and Abdullah Ely
The discovery of the RNA interference (RNAi) pathway in 1998 resulted in intense excitement and interest in elucidating the mechanisms of this gene regulatory pathway. These studies laid the groundwork for manipulating the RNAi pathway to silence genes of interest in functional genomics studies and for therapeutic purposes. Mammalian RNAi is mainly activated by non-coding double stranded RNA molecules called microRNAs (miRNAs). Induction of RNAi can be achieved with exogenous RNA molecules that mimic the various intermediates of the pathway. This review discusses application of RNAi intermediate mimics for therapeutic gene silencing in mammalian cells. Like any other drug development strategy, using gene silencing as a therapeutic approach has not been without challenges. The most daunting of which include off-targeting effects, toxicity and identifying effective delivery vehicles. Intense dedication to overcome these problems has led to the approval of antisense based drugs for clinical use and progression of many more gene silencing lead candidates to clinical trials. The same dedication will see the use of artificial miRNAs in clinical settings.
Harnessing RNAi for the Treatment of Viral Infections
Lorea Blazquez and Puri Fortes
No treatment currently exists for many devastating viral infections. These include infections with emerging viruses or with viruses such as influenza that change very rapidly. Other viral infections can be treated, but the therapies may exert damaging unwanted side effects. Moreover, many viruses may evolve easily to drug resistant variants. Therefore, development of novel therapies for the treatment of viral infections is mandatory. RNA interference (RNAi) is a widely used technique to inhibit gene expression with a tremendous potential as an antiviral. Since RNAi degrades RNA in a sequence-specific manner, the only requirement for the development of RNAi inhibitors is to know the sequence of the target gene or the viral genome. Furthermore, host cell factors that are essential for viral infection but dispensable for cell viability, can be easily identified with genome-wide screenings and targeted by RNAi. The combination of different RNAi inhibitors, or the combination of RNAi with alternative therapies, should avoid the emergence of escape mutants resistant to the treatment. Over the last decade, several studies have used synthetic or gene-expressed short-interfering RNAs (siRNAs) to treat viral infections. Despite promising results in preclinical models, translation to clinical trials has been slow. Delivery remains the main challenge for the therapeutic application of RNAi. Several chemical formulations have been developed that increase delivery, stability and specificity of synthetic RNAi inhibitors or that target them to a specific organ. Several viral and non viral vectors have been employed to increase the delivery or the targeting of gene-expressed siRNAs. However, further efforts to improve delivery and decrease unwanted side effects of RNAi are still required. In the present review we discuss the current status of RNAi as an antiviral for the treatment of viral hepatitis, haemorrhagic fever viruses, respiratory viruses and other viruses.
Roles of miRNAs in Cancers Associated with Human Tumor Viruses
Xianzhi Lin, Deguang Liang and and Ke Lan
Considerable accumulated lines of evidence have shown that microRNAs (miRNAs) play important roles in regulating gene expression in many critical physiological conditions as well as disease progression. The regulation of gene expression by miRNAs could affect mRNA cleavage, mRNA degradation, and inhibition of mRNA translation. Seven tumor viruses have so far been identified in humans. The infection of the host by these human tumor viruses could result in the development of many severe diseases or even several malignant ones like cancers. These human tumor viruses have taken advantage of the pathways that cellular miRNAs have been involved in by encoding their own viral miRNAs and/or hijacking host miRNAs. In this chapter, we will review recent advances on the roles miRNAs played in cancers associated with human tumor viruses, with the focus on human tumorigenic herpesviruses.
MicroRNAs as Cancer Biomarkers
David Otaegui and Charles H. Lawrie
Despite having been only formally recognized for just over ten years, microRNAs (miRNAs) have become one of the hottest topics in biology. In this short time these endogenously produced small (19-22nt) ssRNA post-transcriptional regulators have been found to play crucial roles in many, if not all, physiological and pathological processes including cancer. The importance of miRNAs to carcinogenesis is implied by the fact that many miRNAs are encoded at cancer-associated regions of the genome, and there is now overwhelming evidence that aberrant expression of miRNAs is a ubiquitous characteristic of malignancy. As well as clear therapeutic implications for miRNAs, the remarkable stability of these molecules coupled with their ability to discriminate between different cancers to a degree that surpasses traditional genes, suggests great potential as cancer biomarkers. In this chapter we review the accumulating evidence for this assertion, and provide an review of the major techniques used in miRNA biomarker studies (e.g. microarray, qRT-PCR and next generation sequencing etc.), discussing their relative strengths and weaknesses.
MicroRNA Deregulation in Trinucleotide Repeat Expansion Disorders
Edyta Koscianska, Emilia Kozlowska, Edyta Jaworska, and Wlodzimierz J. Krzyzosiak
Trinucleotide repeat expansion disorders (TREDs) constitute a group of dominantly inherited neurological diseases that are incurable and ultimately fatal. The underlying cause of TREDs is an expansion of trinucleotide repeats that may occur in the coding and non-coding regions of human genes. MicroRNAs (miRNAs) have emerged as potent regulators of gene expression at the posttranscriptional level. They are involved in a variety of physiological and pathological processes in humans, and the alteration of miRNA expression is considered to be a hallmark of many diseases, including TREDs. This review summarizes the current knowledge regarding the involvement of miRNAs in the pathogenesis of TREDs and the experimentally proven associations between specific miRNAs and particular disorders that have been reported to date.
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(EAN: 9781908230430 9781908230676 Subjects: [genomics] [molecular biology] [epigenetics] [virology] )