Gene Therapy for Viral Infections - Patrick Arbuthnot

Gene Therapy for Viral Infections (eBook)

Patrick Arbuthnot (Autor)

eBook Download: PDF | EPUB

2015 | 1. Auflage
392 Seiten
Elsevier Science (Verlag)
978-0-12-411452-4 (ISBN)

Gene Therapy for Viral Infections provides a comprehensive review of the broader field of nucleic acid and its use in treating viral infections. The text bridges the gap between basic science and important clinical applications of the technology, providing a systematic, integrated review of the advances in nucleic acid-based antiviral drugs and the potential advantages of new technologies over current treatment options.ÿ Coverage begins with the fundamentals, exploring varying topics, including harnessing RNAi to silence viral gene expression, antiviral gene editing, viral gene therapy vectors, and non-viral vectors. Subsequent sections include detailed coverage of the developing use of gene therapy for the treatment of specific infections, the principles of rational design of antivirals, and the hurdles that currently face the further advancement of gene therapy technology. - Provides coverage of gene therapy for a variety of infections, including HBV, HCV, HIV, hemorrhagic fever viruses, and respiratory and other viral infections - Bridges the gap between the basic science and the important medical applications of this technology - Features a broad approach to the topic, including an essential overview and the applications of gene therapy, synthetic RNA, and other antiviral strategies that involve nucleic acid engineering - Presents perspectives on the future use of nucleic acids as a novel class of antiviral drugs - Arms the reader with the cutting-edge information needed to stay abreast of this developing field

Patrick Arbuthnot is currently a personal professor and director of the Antiviral Gene Therapy Research Unit at the University of the Witwatersrand in Johannesburg, South Africa. After completing a medical degree and then a PhD, he carried out post-doctoral research on gene therapy for liver diseases at Necker Hospital in Paris. For more than ten years, his primary research focus has been on advancing gene silencing and gene editing technologies to develop improved treatment of chronic hepatitis B virus infection. In addition, he has worked on furthering methods of disabling genes of HIV-1, Rift Valley Fever virus and hepatitis C virus. Dr Arbuthnot has been an author of numerous publications and edited scientific books on topics related to antiviral gene therapy.

Chapter 2

Harnessing RNAi to Silence Viral Gene Expression

Abstract

Since discovery of the RNA interference (RNAi) pathway, there has been rapid progress in research aimed at harnessing this gene silencing mechanism for antiviral therapeutic application. Micro RNAs (miRs) are the prototype natural activators of RNAi. Their maturation entails a regulated stepwise process during which transcripts with hairpin motifs within primary miRs (pri-miRs) are cleaved by the nuclear microprocessor complex to generate precursor miRs (pre-miRs). pre-miRs are exported to the cytoplasm before further processing by Dicer, an RNase III, to generate the mature miR comprising a short duplex RNA sequence. One of the strands is selected for incorporation into the RNA induced silencing complex (RISC), where it naturally serves as a guide to direct translational suppression of mRNA targets. Cellular and viral mechanisms operate to control miR maturation and thereby modulate gene silencing by these short RNA sequences.

Many different exogenous RNAi activators have been used to reprogram RNAi to silence expression of viral genes. Synthetic silencers typically comprise short interfering RNAs (siRNAs) that mimic Dicer substrates or cleavage products. Highly efficient viral gene silencing may be achieved with these RNAi activators, and they have the particularly useful property of being amenable to chemical modification to alter their biological properties. Changes to the 2′-hydroxyl group on the ribose and phosphodiester backbone have been used to augment stability, improve specificity, and diminish immunostimulation. Nonviral vectors, such as lipoplex formulations, are used as carriers of siRNAs, and potential for large-scale synthesis of nonviral vectors is also useful for clinical application. Expressed RNAi activators are typically produced from Pol II or Pol III (e.g., U6 and H1) promoter-containing cassettes. These intracellular transcripts are engineered to mimic pri-miRs or pre-miRs. Cassettes comprising polycistronic pri-miR sequences are useful to augment silencing efficacy and limit viral escape from single gene silencing elements. The stability of the DNA templates that generate the antiviral sequences is useful for achieving sustained silencing that may be required for treatment of chronic viral infection. Another advantage of DNA expression cassettes is that they are compatible with viral vectors for efficient delivery to target tissues.

Although there has been considerable progress in the application of RNAi-based silencing to treat diseases, widespread clinical use for therapeutic inhibition of viral replication has not yet been realized. Achieving safe and efficient delivery to target tissues, minimizing off-target effects, and optimizing pharmacokinetics are particularly challenging.

Keywords

Chemical modification; Expression cassette; microRNA; RNAi; shRNA; siRNA; Viral gene silencing

2.1. Introduction

RNA interference (RNAi) is a process that operates in metazoan cells to regulate gene expression. The mechanism typically involves gene silencing by short RNAs that have their effects by base pairing to cognate complementary mRNA sequences. The report on RNAi action in Caenorhabditis elegans by Fire, Melo, and their colleagues was a significant development in the field [1]. The finding was made after observing that introducing double-stranded RNA (dsRNA) into nematodes resulted in highly effective silencing of genes with homologous sequences. To the authors’ surprise, they noticed that the inhibitory effectiveness was considerably more pronounced than when using the antisense or sense strands alone. Quelling and co-suppression, seemingly unrelated processes that had been described in plants and animals, were shown to operate by the same posttranscriptional RNAi-based gene silencing mechanism. Since publication of Fire and Melo’s article in 1998, RNAi research has advanced very rapidly. In addition to providing important insights into the complexity of gene regulation, it has been shown that RNAi may be exploited to achieve posttranscriptional silencing of almost any intended gene target. The landmark study by Elbashir, Tuschl, and colleagues was the first to prove this [2]. They demonstrated that artificial synthetic short duplex RNAs acted as exogenous activators of RNAi to reprogram the pathway in mammalian cells. Since then, many studies have described use of exogenous RNAi activators that are effective against a wide variety of pathology-causing genes, including those expressed by viruses. DNA expression cassettes and synthetic RNA sequences are being used to silence gene expression, and both classes have been shown to have potential therapeutic utility. microRNAs (miRs) are the prototype endogenous activators of RNAi, and their mechanisms of action have directed the design of potentially therapeutic exogenous silencers. Mature miRs comprise 19–24 nt of noncoding RNA that exert posttranscriptional mammalian gene inhibition by partial base pairing to target mRNA. miRs control most human genes, and individual miRs may be capable of targeting in excess of 300 different transcripts [3]. Bioinformatic analysis suggests that there are more than 45,000 miR target sites within the human genome. Almost all cellular processes, including cell division, immune responses, programmed cell death, differentiation, and development of tissue-specific phenotypes, are subject to control by miRs [4]. There are also other short RNA sequences that are capable of regulating gene expression. An example is the piwi-interacting RNAs (piRNAs) that are enriched in gonadal tissue where they function to inhibit the effects of transposons [5]. Recently, piRNAs have been shown to have broader and important regulatory functions [reviewed in ref. [6]]. These include control of genome rearrangement, epigenetic programming, and possibly cancer etiology. Effects of piRNAs are not restricted to germ cells and also manifest in somatic cells. Understandably, because RNAi plays such an important role in controlling cell functions, the mechanisms that are involved in regulating RNAi are complex and subtle. Moreover, disruption to RNAi may lead to the emergence of disease states such as cancer.

Viruses themselves are capable of expressing miRs to influence the expression or viral and host genes (reviewed in ref. [7]). In addition, viruses may alter the functioning of host miRs to affect their own proliferation. As viral gene controlling elements, miRs have several features that are useful for viruses. miRs are

• Small and require minimal coding capacity within the viral genome,

• Nonimmunogenic,

• Capable of evolving rapidly to adapt to changes in the environment, and

• Able to regulate different target sequences with varying effects.

Given the importance of miRs for viral replication, it is not surprising that many viral miRs have been described. As with miRs detected in metazoan genomes, the number of similar viral candidate regulatory sequences is constantly expanding (http://crdd.osdd.net/servers/virmirna/index.html).

Understanding the details of the processes involved in RNAi-based gene silencing is very important for using the pathway efficiently to treat viral infections. In addition to posttranscriptional silencing by RNAi activators, transcriptional regulation of gene expression by RNA has recently emerged as an important topic [8]. Exploiting this mechanism also has potential application to therapy of viral infections.

2.2. Biogenesis of miRS in Mammalian Cells

Formation of mature miRs involves a series of consecutive steps. The process is initiated by transcription of RNAs that include miR precursors and culminates with silencing that is mediated by sequence-specific interaction between mature miR guide strands and cognates on mRNA targets (Figure 2.1). Various mechanisms control each stage of the process and control of gene expression by mature miRs is subjected to regulation by various influences (reviewed in refs [9–11]; Table 2.1). Exogenous RNAi activators are subject to similar control; therefore, understanding the mechanisms of natural miR biogenesis has relevance to antiviral therapeutic application of gene silencing.

Figure 2.1 Natural biogenesis of miR.

Pol II transcripts, containing poly- or mono-cistronic miRs, are processed in the nucleus by the microprocessor complex to form pre-miR sequences of approximately 70 nt. After export to the cytoplasm, pre-miRs are cleaved by Dicer and TRBP to yield the mature miR duplex of approximately 22 bp. RISC activation follows incorporation of the duplex into the complex and removal of the passenger strand. The retained guide strand directs the complex to complementary mRNA targets. When complementarity between the target mRNA and guide strand is incomplete, translational suppression occurs, which involves mRNA degradation and impairment of ribosome function. Complete base pairing between the target and guide results in mRNA degradation. Expressed pri-miR and pre-miR sequences have been used to inhibit viral replication. Synthetic siRNAs, which enter the pathway at a more distal stage of the pathway, have also been used successfully against viruses.

2.2.1. Transcription of miR Precursors

Canonical miR biogenesis starts with Pol II-mediated transcription of sequences that include primary miRs (pri-miRs) [12]. These pri-miRs are...

Erscheint lt. Verlag	1.6.2015
Sprache	englisch
Themenwelt	Informatik ► Weitere Themen ► Bioinformatik
	Medizin / Pharmazie ► Medizinische Fachgebiete ► Biomedizin
	Studium ► 2. Studienabschnitt (Klinik) ► Humangenetik
	Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
	Naturwissenschaften ► Biologie ► Mikrobiologie / Immunologie
	Technik
ISBN-10	0-12-411452-0 / 0124114520
ISBN-13	978-0-12-411452-4 / 9780124114524

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