RNA Therapeutics: The Evolving Landscape of RNA Therapeutics

By John J. Rossi

RNA Therapeutics: The Evolving Landscape of RNA Therapeutics provides a comprehensive overview of RNA therapeutic modalities, from bench-to-bedside, with an emphasis on the increasingly impactful areas of gene therapy, oligonucleotide therapeutics, gene editing and delivery. International leaders in the field examine RNA-based therapeutics tools that have been developed to-date to modulate cellular processes such as transcription, translation and protein function. Approved RNA-based therapies and lessons learned from failed therapies are discussed in-depth, as are evolving advances in RNA biochemical analysis, and similar advances that are enabling clinical application of RNA-based therapies.

Later sections discuss delivery technologies, remaining hurdles in research and translation, the therapy development process from the lab to the clinic, and novel RNA-based therapies currently in development.

• Features leading experts in the field of RNA therapeutics, spanning all classes of RNA therapies
• Provides a detailed examination of approved RNA therapies and lessons learned from failed therapeutics
• Covers all aspects of therapeutic discovery and preclinical development, as well as clinical translation, manufacturing and regulatory aspects

• Language: English
• Publisher: Academic Press
• Release date: Apr 10, 2022
• ISBN: 9780128217399

Book preview

Section 1

The world of RNA therapeutics: Available RNA tools to modulate cellular processes

Chapter 1: Targeting chromatin: Transcriptional gene activation (saRNA)

Jon Voutilaa; Nagy A. Habiba,b    a MiNA Therapeutics Limited, London, United Kingdom

b Department of Surgery and Cancer, Imperial College London, London, United Kingdom

Abstract

It is well-recognized that siRNA and miRNA have therapeutic potential through their ability to post-transcriptionally downregulate gene expression and translation. Common targets for disease modulation with these small RNAs are mutated or aberrantly overexpressed genes. An emerging technology exploits the endogenous mechanism of transcriptional gene activation to upregulate the expression of genes altered in disease. Called small-activating RNAs (saRNAs), these short duplex RNA oligonucleotides are structurally similar to siRNA and share a dependence on Argonaute-2 (Ago2) for activity but are distinct in their mechanism. In this chapter, we will discuss the discovery and mechanism of action of RNA activation and explore the therapeutic applications that have been demonstrated preclinically and in clinical trials.

Keywords

Small-activating RNA; saRNA; RNA activation; RNAa; Transcriptional gene activation; Oligonucleotide therapy

Discovery and mechanism of action

The ability of small promoter-targeted double-stranded RNAs (dsRNAs) to activate gene expression was first reported by Li et al. in 2006 and was called RNA activation (RNAa) (Li et al., 2006). dsRNAs targeted to the promoter region less than 1000 nucleotides upstream of the transcription start site (TSS) of the genes E-cadherin, p21, and VEGF induced upregulation of the target gene mRNA up to 12.5-fold within 48–72 h. This phenomenon was soon confirmed by the Corey group showing upregulation of the progesterone receptor mRNA 3 days after transfection of promoter-targeted dsRNAs (Janowski et al., 2007). Gene activation triggered by promoter-targeted dsRNAs, later called saRNAs, was soon demonstrated in mouse, rat, and nonhuman primate cells in vitro (Huang, Qin, et al., 2010), and in mice in vivo (Turunen et al., 2009). Mouse hindlimbs injected with lentiviral VEGF-targeted shRNAs showed a significant upregulation of VEGF mRNA and ultrasound analysis showed increased blood flow, demonstrating for the first time that RNAa could drive therapeutic biological changes in vivo. Most of these saRNAs were 21-nucleotide duplexes structurally indistinguishable from siRNA, and it was demonstrated that saRNAs also require Ago2 for activity. Subsequent reports would help to clarify the distinct mechanism of RNAa and demonstrate that it is a true endogenous gene regulatory mechanism.

The introduction of any dsRNA into a mammalian cell, regardless of its intent, can introduce a range of off-target effects through sequence-specific binding to fully or partially complementary transcripts and stimulation of innate immunity (Jackson & Linsley, 2010). Since saRNA and siRNA share a dependence on Ago2 for activity, early studies were met with criticism on the lack of mechanistic distinction between the two (Check, 2007; Garber, 2006), and that the observed effects could be due to off-target siRNA- or miRNA-like downregulation activity. To rule out an RNAi cleavage mechanism, saRNAs can be made with internal sequence mismatches to prevent target cleavage (Elbashir, Martinez, Patkaniowska, Lendeckel, & Tuschl, 2001), and this does not affect the ability to upregulate gene expression (Li et al., 2006; Matsui et al., 2010, 2013; Voutila et al., 2017). The sequence specificity required for saRNA activity is more similar to miRNA binding, with the guide strand 5′ seed region critical for activity, and mismatches outside this region are generally well-tolerated (Li et al., 2006; Matsui et al., 2010, 2013; Meng et al., 2016; Voutila et al., 2017). While increases in steady-state mRNA can be due to changes in mRNA stability and turnover, the increase in saRNA target mRNA is due to an increase in transcription as measured by nuclear run-on or nascent RNA assays (Portnoy et al., 2016; Voutila et al., 2012, 2017). This is supported by ChIP assays demonstrating an increase in RNA PolII at the promoter of genes activated by saRNA (Hu et al., 2012; Matsui et al., 2010, 2013; Place, Li, Pookot, Noonan, & Dahiya, 2008; Portnoy et al., 2016) and changes in histone modifications consistent with active transcription (Janowski et al., 2007; Li et al., 2006; Matsui et al., 2013; Portnoy et al., 2016; Turunen et al., 2009; Zhang, Li, Burnett, & Rossi, 2014). The presence of Ago2 as well as the saRNA itself has also been shown at the target site (Chu, Yue, Younger, Janowski, & Corey, 2010; Hu et al., 2012; Janowski et al., 2007; Meng et al., 2016; Portnoy et al., 2016; Schwartz et al., 2008; Voutila et al., 2017; Zhang et al., 2014), supporting an on-target nuclear transcriptional activation mechanism distinct from RNAi.

Although RNAi activity in the nucleus was reported as early as 2005 (Robb, Brown, Khurana, & Rana, 2005), the presence and activity of RNAi factors in the nucleus remained controversial until RNA-induced silencing complex (RISC) members were clearly shown localized in the nucleus driving target cleavage (Gagnon, Li, Chu, Janowski, & Corey, 2014). Notably, RISC loading factors were found to be restricted to the cytoplasm. The activity of these factors in the nucleus has been linked to transcriptional gene silencing and modulation of splicing, in addition to the transcriptional gene activation described here (Kalantari, Chiang, & Corey, 2016). While most studies have identified promoter-associated noncoding RNA transcripts as the target of Ago2-guided saRNA in the nucleus (Chu et al., 2010; Matsui et al., 2010, 2013; Schwartz et al., 2008; Voutila et al., 2017; Zhang et al., 2014), there remains an argument that Ago2-saRNA could bind directly to chromatin (Meng et al., 2016; Portnoy, Huang, Place, & Li, 2011). The binding of Ago2 to this complementary sequence does not induce cleavage but provides a molecular scaffold for Ago2 partner proteins such as TNRC6A to interface with activating cofactors (Hicks et al., 2017; Liu, Johnson, Zhang, & Corey, 2019). This has been called the RNA-Induced Transcriptional Activation Complex (Portnoy et al., 2016), and has been shown to include histone-modifying factors like WDR5 and transcriptional regulators such as mediator proteins, heterogeneous nuclear ribonucleoproteins, CTR9, and RHA (Hicks et al., 2017; Hu et al., 2012; Matsui et al., 2013; Portnoy et al., 2016). This activation mechanism is an endogenous system of transcriptional regulation. Soon after RNAa was discovered, it was demonstrated that miRNAs target promoters for activation (Matsui et al., 2013; Place et al., 2008), and a growing number of miRNAs have been shown to have direct gene activating activity (Vaschetto, 2018). saRNAs retain activity when chemically modified, as will be required for therapeutic use to prevent innate immune stimulation and increase resistance to nuclease digestion (Place et al., 2012; Voutila et al., 2017; Watts et al., 2010). By exploiting this natural mechanism, researchers now have the ability to upregulate endogenous gene expression for therapeutic benefit.

Preclinical therapeutic saRNA proof-of-concept studies

Metabolic and cardiovascular disease

Oligonucleotide therapeutic development has primarily focused on local or liver delivery due to difficulties in targeting specific organs or tissues (Roberts, Langer, & Wood, 2020). The liver is a vascular organ with a high concentration of receptors that can take up systemic administration of free oligonucleotides, conjugates, or nanoparticle formulations. The saRNA field has the advantage of building upon decades of established work from antisense and siRNA delivery to the liver to impact the growing burden of metabolic and cardiovascular disease. An early example of this potential was demonstrated with the in vitro upregulation of the LDL receptor gene, which resulted in functionally increased LDLR on the surface of HepG2 cells and increased LDL particle binding (Matsui et al., 2010). A unique advantage of activating endogenous gene expression is the upregulation of targets that are traditionally seen as undruggable, such as transcription factors. It has been shown that liver-specific transcription factors such as HNF4A are downregulated in the progression of non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH) (Baciu et al., 2017). Dendrimer delivery of HNF4AsaRNA showed significant upregulation of HNF4A gene expression in the liver, leading to significant functional reduction of serum glucose, increase in HDL/LDL ratio, and reduction of liver cholesterol (Huang et al., 2020). Activation of HNF4A by saRNA may also be beneficial as NASH progresses to fibrosis and cirrhosis, as it has been shown that AAV-overexpression of HNF4A can reverse terminal liver failure in cirrhotic rats (Nishikawa et al., 2015). Activation of the transcription factor CEBPA has also been shown to reverse fibrosis and normalize liver function in mouse and rat models of CCl4- or MCD diet-induced liver injury (Reebye et al., 2018).

Outside of liver targeting, there is potential for saRNA therapies to modulate other metabolic disorders such as diabetes. Transfection of pancreatic β-cell-specific transcription factor MAFA saRNA successfully induced differentiation of CD34  + cells into glucose-sensitive, insulin-secreting cells (Reebye et al., 2013). Other cell therapies may be possible, as saRNAs have been reported to upregulate the reprogramming and pluripotency factors KLF4, OCT4, MYC, and NANOG in stem and somatic cells (Voutila et al., 2012; Wang, Sun, et al., 2016; Wang et al., 2015; Wang, Wang, Huang, Place, & Li, 2012). Looking at complications from diabetes and hyperglycemia, upregulation of iNOS via adenoviral-delivered saRNA significantly increased cGMP production and improved erectile function in diabetic rats (Wang et al., 2013). With the recent development of GLP-1 receptor targeting of oligonucleotides to the pancreas in vivo (Ämmälä et al., 2018), we expect a wave of targeted oligonucleotide therapies for pancreatic disorders over the coming years.

The first demonstration of functional RNAa in vivo was upregulation of VEGF-A leading to increased vasculature and blood flow in the muscle of mice (Turunen et al., 2009). This lentiviral delivery system was later shown to upregulate VEGF-A in the heart in a mouse model of myocardial infarction (Turunen et al., 2014). Treated animals showed a significant reduction in infarct size as measured by MRI compared to control shRNA animals. Downregulation of VEGF-A has also been implicated in preeclampsia, and upregulation by saRNA has been shown to increase trophoblast migration in vitro as a potential therapeutic intervention (Guo, Feng, Jia, Chen, & Yu, 2016). Taken together, these examples show that the activation of a single target with saRNA therapy can modulate both local and systemic complications of metabolic and cardiovascular disease.

Genetic disease

A genetic disease of haploinsufficiency is where one genomic allele of a gene has lost function due to mutation, and expression of the single unaltered allele is not sufficient for normal function. While there are more than 300 such genetic disorders identified, it is estimated that there may be many more unidentified that can cause a disease (Huang, Lee, et al., 2010). Genetic diseases with two nonfunctional alleles may be difficult or impossible to treat by upregulating endogenous gene expression, but haploinsufficiency provides a unique opportunity for saRNA therapies by upregulating the functioning allele. saRNAs have also been generated upregulating the aniridia-linked gene PAX6 which rescues some features of the haploinsufficiency phenotype in vitro (Papadimitropoulos et al., 2019). Aniridia from PAX6 haploinsufficiency can present itself in childhood or adulthood and develop progressively (Lagali et al., 2020), so intervention with upregulation of the functioning allele may prevent progression to blindness. A proof-of-principle study using FOXG1, a haploinsufficiency gene linked to Rett syndrome, showed that Foxg1 could be upregulated in the mouse neonatal brain with saRNA delivered by AAV (Fimiani, Goina, Su, Gao, & Mallamaci, 2016). For haploinsufficient diseases that cause damage in fetal or very early development, it is difficult to know whether upregulation of the normal allele later in life will provide any benefit. A clear understanding of the disease’s etiology will be necessary for any haploinsufficient saRNA target. Furthermore, the exact mutations for each patient will need to be screened and checked against known allele functions. Since both the normal and mutant allele will be upregulated, this approach would not be appropriate in cases where the mutation is a dominant-negative.

For genetic diseases where both copies are altered, there may be some benefit to upregulation where the mutant protein still has some residual activity, or the defect affects normal expression. Friedreich’s ataxia is a recessive genetic disease caused by an intronic GAA repeat expansion that induces transcriptional silencing of the FXN gene locus (Li et al., 2015). By targeting this repeat expansion with dsRNAs, it has been shown that Ago2 directs binding to the mutant transcript and a reversal of epigenetic silencing (Li, Matsui, & Corey, 2016). Target cleavage was not required for activity, but an increase in RNA PolII was not observed, suggesting that the gene activation was due to blocking of the mutation-triggered transcriptional silencing. The field would benefit from a wider examination of transcription rates and RNA PolII binding to determine if this is a common mechanism. For mutations with no residual beneficial activity, researchers can also look for potential compensatory genes that could be upregulated to rescue the effect of a genetic disorder, similar to the mechanism of the antisense drug nusinersen which compensates for SMN1 deficiency by altering productive splicing of SMN2 (Finkel et al., 2016). This would avoid any potential deleterious effects of upregulating expression of the disease-causing gene. Further work remains to establish a clear in vivo proof-of-concept for saRNA activation of endogenous gene expression in genetic disease.

Oncology

The majority of saRNA research published investigating possible therapeutic benefit so far has been its application in oncology, mostly focused on tumor suppressors and anti-metastatic genes. Tumor suppressors are attractive targets because they are frequently downregulated in cancer and their function has been widely studied. One of the first to show therapeutic potential was the p53 target gene p21, which has shown growth suppression of human bladder, prostate, liver, and colorectal cancer cell lines in vitro(Chen et al., 2008; Kosaka, Kang, Yang, & Li, 2012; Place et al., 2012; Wang et al., 2017; Yang et al., 2008). p21saRNA was chemically modified to improve serum stability and reduce innate immune stimulation and formulated in a lipid nanoparticle for intratumoral injection in prostate tumor xenografts (Place et al., 2012). Mice treated with p21 saRNA showed an increase in p21 protein in the tumor by immunohistochemistry and western blot, and had a significant reduction in tumor growth.

A number of other saRNAs targeting tumor suppressor genes have shown growth-suppressive or apoptotic activity in vitro, including KLF4 (Wang et al., 2010), WT1 (Qin et al., 2012), HIC1 (Pan et al., 2013), PAWR (Yang et al., 2013), PTEN (Li et al., 2016), and VHL (Kang et al., 2018). Upregulating the iodine transporter NIS in liver cancer cells may sensitize them to radiotherapy (Xia et al., 2016). Among those saRNA targets that have been evaluated in vivo include NKX31, which reduced tumor volume and increased survival in prostate cancer xenografts (Ren et al., 2013), and p53, which reduced tumor volume and engraftment of bladder cancer xenografts (Wang, Ge, et al., 2016). An alternative strategy is to target the ability of cancer cells to migrate and invade. Upregulation of E-cadherin by saRNA suppresses breast cancer cell migration in vitro and reduces tumor volume in xenografts (Junxia et al., 2010). Similarly, upregulation of the metastasis suppressor DPYSL3 by aptamer-delivered saRNA in orthotopic prostate cancer xenografts prevented lung and liver metastasis and significantly reduced lymph node metastasis compared to a scrambled control oligo (Li et al., 2016).

Many of these genes are reported to be downregulated in clinical samples, which make them ideal targets for saRNA upregulation. However, the frequent mutation of tumor suppressor and apoptosis-inducing genes in cancer will be a significant consideration for the development of saRNA therapeutics in oncology. Even when the target gene is unaltered, mutations in downstream effectors can prevent any benefit of target upregulation. Tumor DNA genotyping may be needed to screen for patients bearing mutations in target pathway genes. Treatment with saRNA drugs activating an intact pathway may produce selective pressure for inactivating mutations, so combination therapies may be necessary for durable responses (Venkatesan, Swanton, Taylor, & Costello, 2017). Opportunities to synergize with existing or emerging treatments may provide a greater chance for success.

Clinical-stage therapeutic saRNA

The first saRNA therapeutic drug to be tested in the clinic was MTL-CEBPA in 2016, developed by MiNA Therapeutics. MTL-CEBPA is a liposomal-formulated saRNA that upregulates the gene CEBPA (Reebye et al., 2018; Voutila et al., 2017). The CCATT/enhancer-binding protein alpha (C/EBPα) is a transcription factor necessary for the differentiation of hepatocyte, adipocyte, and myeloid lineage cells, and acts as a tumor suppressor through cell cycle arrest and induction of apoptosis in cancer cell lines (Lourenço & Coffer, 2017). With the potential ability to both improve liver function and reduce tumor size, CEBPA saRNA was initially tested in rat hepatocellular carcinoma (HCC) models where a significant improvement in liver function tests as well as reduction in tumor volume was observed (Reebye et al., 2014). This improvement in function and reversal of liver damage was later confirmed in mouse and rat models of cirrhosis, fibrosis, and hepatosteatosis (Reebye et al., 2018). The antitumor effect of CEBPA saRNA was explored further by other groups in mouse models of HCC (Huan et al., 2016) and pancreatic cancer (Yoon et al., 2016, 2019) with delivery using dendrimers or RNA aptamers. These promising preclinical results led to the first-in-human study of a saRNA therapeutic (First-in-Human Safety and Tolerability Study of MTL-CEBPA in Patients With Advanced Liver Cancer—Full Text View—ClinicalTrials.Gov, 2021).

In this Phase 1, open-label, dose-escalation study, 38 patients with advanced HCC or liver metastases were treated intravenously with MTL-CEBPA once per week for 3 weeks followed by 1 week rest for each treatment cycle (Sarker et al., 2020). No maximum dose was reached in the three dose levels, and of the 24 patients evaluable for efficacy, 1 patient had a partial response and 12 (50%) had stable disease. Seven patients with progressive disease were treated with tyrosine kinase inhibitors (TKIs) off-study after treatment with MTL-CEBPA. Among these patients, three had a complete response, one had a partial response, two had stable disease, and one had progressive disease. The patients with a complete or partial response had not been previously treated with TKIs. One patient with HCC and metastatic lung lesions was previously treated with transarterial chemoembolization and anti-PDL1. After disease progression with MTL-CEBPA treatment, he was treated with the TKI sorafenib and had a complete radiological response 4 months later (Fig. 1.1). It should be noted that in the trial that led to the approval of sorafenib in HCC, there were no complete responses out of 299 patients (Llovet et al., 2008). Also observed in MTL-CEBPA-treated patients was a significant increase in CEBPA mRNA in circulating white blood cells the day after treatment (Fig. 1.2), and a transient increase in white cell count and neutrophils consistent with CEBPA’s role in myeloid differentiation. Since tumor-infiltrating myeloid cells have been shown to drive resistance to TKIs in preclinical models of HCC (Chang et al., 2018; Zhou et al., 2016), it appears that MTL-CEBPA may function primarily through modulation of myeloid cells. In preclinical models of HCC, myeloid-derived suppressor cells from tumors were found to have reduced CEBPA expression, and deletion of CEBPA increases the proliferation of these cells (Mackert et al., 2017). The activity of MTL-CEBPA in cancer may then be driven by reprogramming of myeloid cell function in the tumor microenvironment, and upregulation of CEBPA may prime the tumor for treatment with TKIs or checkpoint inhibitors. These results have led to a continuation of the trial in combination with TKIs, and a Phase 1a/b study in combination with the checkpoint inhibitor pembrolizumab in patients with advanced solid tumors (A Study of MTL-CEBPA in Combination With a PD-1 Inhibitor in Patients With Advanced Solid Tumors (TIMEPOINT)—Full Text View—ClinicalTrials.Gov, 2021).

Fig. 1.1

Fig. 1.1 Radiological response in liver and lungs after treatment with MTL-CEBPA followed by sorafenib. Red arrow in bottom left image ( dark gray in print version), peritoneal metastasis/hepatic extension (which was irradiated on July 14, 2018, due to intrahepatic bleed and severe pain). Yellow arrow in bottom images ( gray in print version), HCC. Green arrows in top left image ( gray in print version), Lung mets which were no longer present on March 5, 2018 (2 months after was started sorafenib) and July 3, 2018 (2 months after sorafenib was stopped). Figure from Sarker, D., Plummer, R., Meyer, T., Sodergren, M., Basu, B., Chee, C. E., … Habib, N. (2020). MTL-CEBPA, a small activating RNA therapeutic up-regulating C/EBP-α, in patients with advanced liver cancer: A first-in-human, multi-centre, open-label, phase I trial. Clinical Cancer Research. https://doi.org/10.1158/1078-0432.CCR-20-0414. No permission required.

Fig. 1.2

Fig. 1.2 qPCR of CEBPA mRNA levels at days 2, 8, and 15 following first MTL-CEBPA treatment. Pretreatment: day of treatment before the start of MTL-CEBPA infusion; Day 2: 24 h after the first treatment; Day 8: 7 days after first treatment and day of second treatment; Day 15: 7 days after second treatment. Figure from Sarker, D., Plummer, R., Meyer, T., Sodergren, M., Basu, B., Chee, C. E., … Habib, N. (2020). MTL-CEBPA, a small activating RNA therapeutic up-regulating C/EBP-α, in patients with advanced liver cancer: A first-in-human, multi-centre, open-label, phase I trial. Clinical Cancer Research. https://doi.org/10.1158/1078-0432.CCR-20-0414. No permission required.

Conclusions

As of the end of 2020, MTL-CEBPA is the only therapeutic saRNA drug to be clinically tested. Now that saRNA-driven upregulation of target mRNA and on-target activity has been demonstrated in human patients, we expect an increasing interest in the technology. As the field grows, it would be prudent to stress the necessity of the careful evaluation of any candidate saRNAs for potential off-target effects and confirmation of on-target mechanism through the use of proper controls (Gagnon & Corey, 2019). It is critical that therapeutic saRNAs be validated as having a true on-target transcriptional activation mechanism to distinguish from off-target siRNA or miRNA-like activity. It is clear that small RNA-guided Argonaute proteins can direct a wide range of activity in the cytoplasm and nucleus. As more is learned about the distinguishing mechanisms of action, we can design better drugs in more disease areas for the next wave of saRNA therapeutics.

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Chapter 2: Argonaute and TNRC6, partners in RNAi

Samantha T. Johnson; Krystal C. Johnson; David R. Corey    UT Southwestern, Departments of Biochemistry and Pharmacology, Dallas, TX, United States

Abstract

The control of gene expression by RNA interference (RNAi) requires that small RNAs selectively recognize sequences within the transcriptome. Complementary hybridization between the guide strand of small duplex RNAs and their targets is necessary for recognition. Proteins make a critical contribution to target recognition by protecting small RNAs from degradation by enzymes, facilitating efficient recognition, and enhancing discrimination between target sequences and closely related off-target sequences. Here, we focus on the Argonaute (AGO) protein family, the factor that binds directly to small RNAs, and the Trinucleotide repeat-containing protein six (TNRC6) family, scaffolding proteins that help organize gene silencing.

Keywords

RNA interference; AGO protein; Enzyme; eCLIP; Immunoprecipitation; Cytoplasmic nuclear separation; TNRC6 protein

Introduction

RNA interference (RNAi) is a mechanism that drives recognition of mRNA by small RNAs (Gebert & MacRae, 2019). This recognition is a demanding molecular task. Target sequences must be efficiently bound while off-target interactions with other sequences must be minimized. Naturally expressed RNAs cannot efficiently recognize target sequences without assistance; they require proteins to process them, separate them into single strands, protect the strands from degradation by nucleases, and facilitate hybridization to appropriate targets.

The Argonaute (AGO) (Muller, Fazi, & Ciaudo, 2019) and trinucleotide repeat-containing protein 6 (TNRC6) (Niaz & Hussain, 2018) families include some of the most important protein factors involved in RNAi. These proteins facilitate binding to target RNA and help create the network of protein interactions necessary for translational silencing by miRNAs. Our goal in this chapter is to describe features of AGO and TNRC6 protein families and provide practical information central to their study.

Argonaute protein

Argonaute and RNAi

AGO proteins are the critical effector proteins of RNAi. There are four mammalian AGO protein variants, AGO1–4 (Sala, Chandrasekhar, & Vidigal, 2020). AGO proteins bind small guide strand RNAs to form a ribonucleoprotein complex capable of efficiently recognizing complementary nucleic acid sequences. Typically, recognition is thought to occur in the cytoplasm within mRNA, but other types of sequences are also accessible including sequences within cell nuclei (Kalantari, Chiang, & Corey, 2016).

The small duplex RNAs involved in RNA are composed of a guide strand and a passenger strand (Fig. 2.1A). The RNA guide strand directs the complex to specific RNA targets, while AGO protects the RNA and facilitates efficient recognition of target sequences within the transcriptome. AGO protein and the loaded small RNA guide strand are the minimal requirements for the RNA-induced silencing complex (RISC) and RNA interference (RNAi) (Fig. 2.1B). After RISC recognizes a target sequence within an mRNA target, the translation of the mRNA target will either be silenced by direct endonucleolytic cleavage (Fig. 2.1C) or repressed by recruitment of degradation factors (Fig. 2.1D; Huntzinger & Izaurralde, 2011).

Fig. 2.1

Fig. 2.1 RNA interference in the cytoplasm of mammalian cells. (A) Small duplex RNAs are either exogenously introduced, such as siRNAs, or endogenously transcribed and exported to the cytoplasm, such as miRNAs. One strand of the duplex, known as the guide strand, is selected and loaded into Argonaute protein by the RISC loading complex, composed of the RNase III enzyme Drosha and its cofactor DGCR8 (DiGeorge syndrome critical region 8). (B) The minimal requirements for the RNA-induced silencing complex (RISC) and RNA interference (RNAi) are an Argonaute effector protein and a guide RNA. Depending on the degree of complementarity, the target is either (C) cleaved directly by AGO2 or (D) translationally repressed by recruiting deadenylation and decay factors through interaction with scaffolding protein, TNRC6.

The mechanism of gene silencing is determined by the complementarity of the guide RNA to the target mRNA and by the identity of the associated AGO protein. AGO2 is the only mammalian AGO capable of endonucleolytic cleavage (Liu et al., 2004; Meister et al., 2004). Cleavage occurs when the guide RNA is fully complementary to the RNA target.

Full complementarity is often the case when the RNA is an exogenously introduced small interfering RNA (siRNA). siRNAs are typically designed to target specific genes to knock down gene expression. Since their discovery as tools for gene silencing in 2001 (Elbashir et al., 2001), siRNAs have proven their value as common tools for laboratory experimentation and therapeutic drug