key: cord-0738943-zdtxzb7p
authors: Yu, Ai-Ming; Choi, Young Hee; Tu, Mei-Juan
title: RNA Drugs and RNA Targets for Small Molecules: Principles, Progress, and Challenges
date: 2020-10-03
journal: Pharmacol Rev
DOI: 10.1124/pr.120.019554
sha: 91f1bcdc2d1c51d989826008e10d23d99870f16e
doc_id: 738943
cord_uid: zdtxzb7p

RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.

Abstract--RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed.

Significance Statement--With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.

Therapeutic drugs act on corresponding molecular targets, biological pathways, or cellular processes to elicit pharmacological effects for the treatment of human diseases. Small-molecule compounds and proteins/antibodies remain as the major forms of medications for medical use and the preferred modalities in drug development, acting mainly on protein targets such as enzymes, receptors, ion channels, transporters, and kinases (Santos et al., 2017; Usmani et al., 2017; Rock and Foti, 2019; Yin and Rogge, 2019) . With unique physicochemical and pharmacological characteristics complementary to traditional protein-targeted small-molecule and protein drugs (Table 1) , RNA molecules, such as aptamers, antisense oligonucleotides (ASO), small interfering RNAs (siRNA), and guide RNAs (gRNA), have emerged as a new class of modalities in clinical practice and are under active development (Crooke et al., 2018; Yin and Rogge, 2019; Yu et al., 2019) ; RNA molecules may act not only on conventional proteome but also on previously undrugged transcriptome, including mRNAs to be translated into proteins and functional noncoding RNAs (ncRNAs) which largely outnumber mRNAs (Mattick, 2004; Djebali et al., 2012) , as well as the genome. Moreover, some mRNAs and ncRNAs, such as microRNAs (miRNA or miR), are also under preclinical and clinical development for replacement therapy or vaccination ABBREVIATIONS: A, adenine; AHP, acute hepatic porphyria; ALA, d-aminolevulinic acid; ALAS1, d-ALA synthase 1; AMD, age-related macular degeneration; ApoB, apolipoprotein B; A-site, aminoacyl-tRNA site; ASO, antisense oligonucleotide; asRNA, antisense RNA; BERA, bioengineered or biological RNA agents; C, cytosine; Cas, CRISPR-associated protein; DC, dendritic cell; DMD, Duchenne muscular dystrophy; DPQ, 6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine; dsRNA, double-stranded RNA; EphA2, ephrin type-A receptor 2; E-site, exit site; EWS, Ewing sarcoma breakpoint region 1; FDA, Food and Drug Administration; FL, fluorescence; FLI1, Friend leukemia integration 1 transcription factor; FLT-1, fms-like tyrosine kinase; FMN, flavin mononucleotide; G, guanine; GalNAc, N-acetylgalactosamine; gRNA, guide RNA; hATTR amyloidosis, hereditary transthyretin-mediated amyloidosis; HCV, hepatitis C virus; HD, Huntington disease; HIV, human immunodeficiency virus; HoFH, homozygous familial hypercholesterolemia; HPLC, high-performance liquid chromatography; HTT, huntingtin; IL, interleukin; IRES, internal ribosome entry site; IVT, in vitro-transcribed; LC, liquid chromatography; LC-HRMS, LC tandem high-resolution accurate MS; LDL, low-density lipoprotein; LNA, locked nucleic acid; LNP, lipid nanoparticle; LPP, lipopolyplex; LPX, lipoplex; Mage, melanoma-associated antigen; miR or miRNA, microRNA; MMA, methylmalonic acidemia; 29-MOE, 29-O-methoxyethyl; MS, mass spectrometry; MTDB, 2-[ [4-(2-methylthiazol-4-ylmethyl) - (1, 4) diazepane-1-carbonyl]amino]benzoic acid ethyl ester; MUT, methylmalonyl CoA mutase; ncRNA, noncoding RNA; NPET, nascent peptide exit tunnel; NSCLC, non-small-cell lung cancer; NY-ESO-1, New York Esophageal Squamous Cell Carcinoma-1; PBG, porphobilinogen; PD, pharmacodynamics; PD-1, programmed cell death protein 1; pDNA, plasmid DNA; PK, pharmacokinetics; PMO, phosphorodiamidate morpholino oligomers; PO, phosphodiester; pre-miRNA, precursor miRNA; pri-miRNA, primary miRNA; PS, phosphorothioate; P-site, peptidyl-tRNA site; PTC, peptidyl transferase center; qPCR, quantitative polymerase chain reaction; RISC, siRNA-induced silencing complex; RNAi, RNA interference; RNP, ribonucleoprotein; rRNA, ribosomal RNA; SARS-CoV, severe acute respiratory syndrome coronavirus; SDF-1, stromal cell-derived factor 1; shRNA, short or small hairpin RNA; siRNA, small interfering RNA; SMA, spinal muscular atrophy; SMN, survival motor neuron; sRNA, small RNA; TAR, transactivation response; Tat, transactivating regulatory protein; TCR, T cell receptor; TGP, targaprimir; TPP, thiamine pyrophosphate; TTR, transthyretin; U, uracil; UTR, untranslated region; VEGF, vascular endothelial growth factor. RNA-Based Therapies Sahin et al., 2014; Lieberman, 2018) , and new approaches and technologies are emerging to tackle some inherited or overlooked issues, such as the choice of RNA molecules (Ho and Yu, 2016; Yu et al., 2019) . On the other hand, traditional small-molecule compounds may be employed to directly target pathogenic RNAs for the treatment of diseases (Donlic and Hargrove, 2018; Warner et al., 2018; Costales et al., 2020) , providing another unparalleled opportunity to expand the range of therapeutic targets.

In this review, we first provide a classification of RNA-based therapeutics, RNA drugs, and RNA-targeted small molecules and describe their general characteristics. After summarizing the promise of RNA drugs, we review specific types of RNA drugs and their novel mechanisms of action and discuss major barriers in the development of RNA therapeutics, as well as respective proven and potential strategies. Next, we summarize the strategies and potential in developing RNA-targeted small-molecule drugs, including recent advances in structural understanding of antibiotic-ribosomal RNA (rRNA) interactions and identification of novel RNAbinding small molecules. Challenges in the identification of therapeutic RNA targets and determination of the selectivity of RNA-small-molecule interactions for the control of specific diseases are also discussed.

RNA-based therapeutics are classified as two types of entities: RNA molecules or analogs directly used as therapeutic drugs (Kole et al., 2012; Sahin et al., 2014; Crooke et al., 2018; Yu et al., 2019) and RNA-targeted small-molecule medications (Donlic and Hargrove, 2018; Warner et al., 2018; Costales et al., 2020) . Firstly, a number of RNA drugs have been approved by the US Food and Drug Administration (FDA) for the treatment of various human diseases, including RNA aptamers (e.g., pegaptanib) (Gragoudas et al., 2004; Gryziewicz, 2005) , ASOs or antisense RNAs (asRNAs) (e.g., mipomersen, eteplirsen, nusinersen, inotersen, and golodirsen) (Morrow, 2013; Stein, 2016; Syed, 2016; Ottesen, 2017; Keam, 2018) , and siRNAs (e.g., patisiran and givosiran) (Wood, 2018; Scott, 2020) (Table 2 ; this review). RNA molecules have appeared to be highly specific in acting on a wide variety of proven and possible therapeutic targets, including proteins, transcripts, and genes (Table 1) , that may not be accessible by small-molecule compounds and proteins. Nevertheless, RNAs are prone to catabolism by serum RNases and are required to pass the cellular membrane barriers to access intracellular targets. Similar to protein therapeutics (e.g., insulin, trastuzumab, and pembrolizumab, etc.), RNA drugs (e.g., mipomersen and patisiran, etc.) are not orally bioavailable; hence, both RNA and protein drugs are usually administered to patients via other routes, such as intravenous or subcutaneous injection (Table 1) . This is totally different from many smallmolecule inorganic (e.g., lithium carbonate) and organic (e.g., acetaminophen, dextromethorphan, and ibuprofen, etc.) compound drugs, which exhibit favorable or acceptable oral bioavailability and are primarily administered orally to patients.

Secondly, conventional small-molecule compounds with broad structural diversities and drug-like physicochemical and PK properties are preferred entities to bind and manipulate highly structured RNA targets (Hermann, 2016; Donlic and Hargrove, 2018; Warner et al., 2018; Costales et al., 2020) . After the identification of an RNA target, selection of proper drug-like small molecules, and determination of RNA-small-molecule interactions (e.g., binding affinity and selectivity), the RNA-targeted small molecules may be processed for further preclinical and clinical investigations to define efficacy and safety profiles. Supporting this concept, many antibiotic drugs, such as natural and semisynthetic aminoglycosides (e.g., streptomycin, paromomycin, neomycin, etc.) (Fourmy et al., 1996; Ogle et al., 2001; Demeshkina et al., 2012; Demirci et al., 2013) , tetracyclines (e.g., tetracycline, tigecycline, etc.) (Brodersen et al., 2000; Anokhina et al., 2004; Schedlbauer et al., 2015) , and macrolides (erythromycin, azithromycin, telithromycin, etc.) (Vannuffel and Cocito, 1996; Hansen et al., 2002; Berisio et al., 2003; Tu et al., 2005; Bulkley et al., 2010) , as well as synthetic oxazolidinones (e.g., linezolid, etc.) (Ippolito et al., 2008; Wilson et al., 2008) , being approved for clinical use have been revealed to mechanistically bind to rRNAs within the 30S or 50S subunits to interfere with protein synthesis for the control of infections (Wilson, 2009 (Wilson, , 2014 Lin et al., 2018) . Therefore, large efforts are underway to identify viable RNA targets and assess new RNA-targeted small molecules for the treatment of various types of human diseases (Warner et al., 2018) .

A. The Rise and Promise of RNA Therapeutics

With the understanding of new biological processes and development of novel technologies, such as those for gene silencing and genome editing Zamecnik and Stephenson, 1978;  RNA-Based Therapies 865 Lee et al., 1993; Wightman et al., 1993; Fire et al., 1998; Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013) , diverse RNA molecules have been used to interfere with potential therapeutic targets (Fig. 1) . Firstly, RNA aptamers can directly bind to extracellular, cell surface, or intracellular proteins (Gragoudas et al., 2004; Gryziewicz, 2005) that are traditionally targeted by small-molecule and protein drugs. Secondly, ASOs or asRNAs, siRNAs, and miRNA mimics may be delivered into cells to target intracellular mRNAs or functional ncRNAs through complementary base pairings, leading to gene silencing or control of gene expression for the treatment of diseases. Thirdly, a sense RNA or mRNA molecule can be introduced into cells and then translated into target proteins for protein replacement therapy or vaccination (Sahin et al., 2014; Lieberman, 2018) . In addition, the genetic sequences dictating disease initiation and progression may be directly changed by using proper gRNAs and other necessary components to achieve eradication of the disease. As such, RNAs are unique molecules that are able to interact with three major forms of biological macromolecules-DNAs, RNAs, and proteins ( Fig. 1 )-and the development of RNA therapeutics is expected to expand the range of druggable targets, including conventional proteins and previously undrugged or "undruggable" transcripts and genes. The development of novel RNA therapeutics has proven highly challenging given the fact that RNA drugs are anticipated to act primarily on intracellular targets ( Fig. 1 ) and that RNA molecules exhibit "undrug-like" physicochemical and PK properties, especially when compared with small-molecule compounds ( Table 1) .

As a polymeric molecule consisting of a sequence of variable numbers of four major forms of ribonucleotides that differ in their nucleobases, adenine (A), uracil (U), guanine (G), and cytosine (C) , naked and unmodified RNAs are extremely susceptible to hydrolysis by nonspecific RNases (e.g., RNase A) that are highly abundant in the blood (Houseley and Tollervey, 2009) . Furthermore, RNAs are large molecules (e.g., with molecular weights .7 kDa) and are negatively charged (Table 1) ; thus, it is hard for them to cross the cell membrane. In addition, upon entering into cells, exogenous RNAs need to escape from endosomal trapping and/or degradation by large classes of intracellular ribonucleases or RNases (e.g., endonucleases, and 59 and 39 exonucleases), become incorporated into specific complex, and get access to the targets (Fig. 1) to exercise pharmacological effects.

With the development of new strategies to improve the druggability of RNA molecules, as well as the understanding of mechanisms of action, a number of RNA analog drugs have been approved by the FDA for the treatment of human diseases (Table 2) , and many others are under active trials [for recent reviews, see Kowalski et al. (2019) , Yu et al. (2019) ]. The approval of the first RNA aptamer drug, pegaptanib (Gragoudas et al., 2004; Gryziewicz, 2005) , supports the concept of using RNA molecules to inhibit protein targets (Fig. 1) . Through specific chemical modifications to improve metabolic stability, targeting, binding affinity, and silencing efficacy (Eckstein, 1985; Campbell et al., 1990; Wheeler et al., 2012; Summerton, 2017; Crooke et al., 2018) , ASOs [including "gapmers" and phosphorodiamidate morpholino oligomers (PMOs)] have become the most successful class of RNA drugs (e.g., mipomersen, eteplirsen, nusinersen, inotersen, and golodirsen) ( Table 2) . Furthermore, the most recent approval of two siRNA drugs, patisiran (Adams et al., 2018; Wood, 2018) and givosiran (Scott, 2020) , not only testifies to the benefits of developing new approaches to improve the PK and pharmacodynamics (PD) properties of RNA drugs (Nair et al., 2017) but also supports the potential of RNA therapeutics.

B. Types of RNA Drugs and Mechanisms of Action 1. Antisense Oligonucleotides. The use of chemically synthesized, single-stranded oligonucleotide to selectively inhibit target gene expression via complementary base pairings with targeted mRNA was first reported in 1978 Zamecnik and Stephenson, 1978) . Since then, ASOs have been widely used for the study of gene functions and development of novel therapeutics [for reviews, see Kole et al. (2012) , Bennett (2019) , Levin (2019) ]. This is in parallel with the discovery of the presence of natural asRNAs in virtually all species (Spiegelman et al., 1972; Light and Molin, 1983; Simons and Kleckner, 1983; Izant and Weintraub, 1984; Ecker and Davis, 1986) and thus broad recognition of their critical functions in post-transcriptional gene regulation [for reviews, Fig. 1 . RNA therapeutics are expected to expand the range of druggable targets from proteins to RNAs and DNAs. Cell surface, extracellular, and intracellular proteins remain favorable targets for the development of small-molecule and protein (e.g., antibody) therapeutics, as well as RNA aptamer drugs (1) . Actually, the majority of human genome sequences transcribed as functional ncRNAs largely outnumbered mRNAs to be translated into proteins. Both mRNAs and ncRNAs can be directly targeted by RNA drugs such as ASO/asRNAs, miRNAs, and siRNAs (2). Once introduced into cells, mRNA therapeutics (3) may be developed for protein replacement therapy or vaccination. In addition, gRNAs (4) could be used along with other elements to directly edit the target gene sequences for the treatment of particular diseases. 866 see Vanhée-Brossollet and Vaquero (1998) , Pelechano and Steinmetz (2013) , Nishizawa et al. (2015) ]. The knockdown of target gene expression with ASOs includes RNase-dependent cleavage of mRNAs and precursor mRNAs (pre-mRNAs) (RNase H and RNase P), as well as RNase-independent suppression of protein synthesis. Furthermore, ASOs can be employed to modulate RNA splicing to produce functional proteins or preferred genetic products (Condon and Bennett, 1996; McClorey et al., 2006) . Although they differ from siRNAs and miRNAs that rely mainly on cytoplasmic siRNAinduced silencing complexes (RISCs)/miRNA-induced silencing complexes to control target gene expression, ASOs designed to target the same sites of common transcripts could be equally active as siRNAs, with some exceptions (Vickers et al., 2003) . In addition, ASOs seem to be more effective to knock down nuclear targets, whereas siRNAs are superior at suppressing cytoplasmic targets (Lennox and Behlke, 2016) , likely due to the fact that RNases H & P are highly abundant in the nucleus and RISCs are present within the cytoplasm.

To make ASOs druggable, a wide variety of chemical modifications have been developed to improve their metabolic stability and cell penetration efficiency, including the change of phosphodiester (PO) linker to phosphorothioate (PS), protection of the 29-hydroxyl group on ribose with methyl (29-methoxy) or methoxyethyl (29-MOE), or direct substitution with fluorine (29-fluoro), and connection of the 29-O and 49-C with a methylene bridge, namely locked nucleic acid [for reviews, see Ho and Yu (2016) , Khvorova and Watts (2017) , Crooke et al. (2018) , Yu et al. (2019) ]. More extensive modifications are also established for ASOs, among which the nucleobases are retained for base pairings while the ribose 5-phosphate linkages may be fully substituted with morpholino phosphorodiamidate backbones, leading to PMOs (Heasman et al., 2000) . In addition, specific ligands, such as hepatocyte asialoglycoprotein receptor-binding N-acetylgalactosamine (GalNAc) (Nair et al., 2014 (Nair et al., , 2017 , may be covalently attached to ASOs (and siRNAs, miRNAs, aptamers, etc.) to achieve cell-or organ-selective gene silencing. Some chemical modifications are proven to be very useful for the development of ASO drugs, as demonstrated by their utility in FDA-approved RNA drugs (Table 2) .

Since the first ASO drug, fomivirsen, an antisense oligodeoxynucleotide (59-GCG TTT GCT CTT CTT CTT GCG-39) with phosphorothioate linkages, was approved by FDA in 1998 for the treatment of cytomegalovirus retinitis (Roehr, 1998) , a number of ASO therapeutics have been successfully marketed in the United States (Table 2) (Mendell et al., 2013; Morrow, 2013; Robinson, 2013; Syed, 2016; Aartsma-Rus and Krieg, 2017; Ottesen, 2017; Stein and Castanotto, 2017; Benson et al., 2018; Keam, 2018; Wood, 2018) . Among them, mipomersen (59-G*-MeC*-MeC*-MeU*-MeC*-dA-dG-dT-dMeC-dG-dT-dMeC-dT-dT-dMeC-G*-MeC*-A*-MeC*-MeC*-39; * = 29-MOE; Me = 5-methyl, and d = deoxy; all PS linkages) and inotersen (59-MeU*-MeC*-MeU*-MeU*-G*-dG-dT-dT-dA-dMeC-dA-dT-dG-dA-dA-A*-MeU*-MeC*-MeC*-MeC*-39; * = 29-MOE; Me = 5-methyl, and d = deoxy; all PS linkages) are also known as gapmers, consisting of modified antisense oligoribonucleotides at both 59 and 39 ends with a "gap" of oligodeoxynucleotide in the middle. Upon selective binding to a targeted transcript, the resulting DNA-RNA heteroduplex is recognized by RNase H, leading to the cleavage of targeted RNA strand and the knockdown of targeted gene expression. Specifically, mipomersen has been shown to selectively bind to ApoB-100 mRNA to reduce ApoB-100 protein levels, which is the major constituent of low-density lipoprotein (LDL); thus, it exhibits effectiveness for the treatment of patients with homozygous familial hypercholesterolemia (HoFH) (Stein et al., 2012; Crooke and Geary, 2013; Thomas et al., 2013) . Rather, mipomersen was discontinued in 2018 because of competition from other therapeutics and an incapability of achieving marketing success (Yin and Rogge, 2019) . On the other hand, inotersen selectively binds to transthyretin (TTR) mRNA to achieve the suppression of hepatic TTR protein expression levels and thus exerts therapeutic benefits among adults with hereditary transthyretinmediated amyloidosis (hATTR amyloidosis) (Benson et al., 2018; Coelho et al., 2020) .

Nusinersen (59-UCA CUU UCA UAA UGC UGG-39; fully modified with 29-MOE and PS linkages; all Us and Cs are 59-methylated) was approved by FDA in 2016 (Aartsma-Rus, 2017; Ottesen, 2017) for the treatment of a rare autosomal recessive neuromuscular disorder, spinal muscular atrophy (SMA), which is caused by genetic variations in the chromosome 5q11.2-q13.3 locus, affecting survival motor neuron (SMN) gene expression and leading to an insufficient level of SMN protein (Brzustowicz et al., 1990; Lefebvre et al., 1995) . Nusinersen is an extensively modified 18-mer ASO whose PO linkages are completely changed to PS, and all ribose rings are protected with 29-MOE (Table 2) . Through the modulation of alternate splicing of SMN2 pre-mRNA to increase exon 7 inclusion to achieve the expression of full-length functional SMN protein (Rigo et al., 2014) , nusinersen was found to be effective in improving patient survival or motor function Mercuri et al., 2018) .

Eteplirsen and golodirsen are two PMO drugs (Table 2) , approved in 2016 (Stein, 2016; Syed, 2016) and 2019 (Heo, 2020) , respectively, for the treatment of Duchenne muscular dystrophy (DMD), a lethal neuromuscular disorder commonly caused by genetic mutations disrupting the reading frame of the X-linked dystrophin gene and which might be found in one of 3500 newborn boys (Cirak et al., 2011) . Eteplirsen and golodirsen contain 30 and 25 linked PMO subunits whose sequences of bases are 59-CUC CAA CAU CAA GGA AGA UGG CAU UUC UAG-39 and 59-GUU GCC UCC GGU UCU GAA GGU GUU C-39, respectively, among which all Us are methylated or regarded as T. Both eteplirsen and golodirsen are designed RNA-Based Therapies to restore the reading frame of dystrophin gene for the production of an internally truncated, yet functional, dystrophin protein. In particular, eteplirsen selectively binds to the exon 51 of dystrophin pre-mRNA (Popplewell et al., 2010) , leading to exclusion of this exon during mRNA processing among patients with DMD showing genetic mutations amenable to exon 51 skipping (van Deutekom et al., 2007) . With the production of functional dystrophin protein, eteplirsen-treated patients were shown to have a slower rate of decline in ambulation (Cirak et al., 2011; Mendell et al., 2013 Mendell et al., , 2016 Khan et al., 2019) . Likewise, golodirsen selectively binds to the exon 53 of dystrophin pre-mRNA to achieve the exclusion of this exon during mRNA processing (Popplewell et al., 2010) and thus the expression of functional muscle protein dystrophin among patients with confirmed genetic mutations that are amenable to exon 53 skipping (Frank et al., 2020; Heo, 2020) .

Many clinical trials are ongoing to investigate some new ASOs for the treatment of specific diseases, spanning from orphan genetic disorders to infectious diseases and cancers (https://www.clinicaltrials.gov/) . For instance, a deep intronic c.2991+1655A.G mutation in CEP290 underlying Leber congenital amaurosis type 10, an inherited retinal dystrophy, may be corrected by ASO therapy. Indeed, QR-110 was identified to effectively restore wild-type CEP290 mRNA and protein expression levels in CEP290 c.2991+1655A.G homozygous and heterozygous Leber congenital amaurosis type 10 primary fibroblasts as well as induced pluripotent stem cellsderived retinal organoids, and it was tolerated in monkeys after intravitreal injection (Dulla et al., 2018) . Therefore, a double-masked, randomized, multiple-dose phase II/III study (NCT03913143) is underway to evaluate the efficacy, safety, tolerability, and systemic exposure of intravitreally administered QR-110 in patients with Leber congenital amaurosis who are amenable to the CEP290 p.Cys998X mutation. As another example, IONIS-AR-2.5Rx, a next-generation ASO against androgen receptor, has entered into a phase Ib/II single-arm study (NCT03300505) to identify an effective and safe dose level for the treatment of metastatic castrationresistant prostate cancer as combined with a fixed dose of enzalutamide.

2. Small Interfering RNAs. Since the discovery and development of RNA interference (RNAi) technologies with double-stranded RNAs (dsRNAs) (Fire et al., 1998; Zamore et al., 2000; Elbashir et al., 2001) , 18-to 22-bp siRNAs have been routinely used for selective and effective knockdown of target gene expression in basic research, and some have entered clinical drug development [for reviews, see Rossi (2009), Setten et al. (2019) , Yu et al. (2019) ]. Different from the single-stranded ASO, siRNA comprises two strands, in which the guide strand is characterized by two 39-overhang ribonucleotides crucial for the duration of gene silencing (Strapps et al., 2010) . The endoribonuclease Dicer or helicase with RNase motif (Bernstein et al., 2001; Hutvágner et al., 2001) trims dsRNAs and separates the guide and passenger strands within the RISC (Hammond et al., 2000) . The passenger strand is preferentially cleaved by the endonuclease argonaute-2 (Matranga et al., 2005; Rand et al., 2005) , which is the catalytic core of RISC Martinez et al., 2002; Liu et al., 2004; Meister et al., 2004; Okamura et al., 2004; Rand et al., 2004) . The guide strand containing a thermodynamically less stable 59 end retained within the RISC (Khvorova et al., 2003; Schwarz et al., 2003) acts on its targeted mRNA through perfect complementary base pairings, leading to a sequence-specific cleavage of the targeted mRNA by argonaute-2 and consequently the knockdown of target gene. Because RISC is solely located within cytoplasm, whereas RNase H is predominately in nucleus, as mentioned previously, siRNAs are usually more effective than ASOs in knocking down cytoplasmic targets (Lennox and Behlke, 2016) .

To develop siRNA therapeutics, it is essential to achieve potent, specific, and long-lasting gene silencing while minimizing off-target effects (Kim et al., 2005; Ui-Tei et al., 2008; Wang et al., 2009) . With an improved understanding of RNAi mechanisms, some specific guidelines may be followed, and particular software can be used for the design of effective siRNAs [for reviews, see Jackson and Linsley (2010) , Naito and Ui-Tei (2012) , Fakhr et al. (2016) ]. The selection of a proper target site, usually closer to the start codon within the coding sequence, is critical for the effectiveness of siRNA. This is also crucial to ensure selectivity and lessen off-target effects of siRNA. The composition of siRNA, such as the use of specific ribonucleotides at particular locations and overall G/C content, affects not only the stability but also the efficacy of siRNA. As siRNA may induce immune response in sequence-independent and sequence-dependent manners (Alexopoulou et al., 2001) , it is important to avoid immune-stimulatory motifs such as U-rich sequences when designing siRNAs (Kleinman et al., 2008; Goodchild et al., 2009) . In addition, similar to the development of ASO drugs, chemical modification is a common strategy to improve the metabolic stability and PK properties of siRNAs (Bramsen and Kjems, 2012; Yu et al., 2019) . In any case, the effectiveness, selectivity, and safety of individual siRNAs require extensive and critical experimental validation.

The first siRNA drug, patisiran (Table 2) , was approved by the FDA in 2018 for the treatment of the polyneuropathy of hATTR amyloidosis in adults (Wood, 2018; Yu et al., 2019) over 20 years after the discovery of siRNA-controlled gene silencing. hATTR amyloidosis is an autosomal-dominant, life-threatening disease caused by genetic mutations of TTR. As TTR protein is primarily produced in the liver, pathogenic mutations lead to misfolded TTR proteins that deposit as amyloid in peripheral nerves, heart, kidney, and gastrointestinal tract 868 (Adams et al., 2018; Zhang et al., 2020b) . Patisiran is a 21-bp siRNA with extensive chemical modifications (Table 2 ) whose sense sequence is 59-

and antisense sequence is 59-A-U-G-G-A-A-MeU-A-C-U-C-U-U-G-G-U-MeU-A-C-dT-dT-39 (Me = 29-O-methyl, and d = deoxy), formulated as a lipid nanoparticle (LNP) for delivery to hepatocytes . Different from conventional siRNAs that are usually projected to target coding sequence regions, patisiran is designed to follow miRNA mechanisms by selectively binding to a conserved sequence in the 39-untranslated regions (UTR) of mutant and wild-type TTR mRNA, leading to a reduction of circulating TTR protein levels and accumulation in tissues. Clinical studies demonstrated the benefits of patisiran (0.3 mg/kg every 3 weeks, i.v. infusion) for the treatment of patients with hATTR amyloidosis, as indicated by a decrease of the modified Neuropathy Impairment Score +7 from baseline to month 18 among the patisiran treatment group compared with a steady increase in the placebo group (Adams et al., 2018) . Meanwhile, overall incidence and types of adverse events did not differ in patisiran and placebo groups, suggesting that patisiran was tolerated in patients (Adams et al., 2018; Zhang et al., 2020b) . It is also notable that, although both patisiran and inotersen act on the same molecular target for the treatment of the same disease, patisiran is administered less frequently and at much lower doses than inotersen (284 mg once weekly, s.c.), although their routes of administration are different (Table 2) .

In November 2019, givosiran was the second siRNA drug approved by the FDA for the treatment of adults with acute hepatic porphyria (AHP) ( Table 2 ) (de Paula Brandao et al., 2020; Scott, 2020) . AHP is a rare, inherited, and life-threatening disease caused by disruption of hepatic heme biosynthesis. The accumulation of neurotoxic heme intermediates d-aminolevulinic acid (ALA) and porphobilinogen (PBG) in patients leads to acute debilitating neurovisceral attacks and even disabling chronic symptoms (Sardh et al., 2019) . Givosiran is a 19-bp, chemically modified siRNA (Table 2 ) whose sense and antisense sequences are 59-

, respectively (Me = 29-O-methyl and f = 29-fluoro). The sense sequence is covalently attached to a triantennary GalNAc ligand at the 39 end for an improved internalization of GalNacconjugated siRNAs into hepatocytes. Givosiran selectively binds to the mRNA of d-ALA synthase 1 (ALAS1), a rate-limiting enzyme in hepatic heme biosynthesis that is responsible for the formation of ALA from succinyl-CoA and glycine, to induce gene silencing, which subsequently reduces ALA and PBG levels and lessens factors associated with attacks and other symptoms of AHP (de Paula Brandao et al., 2020; Sardh et al., 2019) . Although the results of a phase III clinical trial have not been published yet, a phase I study showed that monthly subcutaneous administration of 2.5 mg/kg givosiran to patients with AHP sharply decreased the ALAS1 mRNA levels and returned ALA and PBG levels to near normal, and it subsequently led to a 79% lower mean annualized attack rate than the placebo group (Sardh et al., 2019) . The approval of givosiran also highlights the utility of a GalNAc-based delivery system for the development of RNA therapeutics for the treatment of hepatic diseases.

The very recent approval of two siRNA drugs by the FDA provides incentives to develop novel siRNA therapeutics, and many are now under active clinical trials (https://www.clinicaltrials.gov/) . Lumasiran, or ALN-GO1, is a GalNAc-conjugated investigative siRNA drug for the treatment of primary hyperoxaluria type 1, an inherited rare disease arising from disruption of glyoxylate metabolism (Liebow et al., 2017) . Preclinical studies have demonstrated that, through selective targeting of the mRNA of hydroxyacid oxidase (glycolate oxidase) 1, lumasiran administered subcutaneously was able to reduce oxalate production in multiple animal models (Liebow et al., 2017) . Presently, an open-label phase III clinical trial (NCT03905694) is underway for the investigation of the efficacy, safety, PK, and PD of lumasiran among infants and young children with primary hyperoxaluria type 1. Inclisiran, or ALN-PCSSC, is another synthetic siRNA with extensive chemical modifications and is covalently connected to a triantennary GalNAc ligand that is designed to target the mRNA of hepatic proprotein convertase subtilisin kexin type 9. Previous clinical studies consistently demonstrated the effectiveness of inclisiran in reducing the plasma proprotein convertase subtilisin kexin type 9 and LDL cholesterol levels in healthy individuals and patients at high risk for cardiovascular disease who had elevated LDL cholesterol levels (Fitzgerald et al., 2014 Ray et al., 2017) . Moreover, a single-dose treatment with inclisiran was shown to cause a durable reduction in LDL cholesterol levels among the subjects over 1 year (Ray et al., 2019) , and inclisiran showed similar efficacy and safety profiles among individuals with normal and impaired renal functions (Wright et al., 2020) . A double-blind, placebo-controlled, and open-label phase II/III study (NCT03851705) is ongoing to evaluate the safety and efficacy of inclisiran in patients with HoFH. In addition, fitusiran (or ALN-AT3SC) is an investigative siRNA drug that is designed to suppress the production of antithrombin (encoded by the gene serpin family C member 1 (SERPINC1) through selective interference with SERPINC1 mRNA in the liver for the treatment of hemophilia A and B, inherited bleeding disorders arising from impaired thrombin production (Machin and Ragni, 2018) . Administered subcutaneously once monthly, fitusiran was shown to reduce plasma antithrombin levels RNA-Based Therapies in a dose-dependent manner and increase thrombin production in patients with hemophilia A or B who did not have inhibitory alloantibodies (Pasi et al., 2017) . Presently, two phase III clinical trials (NCT03549871 and NCT03754790) are underway to evaluate the efficacy and safety of fitusiran among patients with hemophilia A and B.

siRNA drugs under clinical development are also expanded to other therapeutic areas, including oncology, that impact millions of patients. For instance, siG12D-LODER is an siRNA that specifically targets the Kirsten rat sarcoma viral oncogene homolog (KRAS) mutant G12D mRNA, a driver oncogene present in various types of cancers, especially pancreatic cancer (Zorde Khvalevsky et al., 2013; Golan et al., 2015; Ramot et al., 2016) . A previous phase I clinical study showed that combination treatment with siG12D-LODE and gemcitabine was well tolerated, and potential efficacy among patients with locally advanced pancreatic cancer was shown (Golan et al., 2015) . As such, siG12D-LODER is currently under a phase II clinical study to evaluate its efficacy, safety, tolerability, and PK for the treatment of patients with unresectable, locally advanced pancreatic cancer, as combined with standard chemotherapy (i.e., gemcitabine plus nanoparticle albumin-bound paclitaxel) in comparison with chemotherapy alone. As another example, synthetic siRNA targeting a protein-tyrosine kinase named ephrin type-A receptor 2 (EphA2) was shown to be effective in controlling tumor growth in xenograft mouse models (Landen et al., 2005) . Furthermore, EphA2-siRNA encapsulated with 1,2dioleoyl-sn-glycero-3-phosphatidylcholine nanocomplex at tested doses was tolerated in murine and primate models (Wagner et al., 2017) . A phase I clinical trial (NCT01591356) is now recruiting patients with advanced solid tumors to evaluate the safety and toxicity profiles of an 1,2-dioleoyl-sn-glycero-3-phosphatidylcholineencapsulated, EphA2-targeted siRNA drug administered intravenously.

3. MicroRNAs. MicroRNAs are a superfamily of genome-derived small ncRNAs governing posttranscriptional gene regulation that was first discovered in Caenorhabditis elegans in 1993 (Lee et al., 1993; Wightman et al., 1993) . More than 1900 miRNAs have been identified in humans (Kozomara and Griffiths-Jones, 2014; Kozomara et al., 2019) . Canonical biogenesis of miRNAs starts from the transcription of miRNA-coding sequences by RNA polymerase II to long primary miRNA (pri-miRNA) transcripts within the nucleus . The pri-miRNA is then cleaved to shorter precursor miRNA (pre-miRNA) by the RNase III Drosha complexed with RNA-binding protein DiGeorge syndrome chromosome region 8 (Lee et al., 2003; Denli et al., 2004; Gregory et al., 2004; Han et al., 2004) . After being transported into the cytoplasm by RAS-related nuclear protein-GTP-dependent exportin-5 (Bohnsack et al., 2004; Lund et al., 2004) , pre-miRNAs are cleaved, double-stranded miRNA duplexes by cytoplasmic RNase III Dicer complexed with transactivation-responsive RNA-binding protein (Hutvágner et al., 2001; Lee et al., 2002; Zhang et al., 2004a; Haase et al., 2005) . The miRNA duplex associated with miRNA-induced silencing complex is then unwound to offer two strands, among which the guide strand mature miRNA binds to the target mRNA through partial complementary base pairings, with corresponding miRNA response element usually present within the 39UTR, leading to translational inhibition or transcript degradation or cleavage (Hutvágner and Zamore, 2002; Bagga et al., 2005; Pillai et al., 2005; Petersen et al., 2006) . On the other hand, functional miRNAs may be generated through noncanonical pathways, such as those pre-miRNAs directly excised from introns not dependent upon Drosha (Okamura et al., 2007; Ruby et al., 2007) , Dicer-independent miRNAs Cifuentes et al., 2010; Ho et al., 2018) 3), and miRNAs or small RNAs (sRNAs) derived from small nucleolar RNAs (Ender et al., 2008; Pan et al., 2013) or transfer RNAs (tRNAs) (Maute et al., 2013; Kuscu et al., 2018) .

One miRNA can simultaneously regulate the expression of multiple transcripts, since the recognition of a target mRNA by miRNA does not require perfect base pairing. Through the control of multiple genes involved in the same biological processes, many miRNAs have been shown to play important roles in pathogenesis of human diseases, including lethal cancers [for reviews, see Ambros (2004) , Bader et al. (2010) , Esteller (2011), Rupaimoole and Slack (2017) ]. In addition, compared with normal tissues, there is generally a decrease or loss of oncolytic miRNAs (e.g., let-7a/b/c-5p, miR-34a-5p and miR-124-3p) and overexpression of oncogenic miRNAs (e.g., miR-21-5p) in tumor tissues and carcinoma cells [for reviews, see Esteller (2011) , Rupaimoole and Slack (2017) ], although there are some exceptions. Therefore, some tumor-suppressive miRNAs lost in cancer cells may be restored, e.g., by using synthetic miRNA mimics and virus-or plasmid-based expression, and oncogenic miRNAs overexpressed in cancer cells may be inhibited, e.g., with chemically synthesized single-stranded ASO (termed antagomirs or miRNA inhibitors) for the control of tumor progression (Ho and Yu, 2016; Yu et al., 2019) . The "miRNA replacement therapy" strategy is of particular interest, compared with "antagonism" of oncogenic miRNAs, because miR-NAs are endogenous components and reintroduced miRNAs may be well tolerable in cells. The premise of an miRNA replacement therapy strategy has been demonstrated by many preclinical studies Rupaimoole and Slack, 2017; that are amendable to clinical investigations (Beg et al., 2017; Hong et al., 2020) .

Human miR-34a-5p is one of the most promising miRNAs for replacement therapy, with well established tumor-suppressive functions while downregulated in a wide range of solid tumors [for reviews, see Bader (2012), 870 Yu et al. (2019) ]. Through effective interference with various oncogenes underlying tumor progression and metastasis, the efficacy of miR-34a-5p was consistently documented in many types of xenograft tumor mouse models (Wiggins et al., 2010; Liu et al., 2011; Pramanik et al., 2011; Craig et al., 2012; Kasinski and Slack, 2012; Wang et al., 2015b; Zhao et al., 2015 Zhao et al., , 2016 Jian et al., 2017; Ho et al., 2018) . Therefore, a liposomeencapsulated synthetic miR-34a mimic, MRX34, became the first miRNA entering phase I clinical trial for the treatment of advanced solid tumors, including unresectable liver cancer (Beg et al., 2017; Hong et al., 2020) . As the maximum tolerated dose was revealed to be 110 mg/m 2 in patients without hepatocarcinoma, a high incidence of adverse events (e.g., 100% all grades and 38% grade 3 among all patients), such as fever, fatigue, back pain, nausea, diarrhea, anorexia, and vomiting, was found among patients with liver cancer receiving treatment with MRX34 (10-50 mg/m 2 , i.v., biweekly) who required palliative management with dexamethasone premedication. Nevertheless, MRX34 did exhibit antitumor activity among patients with refractory advanced solid tumors (Beg et al., 2017; Hong et al., 2020) , offering valuable insight into the development of miRNA therapeutics.

One randomized, double-blind, phase I/II clinical trial (NCT04120493) is underway to explore the safety, tolerability, and efficacy signals of multiple ascending doses of striatally administered, adenoassociated viral vector-carried miR-155, which targets the total huntingtin (HTT) transcripts-namely, rAAV5-miHTT or AMT-130-in early manifest Huntington disease (HD). This strategy is based on previous findings on the efficacy and safety of miHTT therapy in preclinical models, including the use of an Hu128/21 HD mouse model (Miniarikova et al., 2016) , acute HD rat model (Miniarikova et al., 2017) , transgenic HD minipig model (Evers et al., 2018) , neuronal and astrocyte cells derived from patients with HD (Keskin et al., 2019) , and humanized Hu128/21 mouse model of HD (Caron et al., 2020) .

Although there is no single miRNA drug that has yet to be approved by the FDA for medical use, the first siRNA drug, patisiran, seems to mimic miRNA mechanisms of action aforementioned, i.e., through selective binding to the 39UTR of TTR mRNA. In addition, many studies have been conducted, and others are still underway, for the identification of miRNAs as potential diagnostic or prognostic biomarkers for patients with particular diseases or treatments (Hayes et al., 2014; Kreth et al., 2018; Pogribny, 2018) , besides the use of miRNAs as interventional agents.

4. RNA Aptamers. Aptamers are single-stranded, highly structured DNA or RNA oligonucleotides that can bind to a wide variety of molecular targets, including proteins, peptides, DNAs, RNAs, small molecules, and ions, with high affinity and specificity. Upon binding to target protein, RNA aptamer behaves like a nucleic acid antibody or chemical inhibitor to modulate protein function (Fig. 1) for the control of disease (Bunka and Stockley, 2006; Bouchard et al., 2010; Kaur et al., 2018) . Actually, natural RNA-protein complex was first identified in bacteria, among which the RNA molecule is an essential component for the activity of RNase P complex in the processing of precursor tRNA into active tRNA, which could also be inhibited by various RNAs or aptamers (Stark et al., 1978; Kole and Altman, 1979) . There is also autocatalytic RNA or ribozyme, which undergoes self-splicing upon binding with monovalent and divalent cations (Kruger et al., 1982) . Highly structured RNA elements, or "aptamers," are also present within human immunodeficiency virus (HIV)-1 to interact with target proteins for gene expression and viral replication (Feng and Holland, 1988; Marciniak et al., 1990) . Moreover, intrinsic RNAs or riboswitches can sense small-molecule metabolites and then control target gene expression (Mironov et al., 2002; Nahvi et al., 2002; Winkler et al., 2002a) . Ligand binding ribozymes and riboswitches have been identified in humans, as well (Salehi-Ashtiani et al., 2006; Ray et al., 2009) .

With the understanding of the interactions of functional RNAs with proteins as well as other ligands, a high-throughput technology, systematic evolution of ligands by exponential enrichment, was also developed for the identification and development of selective and potent RNA aptamers or ribozymes (Ellington and Szostak, 1990; Robertson and Joyce, 1990; Tuerk and Gold, 1990) . Chemical modifications of selected RNA aptamers may increase metabolic stability and improve PK properties, similar to ASOs and siRNAs (Khvorova and Watts, 2017; . Furthermore, mirror-image L-ribonucleic acids resistant to degradation by RNases have been used for the synthesis and development of artificial aptamers called Spiegelmer (Vater and Klussmann, 2015) .

In 2004, pegaptanib was the first RNA aptamer drug approved by the FDA for the management of neovascular age-related macular degeneration (AMD) ( Table 2) (Gryziewicz, 2005) , supporting the utility of aptamers to interfere with protein targets for the control of human diseases (Fig. 1) .

AMD is a leading cause of low vision in the elderly in developed countries, and neovascular AMD, accounting for approximately 10% of all forms, is responsible for 90% of the severe loss of vision (Gragoudas et al., 2004) . With the understanding of the role of vascular endothelial growth factor (VEGF) in pathogenesis of neovascular AMD, ocular VEGF has become an attractive target for the treatment of neovascular AMD (Hubschman et al., 2009; Miller, 2019) . Pegaptanib, a 28-nt RNA aptamer (Table 2) 

RNA-Based Therapies covalently linked to two branched 20-kDa polyethylene glycol moieties was designed to selectively bind and block the activity of extracellular VEGF-in particular, the 165-amino-acid isoform (VEGF 165 ) (Gragoudas et al., 2004) . The benefits of pegaptanib in improving visual acuity were demonstrated in patients with neovascular AMD, and intravitreal injection could induce some potentially modifiable risk of adverse events (Gragoudas et al., 2004; Gonzales, 2005) . Nevertheless, the market share of pegaptanib declined since 2011 because of competition from anti-VEGF antibody drugs such as ranibizumab and bevacizumab (Yin and Rogge, 2019) .

Olaptesed pegol (NOX-A12), a pegylated 45-nt RNA Spiegelmer designed to selectively target the small chemokine stromal cell-derived factor 1 or C-X-C motif chemokine 12 with high affinity, was effective at preventing the binding of stromal cell-derived factor 1 to its receptors CXC receptor 4 and CXC receptor 7 and thus inhibiting the subsequent signal transduction to achieve control of angiogenesis and metastasis, as well as improvement of other anticancer therapies (Roccaro et al., 2014; Deng et al., 2017) . Two phase IIa studies showed that patients with relapsed/refractory multiple myeloma (Ludwig et al., 2017) and chronic lymphocytic leukemia (Steurer et al., 2019) were highly responsive to olaptesed pegol therapy in combination with bortezomibdexamethasone and bendamustine-rituximab, respectively. One active clinical trial (NCT04121455) is underway to evaluate the safety and efficacy of olaptesed pegol in combination with irradiation among patients with inoperable or partially resected first-line glioblastoma. Another pegylated Spiegelmer, lexaptepid pegol (NOX-94), binds to human hepcidin with high affinity and thus inhibits its biological function (Schwoebel et al., 2013) for the treatment of anemia of chronic disease. A firstin-human study (NCT01372137) showed that lexaptepid pegol was able to inhibit hepcidin and dose dependently elevate serum iron and transferrin saturation, and it was generally safe and tolerated in healthy subjects, with mild and transient transaminase increases at higher doses (Boyce et al., 2016) . After additional investigations in patients (e.g., NCT02079896), no clinical trial is currently open to evaluate the safety and efficacy of lexaptepid pegol. Although clinical development of new aptamer drugs seems less active in recent years, there is growing interest in developing aptamers for drug delivery and as diagnostic agents (Bouvier-Müller and Ducongé, 2018; Kaur et al., 2018) .

5. Messenger RNAs. The development and use of mRNAs as a novel class of drug modalities has great potential in vaccination, protein replacement therapy, and antibody therapy for the treatment of a wide variety of human diseases, including infections, cancers, and genetic disorders (Sahin et al., 2014; Weissman and Kariko, 2015; Pardi et al., 2018; Kowalski et al., 2019) . This concept was first demonstrated by the findings on efficient expression of target proteins in mouse tissues in vivo after the administration of in vitro-transcribed (IVT) mRNAs, which was reported in 1990 (Wolff et al., 1990) . After extensive preclinical studies, many IVT mRNA therapeutics have already entered clinical trials (Heiser et al., 2002; Weide et al., 2009; Rittig et al., 2011; Allard et al., 2012; Van Gulck et al., 2012; Maus et al., 2013; Wilgenhof et al., 2013; Bahl et al., 2017; Leal et al., 2018; de Jong et al., 2019; Papachristofilou et al., 2019) . Different from plasmid DNA or virus-based gene therapy, mRNA drugs are translated into target proteins by the cellular machinery without interference with the genome (Fig. 1) . To ensure translation ability and efficiency, an mRNA needs to contain not only the whole open reading frame of target protein but also intact 59 and 39UTRs as well as 59 cap and 39 poly(A) tail. Therefore, the mRNA drug molecule is much bigger than other types of RNA therapeutics (Fig. 1) . Likewise, direct administration of mRNA therapeutics to patients requires efficient delivery systems to protect mRNAs from degradation by RNases and cross-cellular barrier in vivo. Alternatively, patients may be treated with autologous transplantation of T cells or dendritic cells (DCs) that are reprogrammed with mRNA drugs ex vivo.

Because of the high sensitivity of immune cells in recognizing antigens that can be coded by exogenous mRNAs, as well as their intrinsic immune-stimulatory effects (Hoerr et al., 2000; Weissman et al., 2000; Fotin-Mleczek et al., 2011) , mRNA therapeutics hold great promise as vaccines for the treatment of infectious and cancerous diseases. For instance, a phase I study revealed the effectiveness and safety of autologous transplantation of DCs transfected with mRNA encoding prostate-specific antigen in the induction of prostate-specific antigenspecific immunity and impact on surrogate clinical endpoints among patients with metastatic prostate cancer (Heiser et al., 2002) . Another phase I/II study showed the impact of intradermal injection of protamineformulated mRNAs coding multiple tumor-associated antigens, e.g., melan-A, tyrosinase, glycoprotein 100, melanoma-associated antigen (Mage)-A1, Mage-A3, and survivin, in patients with metastatic melanoma (Weide et al., 2009) . A very recent phase Ib clinical trial also established the benefits of immunotherapy consisting of protamine-protected, sequence-optimized mRNA (BI1361849 or CV9202) encoding six non-small-cell lung cancer (NSCLC)-associated antigens [New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1), MAGE-C1, MAGE-C2, survivin, 5T4, and Mucin-1] among patients with stage IV NSCLC (Papachristofilou et al., 2019) . As such, a phase I/II study (NCT03164772) is underway to evaluate the safety and efficacy of combination therapy with CV9202 mRNA vaccine and checkpoint inhibitors (e.g., anti-programmed death-ligand 1 durvalumab and anti-cytotoxic T-lymphocyte antigen 4 tremelimumab) for the treatment of NSCLC. Other ongoing clinical trials include a dose-escalation and efficacy study of intratumoral administration of LNP-encapsulated 872 mRNA-2416 encoding human OX40L (NCT03323398) and mRNA-2752 encoding human OX40L, IL-23, and IL-36g (NCT03739931), alone or combined with durvalumab, for patients with advanced malignancies.

Managing infectious diseases through mRNA vaccine is also actively assessed in a clinical setting. One investigation demonstrated that autologous transplantation of monocyte-derived DCs pretreated with mRNAs encoding Group-specific antigen and a chimeric transactivating regulatory protein (Tat)-anti-repression trans-activator (Rev)-negative regulatory factor (Nef) protein was tolerated and effective at enhancing antiviral responses in six patients infected with HIV-1 and under stable and highly active antiretroviral therapy (Van Gulck et al., 2012) . Another phase I/IIa study showed that vaccinations with autologous DCs electroporated with mRNA encoding Tat-Rev-Nef were well tolerated and able to induce vaccine-specific immune responses among 17 HIV-1-infected patients who were stable on combined antiretroviral therapy, after which combined antiretroviral therapy was interrupted (Allard et al., 2012) . The benefits of this vaccination therapy with mRNA-transfected DCs were further demonstrated by very recent clinical studies (Gandhi et al., 2016; Leal et al., 2018; de Jong et al., 2019) ; however, the exact mRNA vaccines are different. Furthermore, clinical studies revealed a robust prophylactic immunity of LNP-carried, specifically modified mRNA vaccines encoding hemagglutinin proteins of avian influenza virus A H10N8 induced in humans, although some mild to severe adverse events were noted (Bahl et al., 2017) . In addition, a randomized, observer-blind, placebo-controlled, and dose-ranging phase I study (NCT04064905) is recruiting healthy flavivirus seropositive and seronegative adults for the evaluation of the safety and immunogenicity of a Zika vaccine (mRNA-1893).

To combat against the ongoing global severe acute respiratory syndrome coronavirus (SARS-CoV)-2 pandemic, or coronavirus disease 2019 crisis, which as of the acceptance of this paper for publication, has caused more than 17 million confirmed cases and over 673,000 deaths worldwide and more than 4.6 million cases and over 154,000 deaths in the United States (https:// www.worldometers.info/coronavirus/), enormous efforts are underway to develop treatment and preventive strategies, including mRNA vaccines (Corey et al., 2020; Yi et al., 2020a) . The SARS-CoV-2 virus belongs to a family of positive-sense, single-stranded RNA coronaviruses whose replication depends on the translation of viral RNA into proteins and reproduction of viral RNAs, as well as assembly of the capsid within host cells, after the interactions between viral spike proteins and host cellular membrane proteins (Hoffmann et al., 2020; Letko et al., 2020) . Given the important role in SARS-CoV-2 viral infection, the spike proteins have emerged as potential targets for the development of smallmolecule and protein drugs as well as vaccines. Indeed, one LNP-encapsulated mRNA vaccine encoding a 103 transmembrane-anchored SARS-CoV-2 spike protein with the native furin cleavage site (mRNA-1273) has quickly entered into clinical trials (NCT04405076 and NCT04283461), attributable to the structure-guided design of target protein/mRNA, fast LNP/mRNA vaccine platform technology, and some promising preclinical observations (K. S. Corbett et al., preprint, https://doi.org/ 10.1101 Corbett et al., preprint, https://doi.org/ 10. /2020 . Although the unprecedented need for vaccination against SARS-CoV-2 is clear, establishing the safety and efficacy of a vaccine takes time before it can be used to immunize a large population to protect global public health.

IVT mRNAs encoding target proteins and antibodies may be developed for protein replacement therapy and antibody therapy, respectively. This is an alternative strategy to classic gene therapy using DNA materials, protein/antibody molecules, and the most recent gene editing technology for the treatment of monogenic disorders caused by impaired or disrupted protein synthesis in body (Martini and Guey, 2019) , as well as some common diseases such as infection and cancer (Schlake et al., 2019) . As an example, methylmalonic acidemia (MMA) is an inherited metabolic disorder usually found in early infancy that ranges from mild to life-threatening, and about 60% of MMA cases are attributed to the deficiency of hepatic methylmalonyl CoA mutase (MUT) synthesis, caused by mutations in the MUT gene. A pseudouridine-modified, codonoptimized mRNA encoding human MUT formulated with LNP has been developed as mRNA replacement therapy for the treatment of MMA, and its efficacy and safety profiles have been established very recently in murine models . Currently, an open-label, dose-escalation phase I/II clinical study (NCT03810690) is underway to evaluate the safety, PK, and PD of mRNA-3704 encoding functional MUT enzyme among patients with isolated MMA due to MUT deficiency between 1 and 18 years of age with elevated plasma methylmalonic acid.

With the discovery of gene editing technologies and development of novel therapeutic strategies, there is also growing interest in using mRNAs to introduce target proteins to achieve gene editing. They include the use of mRNAs encoding zinc finger nucleases (Geurts et al., 2009; Wood et al., 2011; Huang et al., 2014; Wang et al., 2015a; Conway et al., 2019) , transcription activator-like effector nucleases (Tan et al., 2013; Wefers et al., 2013; Poirot et al., 2015; Nanjidsuren et al., 2016) , transposases (Wilber et al., 2006; Ivics et al., 2014a,b; Ellis et al., 2017) , CRISPR-associated proteins, or endonucleases (e.g., Cas9 and Cas12a) Wu et al., 2013; Yin et al., 2016; Ren et al., 2017; Cromer et al., 2018; Xu et al., 2018; Gurumurthy et al., 2019) to enable genome editing or alteration of specific gene sequences. Rather, the specificity and safety of editing a genome with such new modalities warrant more extensive and critical RNA-Based Therapies studies, and their utility for the treatment of human diseases is mainly under preclinical investigations thus far.

6. Guide RNAs. The prokaryotic CRISPR/Cas immune system (Jansen et al., 2002; Makarova et al., 2006; Barrangou et al., 2007) has been developed as a novel and accessible technology to precisely edit genome sequence toward irreversible knockout or knockin of a target gene in mammalian cells and organisms (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013; Mashiko et al., 2013; Kim et al., 2017; Anzalone et al., 2019) , as compared with RNAi, which does not completely eradicate gene expression, and mRNA therapy, which transiently introduces functional proteins. The CRISPR/Cas-based gene editing technology relies on two essential components, a designed gRNA and the RNA-guided Cas nuclease. Through its hairpin scaffold binding to Cas to form Cas-gRNA ribonucleoprotein (RNP) complex, the gRNA recognizes a protospacer-adjacent motif element and a 20-nucleotide sequence in the genome through complementary base pairings and thus directs the Cas nuclease to generate a double-stranded DNA break or a single-stranded break (nick) to achieve genome engineering. As such, the CRISPR/Cas technology has been actively evaluated toward the development of new therapies for the treatment of human diseases, including monogenetic disorders, infection, and cancer (Xue et al., 2014; Dever et al., 2016; Long et al., 2016; Nelson et al., 2016; Tabebordbar et al., 2016; Eyquem et al., 2017; Zhang et al., 2017; Georgiadis et al., 2018; Xu et al., 2019b) .

Different from other types of RNA therapeutics, the success of CRISPR/Cas-based genome editing and therapy relies not only on exogenous gRNA but also foreign Cas nuclease, that latter of being a large protein around 160 kDa in size. Moreover, both the gRNA and Cas9 protein need to get into the nucleus and form RNP to exercise genome editing (Fig. 1) . Besides conventional intracellular expression using plasmid DNA (pDNA) or virus vector-based materials, the gRNAs may be produced by IVT or chemical synthesis and directly introduced into cells or organisms with particular delivery systems, alone or combined with Cas nuclease as RNP. Each approach has its own advantages and disadvantages regarding the cost, stability, efficiency, specificity, and safety [for reviews, see Sahel et al. (2019) , Chen et al. (2020) ]. With the knowledge of chemical modifications in protecting against RNase digestion and avoiding immunogenicity, chemically modified gRNAs have been shown to enhance genome editing efficiency and target specificity in mammalian cells (Hendel et al., 2015; Rahdar et al., 2015; McMahon et al., 2018) . Thus, chemoengineered gRNAs are expected to improve the development of CRISPR/Cas-based therapies.

Multiple clinical trials have been launched to investigate the safety and effectiveness of CRISPR/Cas-based therapies (https://www.clinicaltrials.gov/), but no results are reported yet. The first clinical trial involving CRISPR/Cas gene editing (NCT02793856), open in 2016, was a dose-escalation study on autologous implantation of programmed cell death protein 1 (PD-1; coded by PDCD1 gene) knockout T cells for the treatment of patients with advanced NSCLC that has progressed after all standard treatments. Immune checkpoint regulator PD-1 is a membrane receptor responsible for the inhibition of T cell activation, thereby decreasing autoimmune reactions and allowing immune escape of cancers. Antibodies against PD-1 or its ligand have been successfully used for the treatment of various types of cancers, and CRISPR/Cas-based PD-1 immunotherapy represents a novel strategy to combat cancer. Another phase I trial (NCT03399448) was initiated in 2018 to define the safety profile of NY-ESO-1 redirected autologous T cells with CRISPR-edited endogenous T cell receptor (TCR) and PD-1 autologous T cells, in particular, transduced with a lentiviral vector to express cancer/ testis antigen 1 or NY-ESO-1 and electroporated with CRISPR gRNA to disrupt expression of endogenous T cell receptor TCRa and TCRb, as well as PD-1 (NYCE T Cells), among subjects with a confirmed diagnosis of relapsed refractory multiple myeloma, melanoma, synovial sarcoma, or myxoid/round cell liposarcoma. Presently, there are a number of phase I/II studies on the safety and efficacy of autologous CD34+ human hematopoietic stem and progenitor cells modified with CRISPR/Cas at the erythroid lineage-specific enhancer of the B-cell lymphoma/leukemia 11A gene (CTX001) in patients with transfusion-dependent b-thalassemia (NCT03655678) and patients with severe sickle cell disease (NCT03745287). In addition, the safety and efficacy of CD19-directed T cell immunotherapy comprising allogeneic T cells modified with CRISPR/Cas (CTX110) for the treatment of patients with relapsed or refractory B cell malignancies are currently under clinical evaluation (NCT04035434).

7. Other Forms of RNAs. There are also efforts devoted to develop other forms of RNAs for the treatment of human diseases, such as short or small hairpin RNAs (shRNAs) (Brummelkamp et al., 2002; Paddison et al., 2002) , ribozymes or catalytic RNAs (Burnett and Rossi, 2012) , and circular RNAs (Holdt et al., 2018; Santer et al., 2019) . Like siRNAs and miRNAs, shRNAs can be used to achieve selective gene silencing effects via the RNAi mechanism. They are usually introduced into cells through viral vectors or pDNA, and shRNAs can be chemically synthesized with desired modifications. Similar to a pre-miRNA in size and with hairpin structure, shRNA follows the miRNA biogenesis pathway once shRNA precursor is transcribed from the coding sequence integrated into the host genome in the nucleus. The guide strand derived from shRNA in the cytoplasm is loaded into the RISC to silence target gene expression in the same manner as synthetic siRNAs. Likewise, there are some ongoing clinical studies on the 874 benefits of new modalities involving shRNAs (https:// www.clinicaltrials.gov/). For instance, an open-label phase I study (NCT03282656) is recruiting patients with sickle cell disease to evaluate the feasibility of autologous bone marrow-derived CD34+ HSC cells transduced with the lentiviral vector containing an shRNA targeting B-cell lymphoma/leukemia 11A. As another example, a plasmid named pbi-shRNA Ewing sarcoma breakpoint region 1 (EWS)/Friend leukemia integration 1 transcription factor (FLI1) was developed to target the EWS/FLI1 fusion gene, which is a driver in the pathogenesis and maintenance of Ewing's sarcoma (Rao et al., 2016) . Formulated with lipoplex (LPX), the pbi-shRNA EWS/FLI1 LPX was found effective in type 1 Ewing's sarcoma xenograft mouse models (Rao et al., 2016) , leading to the opening of a phase 1 clinical study (NCT02736565) on pbi-shRNA EWS/FLI1 LPX in patients with advanced Ewing's sarcoma.

Ribozymes are a specific group of RNA molecules that are able to catalyze biochemical reactions (Kruger et al., 1982) . The hammerhead or hairpin structures facilitate a ribozyme to cleave target RNAs in specific sequences, and the substrate recognition domain of the ribozyme can be artificially engineered to stimulate site-specific cleavage in cis (the same nucleic acid strand) or trans (a noncovalently linked nucleic acid) (Scherer and Rossi, 2003) . Moreover, ribozymes are amenable to in vitro selection or evolution, e.g., by systematic evolution of ligands by exponential enrichment approaches (Ellington and Szostak, 1990; Robertson and Joyce, 1990; Tuerk and Gold, 1990 ), toward improved properties or new functions for therapeutic and diagnostic purposes. As with enzymes, catalytic RNAs often require cofactor magnesium ions to exert biotransformations (Ban et al., 2000) . The development of ribozymes as therapeutic molecules has been largely dependent on the improvement of PK properties via chemical modifications (Burnett and Rossi, 2012) . Indeed, a chemoengineered, antiangiogenic ribozyme against the oncogene fms-like tyrosine kinase (FLT-1) by targeting the vascular endothelial growth factor receptor-1 mRNA was effective at cleaving FLT-1/vascular endothelial growth factor receptor-1 mRNA and dose dependently inhibiting lung metastasis in an animal model (Pavco et al., 2000) . Although anti-FLT-1 ribozyme (RPI.4610 or Angiozyme) was well tolerated among patients in both phase I and II studies (Kobayashi et al., 2005; Morrow et al., 2012) , there was a lack of clinical efficacy for the treatment of patients with metastatic breast cancer, precluding RPI.4610 from further development (Morrow et al., 2012) . In addition, a phase II clinical study (NCT01177059) was conducted to evaluate the potential benefits of an anti-HIV-1 ribozyme (OZ1) for the treatment of patients with HIV-1 infection; however, the results have not been published yet.

Although they offer an unprecedented opportunity to expand the range of druggable targets for the control of potentially all kinds of human diseases, there were only fewer than 10 RNA drugs approved by the FDA over the past 2 decades (Table 2) . Most RNA drugs are designed to act on intracellular pharmacological targets (Fig. 1) , whereas RNA molecules are intrinsically unstable and are unable to freely cross cellular membranes, unlike traditional small-molecule and protein medications ( Table 1 ). In addition, RNA drugs may not be highly selective toward their targets, as expected. Indeed, exogenous RNAs are commonly recognized by cellular defense systems, which could lead to acute immune response, cytokine release syndrome, or even severe cytokine storms. Therefore, the development of efficacious and safe RNA therapeutics has proven to be highly challenging.

1. Choice of RNA Substances. The chemical nature of RNA molecules makes them highly susceptible to ubiquitous RNases (Houseley and Tollervey, 2009) , and chemically modified RNA analogs dominate RNA drug discovery and development. Indeed, a wide variety of chemical modifications may be introduced into an RNA molecule, the major strategy to improve RNA metabolic stability and PK properties (Bramsen and Kjems, 2012; Khvorova and Watts, 2017; Yu et al., 2019) . For example, the change of PO linkage to PS makes the resulting RNA analog resistant to RNase degradation, and its PK properties can be further improved when its 29-hydroxyl group on ribose is protected or directly substituted with fluorine. This approach has found ultimate success in the development of sRNA drugs such as ASOs, siRNAs, miRNAs, and aptamers, as all RNA drugs approved by the FDA thus far are chemoengineered RNA analogs (Table 2) , supporting the utility of chemical modifications. Additionally, although it becomes much more expensive to chemically synthesize and modify longer RNAs, those chemoengineered molecules, such as gRNAs, can exhibit greater metabolic stability and biological function in human cells (Hendel et al., 2015; Rahdar et al., 2015) .

The mRNA drugs are usually much bigger in size than other sRNAs used for gene silencing or genome editing (Fig. 1 ). In addition to the entire open reading frame of encoded protein, the therapeutic mRNA needs to contain the complete 59 and 39UTRs as well as 59 cap and 39 poly(A) tail to ensure an efficient translation within cells. Therefore, mRNA drug molecules are generally produced by a conventional IVT method using the T7 or SP6 RNA polymerase (Milligan et al., 1987; Beckert and Masquida, 2011) . Compared with chemical synthesis, IVT represents an efficient and economic approach to generating a large therapeutic mRNA molecule that consists of essential components for intracellular translation. Although IVT is unable to specifically assemble post-transcriptionally modified or natural nucleosides into an mRNA molecule at particular sites, and the effects of such modifications on the efficiency of intracellular translation remain obscure, systemic incorporation RNA-Based Therapies of pseudouridine (Karikó et al., 2008) or N1-methylpseudouridine (Svitkin et al., 2017) into mRNA substances could be achieved by IVT reactions, leading to an improvement of translational capacity and biological stability, as compared with unmodified counterparts.

The primary sequence, particularly a series of nucleobases, is critical for the RNA molecule to act on its RNA or DNA therapeutic target. This notion is also supported by the successful marketing of PMO drugs (Table 2 ), in which nucleobases are linked by morpholino phosphorodiamidate bonds. However, RNA secondary (e.g., helices or stems, loops, and bulges), tertiary (e.g., junctions, pseudoknot, and motifs), and quaternary (e.g., complexes) structures formed by Watson-Crick complementary base pairings and/or other types of physicochemical interactions (Butcher and Pyle, 2011; Jones and Ferré-D'Amaré, 2015; Schlick, 2018) are essential for its stability, plasticity, interactions with cofactors, function, and safety. There are also various types and unique post-transcriptional modifications (Limbach et al., 1994; Cantara et al., 2011; Yu et al., 2019) that are critical for the folding and functions of natural RNAs produced in living cells. In addition, post-transcriptional modifications have been shown to suppress immune responses in cells (Nallagatla et al., 2008; Gehrig et al., 2012) , whereas many chemical modifications induce immunogenicity. Therefore, there are growing interests in developing bioengineering technologies to produce true biological RNA molecules in living cells for research and development (Ho and Yu, 2016; Yu et al., 2019 Yu et al., , 2020 , similar to protein research and drug development that create and use recombinant or bioengineered proteins.

Two novel approaches have been developed very recently to offer high-yield and large-scale fermentation production of bioengineered or biological RNA agents (BERAs), e.g., tens of milligrams target RNAs from 1 l of bacterial culture. One strategy involves the use of stable RNA carriers (Ponchon and Dardel, 2007; Ponchon et al., 2009; Li et al., 2014 Li et al., , 2015 Li et al., , 2018b Chen et al., 2015; Ho et al., 2018) , and the other method seeks direct overexpression in RNase III-deficient bacteria (Hashiro et al., 2019a,b) . Among them, stable hybrid tRNA/pre-miRNA molecules have been identified and proven as the most robust and versatile carriers to accommodate various types of warhead RNAs, including miRNAs, siRNAs, aptamers, and other sRNAs Wang et al., 2015b; Ho et al., 2018; Li et al., 2018b) . This approach follows a similar workflow as protein bioengineering (Fig. 2) . After a BERA/miRNA, siRNA, or sRNA substance of interest is designed, the corresponding coding sequence is cloned into a vector. Overexpression of target BERA in pDNA-transformed bacteria can be assessed by RNA gel electrophoresis analysis of total bacterial RNAs. Recombinant BERA may be isolated with different methods (e.g., anion exchange fast protein liquid chromatography), and the quality of purified BERA can be controlled by high-performance liquid chromatography (HPLC) analysis and endotoxin pyrogen testing Ho et al., 2018; .

BERAs produced in living cells have been revealed to carry no or minimal post-transcriptional modifications (Ponchon and Dardel, 2007; Li et al., 2015; Wang et al., 2015b) . Although naked BERAs are still susceptible to degradation by serum RNases, BERAs are readily Fig. 2 . The tRNA/pre-miRNA-based RNA bioengineering technology for the production of biological RNAi agents (BERA) for research and development. After the design of a target BERA/sRNA (e.g., miRNA, siRNA, and asRNA, etc.), the corresponding coding sequence is cloned into a vector. Expression of target BERA in bacterial culture is readily verified through RNA gel electrophoresis, and BERA can be purified to a high degree of homogeneity using different methods [e.g., anion exchange fast protein liquid chromatography (FPLC)]. Purity of isolated BERA is determined by HPLC analysis and endotoxin pyrogen testing. These bioengineered RNA molecules should better recapitulate the properties of natural RNAs to exert biological or pharmacological actions, as both are produced and folded in living cells. Indeed, BERA/sRNAs can be selectively processed to warhead sRNAs for the modulation of target gene expression in human cells and control of diseases in animal models. WT, wild type. delivered into human carcinoma cells and xenograft tumor tissues by lipid or polymer-based materials; selectively processed to target warhead miRNAs or siRNAs to modulate target gene expression; and consequently inhibit cancer cell proliferation, tumor progression, and metastasis Zhao et al., 2016; Jian et al., 2017; Jilek et al., 2017 Jilek et al., , 2019 Ho et al., 2018; Zhang et al., 2018; Tu et al., 2019; Xu et al., 2019a; Yi et al., 2020b) . In addition, these BERA-carried miRNAs and siRNAs have been shown to be equally or more effective than synthetic counterparts in the regulation of target gene expression and suppression of cancer cell growth Wang et al., 2015b) . These bioengineered RNA molecules should better recapitulate the properties of natural RNAs to exert structural, biological, or pharmacological actions, as both are produced and folded in living cells.

2. RNA Delivery Systems. A major challenge in the development of RNA drugs is to overcome the degradation by serum RNases and make RNAs to cross the membranes of targeted cells so that a sufficient number of RNA molecules can access intracellular targets to exert pharmacological effects (Fig. 3) . As mentioned previously, some chemical modifications can greatly improve the metabolic stability and PK properties (Bramsen and Kjems, 2012; Ho and Yu, 2016; Khvorova and Watts, 2017; Yu et al., 2019) and thus make the resulting RNA substances more druggable. All the ASO therapeutics approved by the FDA carry extensive chemical modifications (Table 2) . Without additional excipients, these ASOs can be distributed to targeted cells to achieve efficacy, like small-molecule drugs. On the other hand, RNA drugs may be "actively delivered" to targeted cells or tissues through encapsulation/ formulation with specific materials (Fig. 3) or carried by viral vectors, pDNAs, or intact cells (Tibbitt et al., 2016; Dowdy, 2017; Kaczmarek et al., 2017; Kowalski et al., 2019) . The latter tends to switch the modalities to DNA/gene materials or engineered cells, whereas the former keeps the RNA substances as the active ingredients.

Lipids and lipid-like materials that resemble the lipid component of cell membrane and offer great biocompatibility are commonly used for the delivery of RNA agents besides peptides, hydrogels, dendrimers, and synthetic (e.g., polyamidoamine and polyethyleneimine) and natural polymers (e.g., chitosan) (Tibbitt et al., 2016; Kaczmarek et al., 2017) . Similar to positively charged polymers or other substances, cationic lipids can electrostatically bind to RNAs to protect them against RNase cleavage and facilitate endocytosis. Interestingly, ionizable lipids that only become positively charged in acidic conditions have been shown to enhance endosomal release and reduce cytotoxicity (Kanasty et al., 2013) . The successful use of LNP in RNA drug development is evident in the first FDAapproved siRNA drug, patisiran (Table 2) . LNP is also used for the delivery of novel mRNA therapeutics, such as human MUT mRNA replacement therapy for the treatment of MMA, which has been evaluated in murine models and entered clinical trials. In addition, LNP has been employed Fig. 3 . Common approaches for the formulation and delivery of RNA therapeutics. RNA drugs administered intravenously need to be protected against excessive degradation by serum RNases and overcome the cell membrane barriers to gain access to intracellular targets. After entering cells through endocytosis or other mechanisms, RNA therapeutics are released in the cytoplasm, translocated, and incorporated into corresponding ribonucleoprotein complexes to silence target transcripts. As chemical modifications largely improve the metabolic stability and PK properties, RNA drug may be encapsulated in nanoparticle using different systems such as LNP and LPP. In addition, conjugation of a trivalent GalNAc to the RNA drug or use of antibody can improve the efficiency of delivery to targeted cells.

RNA-Based Therapies for the delivery of the gRNA-Cas9 RNP complex for genome editing .

To optimize LNP formulations, some hydrophobic moieties, such as cholesterol-lipid poly(ethylene glycol)-lipid, may be included to improve RNA delivery efficiency, and the ionizable property of LNP seems to be a determinant factor (Akinc et al., 2008; Love et al., 2010; Dahlman et al., 2014) . Furthermore, as polyethyleniminebased polyplexes provide excellent delivery efficiency but pose a high risk of toxicity (Lv et al., 2006) , lipidation of the polyethylenimine-RNA polyplex offers lipopolyplex (LPP), which may retain the benefits of both materials while reducing the toxicity of polyplex (Rezaee et al., 2016) . Indeed, some recent studies have demonstrated that LPP showed more favorable biological properties for the delivery of nucleic acid agents (Schäfer et al., 2010; Ewe et al., 2014 Ewe et al., , 2017 , including these novel BERAs produced and folded in living cells (Zhang et al., 2018; Jilek et al., 2019) .

RNAs may be covalently conjugated to specific ligands, ranging from relatively smaller molecules (e.g., aptamers) to large molecules (e.g., lipids, peptides, and antibodies). Ligand-directed delivery is expected to improve targeting toward particular types of cells. The conjugation of RNA drug to GalNAc is the most successful example to target hepatocytes through interactions with the cell surface receptor (Nair et al., 2014 (Nair et al., , 2017 . Givosiran is a GalNAcconjugated siRNA drug (Table 2) in which the GalNAc facilitates hepatocyte uptake of givosiran to act on intracellular TTR mRNA. In other cases, aptamers can be used to facilitate targeted delivery of RNAs to specific cells, including siRNAs, miRNAs, and shRNAs (Dassie and Giangrande, 2013; Aaldering et al., 2015) . Interestingly, ligand- and aptamermediated RNA delivery (Pastor et al., 2010; Neff et al., 2011 ) not only increased drug efficacy but also reduced off-target effects.

As targeted delivery may facilitate the internalization of RNA drugs into targeted tissues and cells (e.g., tumor tissues and carcinoma cells), RNAs entering the systemic circulation can still be distributed into other tissues, especially liver and kidney, like small-molecule drugs. Indeed, liver and kidney are the most important organs for the elimination of xenobiotics and exogenous medications including RNA drugs (via hepatic metabolism and renal excretion, respectively), commonly showing higher levels of drug accumulation than other tissues. Interestingly, many novel RNA drugs approved by the FDA (Table 2) and currently under development are designed to act on hepatic targets. In any case, the overall efficacy and safety profiles determine whether a drug can be marketed or not, and both are attributed to the levels of drug exposure. In addition, caution must be exercised when interpreting RNA delivery/distribution/ PK data obtained from studies using small-molecule dye supplemented in the RNA formulation or covalently attaching a fluorescent core to RNA as a tracer or surrogate for the convenience of analyses. On the one hand, upon release from the formulation, a smallmolecule dye with its own physicochemical and PK properties that are totally different from RNA substances will be distributed or redistributed in the body in its own way, e.g., accumulated in highly proliferative tissues/cells such as tumors and carcinoma cells, whereas the RNA drugs of interest might not. On the other hand, conjugation of fluorescent core to an RNA molecule shall result in a new, different, conjugated RNA substance that undoubtedly has its own physicochemical and PK properties. Moreover, the conjugate may be metabolized in the body, and the cleaved fluorescent core will exhibit its own distribution/PK profiles. Therefore, it is vital to define the validity of such tracing approaches in related studies or directly examine the actual warhead RNAs.

3. RNA Analytical Methods. Reliable bioanalytical methods are pivotal for the quantification of drugs during discovery and development. Method validation including RNA stability, calibration range, matrix effects, sensitivity, selectivity, accuracy, and precision should be conducted in line with the regulatory guidelines on bioanalytical method validation, despite there being a lack of specific recommendations for RNAs (Kaza et al., 2019) . Rather, challenges in the development of an accurate method for quantitative analysis of target RNA drug in complex biological samples seem to be overlooked. Two major strategies are commonly used for RNA analyses: hybridization-based (or biology) methods and chromatographic (or chemistry) methods (Wang and Ji, 2016) . Hybridization-based assays, which may [e.g., quantitative polymerase chain reaction, (qPCR)] or may not (e.g., ELISA) involve the amplification of RNA analytes (Table 3) , indirectly analyze RNAs through the use of complementary, fluorophore-labeled, or ligand-labeled oligonucleotide probes or antibodies to interact with target RNA analytes and then quantify the hybridization complexes through different readouts. On the other hand, chromatography methods aim at direct analysis of target RNAs through liquid chromatographic separation followed by detection of intrinsic UV or mass spectrometry (MS) signals (Table 3) . Hybridization methods are usually highly sensitive and applicable to the analyses of all types of RNAs, whereas chromatography methods are relatively more specific and accurate in absolute quantification of sRNAs such as ASOs and siRNAs. Hybridization and chromatography may be used together in the same method, e.g., hybridization-based HPLC-fluorescence (FL) assay (Table 3) .

Classic Northern blot and in situ hybridization methods are highly sensitive in detecting target RNAs through radioactivity or fluorescence detections without polymerase chain reaction-based amplification; however, these methods are semiquantitative. Conventional microarray methods and modern RNA sequencing technologies require amplification and conversion of RNAs to cDNAs, 878 which are both high-throughput approaches but offer merely relative quantification. It is also noteworthy that the efficiencies of individual RNAs can be widely variable and affected by the matrices, whereas they are generally not evaluated. For quantitative bioanalysis of RNAs, realtime qPCR (Kelnar et al., 2014) and ELISA (Humphreys et al., 2019; Thayer et al., 2019) methods (Table 3) have been commonly used. The former approach is applicable to both longer RNAs (e.g., mRNAs) and various types of sRNAs [e.g., miRNAs and siRNAs by stem-loop reverse transcription, real-time polymerase chain reaction (Chen et al., 2005) ]. Nevertheless, these biological methods largely rely on the robustness of hybridization probe, amplification primers, or antibody used in the assays. Another major caveat is that these hybridization-based methods may not be able to distinguish the RNA drug from its metabolites, e.g., 1 nt deleted at the 39 end, which the latter has been able to recognize (Ewles et al., 2014; Husser et al., 2017; Liu et al., 2019; Post et al., 2019) .

After chromatographic separation, target RNA drugs can be accurately quantified by UV detection (McGinnis et al., 2012 (McGinnis et al., , 2013 . As UV absorbance is common for nucleic acids, as well as proteins and many smallmolecule compounds, the robustness of LC-UV analysis of RNA drug is dependent on efficient RNA isolation and chromatographic separation of target RNA from other components ( Table 3 ). The use of LC tandem mass spectrometry and high-resolution accurate MS can greatly improve the specificity in quantitative analysis of ASOs and siRNAs (Watanabe et al., 2016; Liu et al., 2019) that are chemically modified and different from natural RNAs. LC-MS methods often provide good accuracy, precision/reproducibility, and wide dynamic range while offering a limited sensitivity (Table 3) , dependent on the structures of RNAs and biological matrices. Compared with hybridization-based methods, LC-MS methods, especially high-resolution accurate MS, may improve the fidelity of RNA bioanalysis because they can separate and/or distinguish the metabolites from the RNA drug. With a highly demanded specificity in drug quantification, a lower sensitivity might be compromised.

4. Specificity and Safety of RNA Drugs. The specific interaction between a drug and target is critical to not only induce selective, on-target, therapeutic effects but also avoid nonspecific, off-target, adverse events. This is of particular concern because exogenous RNAs may trigger sequence-dependent and -independent immune responses. Indeed, immune-related and dose-limiting toxicities are major reasons for the failure of many RNA drugs during clinical investigations (Kleinman et al., 2008; Davis et al., 2010; DeVincenzo et al., 2010; Beg et al., 2017) . The underlying targets are known as cell membrane or endosomal Toll-like receptors and cytosolic sensors (e.g., RNA-dependent protein kinase) (Karikó et al., 2005; García et al., 2006; Hornung et al., 2006; Anderson et al., 2010) . The recognition of specific sequences or structures of RNA molecules by their corresponding immune receptors triggers immune events such as cytokine secretion, immune cell proliferation and survival, and adaptive immunity activation as the host's natural defense. As such, administration of pharmacological RNA agents may cause immunogenic response or cytokine release syndrome depending on the doses and structures (e.g., size, sequence, etc.) of RNA molecules (Dalpke and Helm, 2012; Tanji et al., 2015) . Interestingly, although natural post-transcriptional modifications may suppress immune responses (Karikó and Weissman, 2007;  RNA-Based Therapies Robbins et al., 2007; Nallagatla et al., 2008; Gehrig et al., 2012) , various and extensive chemical modifications can induce immunogenicity (Robbins et al., 2007; Bramsen and Kjems, 2012) . Therefore, chemoengineering of RNA drugs using naturally occurring modifications (e.g., 29-methoxyribonucleoside, pseudouridine, methyladenosine, methylcytidine, etc.) may help to reduce immunogenicity (Robbins et al., 2007; Kauffman et al., 2016) . The development and use of natural RNAs made in living cells is also expected to minimize the risk of induction of immune responses (Ho and Yu, 2016; Yu et al., 2019) .

While acting on targets through designed complementary base pairing, RNA agents may induce other off-target effects, particularly the interactions with unintended homologous targets. This issue is present not only in using ASOs and siRNAs for target gene silencing (Chi et al., 2003; Jackson et al., 2003; Semizarov et al., 2003) but also in utilizing gRNAs for gene editing (Frock et al., 2015; Liang et al., 2015; Filippova et al., 2019) . Compared with miRNAs that may act on multiple targets, siRNAs (and ASOs) are usually postulated as specific agents in knocking down the expression of targeted genes that are rarely assessed. By contrast, genome-wide experiments showed that many transcripts with partial complementarities to siRNAs were altered in cells (Chi et al., 2003; Jackson et al., 2003; Semizarov et al., 2003) . Based upon cellular miRNAgoverned post-transcriptional regulation mechanism, it is hard to imagine that siRNAs do not affect other transcripts through seed-dependent complementary interactions with their 39UTRs. Indeed, the 39UTR matches or miRNA-like "off-target" effects have been confirmed experimentally (Birmingham et al., 2006; Jackson et al., 2006) . Rather, proper design of RNAi agents can achieve relatively specific targeting and desired efficacy with minimal or manageable adverse effects, and natural miRNAs acting on multiple targets in the common pathways could be helpful for the control of particular diseases.

It is noteworthy that, like all other classes of drugs (Table 1) , the selectivity and safety of RNA therapeutics are dose-dependent. Dose-limited off-target effects were commonly observed when higher or exaggerated doses of RNAi agents were used (Semizarov et al., 2003; Janas et al., 2018) . Therefore, in addition to the design of optimal sequence and use of proper chemical/natural modifications to avoid or minimize immunogenicity and off-target effects, identification of the right dose to achieve therapeutic efficacy and safety is critical for the development of RNA drugs.

Since RNAs are usually delivered with particular carriers, the safety of carrier materials as well as the final formulated drug product should be critically assessed. Toxicities may come from the degradation of these materials during storage, infusion reactions of the modality, and accumulation in nontarget tissues (Vogel, 2010; Zuckerman and Davis, 2015; Szebeni et al., 2018) .

The use of clinically verified safe carriers, such as LNPs, and the development of targeted delivery approaches may help to minimize the risk of toxicity caused by delivery vehicles or induced in nontarget tissues. Actually, some medications can be used proactively to protect against possible adverse effects or afterward to lessen the toxicity. This is extremely important during clinical practice because all medications may cause toxicity while also offering benefits. In terms of RNA therapies, patients may be treated with corticosteroids or antiallergy medications to manage infusion reactions to FDA-approved patisiran and investigational RNA drugs (Adams et al., 2017; Beg et al., 2017) .

Although disease-related mRNAs and ncRNAs may be targeted by sRNAs such as ASOs or asRNAs, siRNAs and miRNAs described above ( Fig. 1; Table 2 ), which are conventional small-molecule compounds, are still the preferred entities in drug discovery and development. With the understanding of mechanistic actions of many natural antibiotic drugs, including aminoglycosides, tetracyclines, macrolides, and oxazolidinones (Fig. 4) , on rRNAs in the inhibition of microbial protein synthesis (Table 4) (Wilson, 2009 (Wilson, , 2014 Lin et al., 2018) , there is growing interest in discovering and developing RNAtargeted small-molecule drugs [for recent reviews, see Hermann (2016) , Donlic and Hargrove (2018) , Warner et al. (2018) ]. The notion of developing small-molecule drugs targeting RNAs is also supported by the fact that riboswitches can be selectively bound by small-molecule ligands in the control of gene expression (McCown et al., 2017) . Indeed, a number of novel classes of rRNA-targeted synthetic or semisynthetic antibiotics, including oxazolidinone (e.g., linezolid and tedizolid, etc.), ketolides (e.g., telithromycin), phenicols (e.g., chloramphenicol), and lincosamides (e.g., clindamycin), have been approved for medical use (Deak et al., 2016; Lin et al., 2018) . Moreover, the first ribosome-targeted ataluren was approved in Europe for the treatment of patients with DMD with a nonsense mutation in the dystrophin gene (Haas et al., 2015) , and it is undergoing phase III clinical trials in the United States (Table 4 ). There are also many other small molecules under clinical and preclinical development that act on existing and new RNA targets for the treatment of various human diseases, including genetic disorders, infections, and cancer (Table 4) .

Similar to protein targets, macromolecule RNAs are folded into highly structured entities for their interactions with small molecules (Cruz and Westhof, 2009) or proteins (Hentze et al., 2018) . Through complementary base pairings and other forms of physicochemical 880 interactions, RNAs are folded into secondary (e.g., helices or stems, loops, and bulges), tertiary (e.g., junctions, pseudoknot, and motifs), and quaternary (e.g., complexes) structures (Butcher and Pyle, 2011; Jones and Ferré-D'Amaré, 2015; Schlick, 2018) ; thus, small-molecule compounds have the potential to directly interact with unique higher-order structures rather than the primary sequences. Specific RNA structural elements or motifs, such as bulges, loops, junctions, pseudoknots, and complexes, may be recognized and bound by particular small molecules beyond the attenuation by sRNAs with complementary sequences. Indeed, highly structured RNAs tend to contain pockets permitting specific and high-affinity binding by small molecules with particular functional groups and electrostatic surfaces, similar to those of protein targets. The interactions between small-molecule and disease-related RNA or RNA motif, therefore, could lead to the inhibition or activation of RNA functions or change of gene expression and cellular processes toward the control of disease.

Different from proteins as drug targets, which comprise a total of 22 proteinogenic amino acids, RNAs consist of only four primary nucleotide monomeric units (A, U, G, and C) as building blocks. In comparison with 22 proteinogenic amino acids, showing a combination of physicochemical properties (e.g., basic and acidic, positively and negatively charged, hydrophilic and hydrophobic amino acids, etc.), the four monomeric nucleotides seem less variable. However, with each containing a nucleobase, ribose, and phosphate group, nucleotide monomers are much more complex than individual amino acids. As each phosphate unit carries one negatively charged residue, RNAs are highly charged macromolecules. Under low-salt conditions, many functional RNA molecules may not fold. The addition of cationic ions (e.g., magnesium ion) can induce RNA folding, whereas salt concentrations that are too high will reduce the electrostatic interactions (Lipfert et al., 2014) . In addition, RNAs are generally more hydrophilic than a typical protein (McCown et al., 2017; Mustoe et al., 2018) . Surrounded by an ion atmosphere under physiologic conditions, the folding and structure of electrostatic RNA, especially the sites for ligand binding, are relatively less understood, and they are very different from those found in proteins (Hermann, 2016; Morgan et al., 2017 Morgan et al., , 2018 .

To identify and develop RNA-targeting small molecules, it is also necessary to understand the structural and physicochemical properties of known small-molecule ligands and recognize the principles of RNA-ligand interactions in addition to the characteristics of diseaserelated RNAs or RNA motifs. Indeed, most RNA-targeted Fig. 4 . Chemical structures of some small-molecule antibiotics that are known to target bacterial ribosomal RNAs to inhibit protein synthesis for the treatment of infections. Among them, aminoglycosides (e.g., streptomycin, neomycin, and paromomycin) and tetracyclines (e.g., tetracycline and tigecycline) directly act on 16S rRNAs, whereas natural and semisynthetic macrolides (e.g., erythromycin, spiramycin, and telithromycin) as well as synthetic oxazolidinones (e.g., linezolid and eperezolid) bind to 23S rRNAs to exert antimicrobial activities. Aminoglycosides (e.g., streptomycin, neomycin, paromomycin, etc.) Binds to 16S rRNA in the decoding region A-site on the 30S subunit to induce misreading of the genetic code and thus inhibit protein synthesis Antibiotics to treat infections Approved for medical use Fourmy et al., 1996; Ogle et al., 2001; Demeshkina et al., 2012; Demirci et al., 2013 Tetracyclines (e.g., tetracycline, tigecycline, etc.) Binds to 16S rRNA in the A-site on the 30S subunit to block tRNA binding and thus inhibit protein synthesis Antibiotics Approved for medical use Brodersen et al., 2000; Anokhina et al., 2004; Schedlbauer et al., 2015 Macrolides (e.g., erythromycin, telithromycin, etc.) Binds to 23S rRNA in the NPET of the 50S subunit to block egress of nascent polypeptide and thus inhibit protein synthesis Antibiotics Approved for medical use Vannuffel and Cocito, 1996; Hansen et al., 2002; Berisio et al., 2003; Tu et al., 2005; Bulkley et al., 2010 Oxazolidinones (e.g., linezolid, tedizolid, etc.) Binds to 23S rRNA in the A-site cleft near PTC of 50S subunit to interfere with tRNA accommodation and thus inhibit protein synthesis Antibiotics Approved for medical use Ippolito et al., 2008; Wilson et al., 2008; Deak et al., 2016 Translarna (ataluren; PTC124) Targets ribosome to promote insertion of near-cognate tRNAs at the site of the dystrophin gene toward nonsense suppression Treatment of patients with DMD with nonsense mutation Approved in Europe; phase III trial in the United States (NCT03179631) Bushby et al., 2014; Ryan, 2014; Haas et al., 2015; Roy et al., 2016; McDonald et al., 2017 Risdiplam (RG7916; RO7034067) Interacts with SMN2 pre-mRNA and modifies RNA splicing to increase SMN protein expression levels

Phase II/III trial (NCT02913482) Ratni et al., 2016 Ratni et al., , 2018 Poirier et al., 2018; Sturm et al., 2019 Branaplam (LMI070; NVS-SM1) Stabilizes the spliceosome and SMN2 pre-mRNA interactions to enhance SMN2 pre-mRNA splicing and thus increase expression of full-length SMN mRNA and functional protein Treatment of patients with SMA Phase I/II trial (NCT02268552) Palacino et al., 2015; Cheung et al., 2018; Dangouloff and Servais, 2019 Anthraquinones Binds to HIV TAR element to inhibit viral replication Treatment of HIV infection Preclinical development Ganser et al., 2018 Benzimidazoles (e.g., Isis-11) Binds to HCV IRES to inhibit viral replication Treatment of hepatitis C Preclinical development Seth et al., 2005; Paulsen et al., 2010; Dibrov et al., 2012 Ribocils Binds to FMN riboswitch selectively to suppress riboswitchtargeted gene expression and thus inhibit bacterial growth Antibiotics Preclinical development Howe et al., 2015 Howe et al., , 2016 Wang et al., 2017; Rizvi et al., 2018 Targaprimir-96 and its bleomycin A5 conjugate Binds to pri-miR-96 to inhibit Drosha-mediated processing or induce cleavage of pri-miR-96

Anticancer Preclinical development Velagapudi et al., 2016; Li and Disney, 2018 Targapremir-210 and its conjugate Binds to pre-miR-210 to inhibit Dicer-mediated processing or recruit RNase L for cleavage Anticancer Preclinical development Costales et al., 2017 Costales et al., , 2019b 882 antibiotics (Fig. 4) derived from natural products and given to patients via intravenous administration are more complex molecules, with each containing a large number of hydrogen bond donors and acceptors, having a molecular mass over 500 Da, and being hydrophilic. This is in contrast to the majority of protein-targeted, orally bioavailable, small-molecule drugs that usually follow empirical Lipinski's rule of five (Lipinski, 2004; Ritchie and Macdonald, 2014) . Therefore, caution should be exercised when adopting the rules of protein-ligand interactions for the design of RNA-targeted small molecules, although there are common principles.

Moreover, some small-molecule ligands identified as highly basic and polar molecules tend to bind to RNAs with high affinity but low selectivity (Guan and Disney, 2012; Connelly et al., 2016) . Thus far, a number of strategies have been developed and used to design and/or identify small molecules that can bind to RNA targets, such as binding-or phenotype-based screening (Mei et al., 1997; Sztuba-Solinska et al., 2014; Howe et al., 2015; Prado et al., 2016; Rizvi et al., 2018; Velagapudi et al., 2018; Green et al., 2019) and computational modeling and structure-or chemoinformatics-based design or virtual screening (Stelzer et al., 2011; Parkesh et al., 2012; Nguyen et al., 2015; Disney et al., 2016; Luu et al., 2016) . These efforts have led to the discovery and development of chemically diverse small molecules targeting RNAs, with many exhibiting more drug-like structural and physicochemical properties (Fig. 6) . In this section, representative small molecules that target various RNAs, including rRNAs, viral RNA motifs, riboswitches, pre-mRNAs, and pri or pre-miRNAs, are introduced, followed by the discussion of challenges in the identification of therapeutic RNA targets and verification of ligand binding specificity.

B. Classes of RNA Targets 1. Ribosomal RNAs. Discovery of the mechanistic actions of natural antibiotics on bacterial ribosome is a major driver for the discovery and development of RNA-targeted drugs. Bacterial ribosome (70S) catalyzing protein synthesis comprises two subunits (50S and 30S) that are assembled from three rRNA chains (16S, 23S, and 5S rRNAs) and around 55 ribosomal proteins (e.g., L16) (Steitz, 2008; Wilson, 2014; Lin et al., 2018) . After the binding of 70S ribosome with the initiator tRNA and start codon of the mRNA positioned at the peptidyl-tRNA site (P-site), protein synthesis is initiated. Going through the cycle of accommodating aminoacyl-tRNA at the A-site, forming peptide bond between the A-and P-site tRNAs, translocating the peptidyl-tRNA from the A-to the P-site, and releasing the deacylated tRNAs from the exit site (E-site) that are stringently controlled by corresponding factors, a nascent polypeptide is synthesized. The termination of translation involves the hydrolysis of mature polypeptide from tRNA at the P-site, and the 70S ribosome is recycled after the release of deacylated tRNA. The rRNA-assembled ribosome is highly structured for its interactions with individual components required for protein synthesis, in which specific clefts and pockets can be directly blocked by small molecules, leading to the inhibition of translation (Table 4 ).

X-ray crystallography and NMR spectroscopy studies have revealed that many aminoglycoside antibiotics, such as paromomycin and streptomycin, directly bind to 16S rRNA in the decoding region A-site on the bacterial 30S subunit to induce misreading of the genetic code and inhibit protein synthesis (Fourmy et al., 1996; Lynch et al., 2003; Demirci et al., 2013) . The drugtarget interactions include the involvement of crucial nucleotides, such as A1408 in 16S rRNA (Fig. 5) , which when mutated (e.g., A1408G) or methylated at the N1 position (A1408m1A), confers resistance to aminoglycosides (Kondo, 2012; Kanazawa et al., 2017) . The G1408 also distinguishes between prokaryotic and eukaryotic ribosomes, leading to a 25-to 50-fold lower binding affinity for paromomycin to the eukaryotic decoding site and thus providing structural explanation for the selectivity of aminoglycosides for prokaryotic ribosomes (Lynch and Puglisi, 2001) . A recent study using a cryogenic electron microscopy technique has shown that the unfavorable base pairing between C1409 and A1491 in human rRNA versus perfect U1409-A1491 pairing in Leishmania donovai may contribute to the selective inhibition of pathogenic organisms by paromomycin instead of human ribosome . Similarly, antibiotic tetracyclines (e.g., tetracycline and tigecycline) have been revealed to inhibit protein synthesis by sterically hindering tRNA binding to the A-site on the 30S subunit through direct interactions with the 16S rRNA (Brodersen et al., 2000; Anokhina et al., 2004; Schedlbauer et al., 2015) . On the other hand, many natural macrolides (e.g., erythromycin, azithromycin, etc.) bind to the 23S rRNA in the nascent peptide exit tunnel (NPET) of the 50S subunit to block passage of newly synthesized polypeptides and thus interrupt translation elongation (Vannuffel and Cocito, 1996; Hansen et al., 2002; Tu et al., 2005; Bulkley et al., 2010; Dunkle et al., 2010) . In addition, a lot of synthetic antibiotics designed as translation inhibitors are found to target specific sites on ribosomes (Deak et al., 2016; Lin et al., 2018) . For instance, oxazolidinones such as linezolid and tedizolid directly interact with the bacterial 23S rRNA (Fig. 5) and bind in the A-site pocket near peptidyl transferase center (PTC) of the 50S subunit to exclude tRNA accommodation and thus inhibit protein synthesis (Ippolito et al., 2008; Wilson et al., 2008; Deak et al., 2016) .

With the understanding of species differences in drug-ribosome interactions mentioned above, and given the importance of dysregulated protein synthesis in highly proliferative human carcinoma cells, efforts have RNA-Based Therapies been made to elucidate the structural characteristics of human 80S ribosome and its detailed interactions with eukaryote-selective protein synthesis inhibitors (e.g., cycloheximide) (Myasnikov et al., 2016) . These studies are expected to offer clues to the discovery and development of rRNA-or ribosome-targeted anticancer drugs. In addition, ribosome-targeted small molecules may be identified to interfere with the synthesis of specific protein for the treatment of genetic disorders. For example, mutations of the dystrophin gene leading to the disruption of its reading frame is a major cause of neuromuscular disorder DMD (Cirak et al., 2011) , and the approval of ASO/PMO drugs eteplirsen and golodirsen (Table 2 ) supports the restoration of functional dystrophin protein production as a therapeutic strategy for the treatment of DMD. A small molecule, Translarna (ataluren; PTC124) ( Fig. 6) , has been developed to target ribosome to promote the insertion of near-cognate tRNAs at the site of the dystrophin gene and thus achieve nonsense suppression and functional protein production (Bushby et al., 2014; Ryan, 2014; Roy et al., 2016; McDonald et al., 2017) . Ataluren has been approved in Europe for the treatment of patients with DMD with a nonsense mutation in the dystrophin gene (Haas et al., 2015) , and it is currently under phase III clinical trials (NCT03179631) in the United States (Table 4) .

2. Viral RNA Motifs. Viral genomes consist of some highly conserved RNA elements that play pivotal roles in gene regulation and viral replication. Being highly structured, viral RNA motifs have emerged as potential targets for the development of small-molecule antiviral drugs (Hermann, 2016; Di Giorgio and Duca, 2019) . The HIV transactivation response (TAR) element located within the first 59 nt of the viral genome is one of the most studied RNA elements (Stevens et al., 2006) . The TAR RNA element, folded into a hairpin structure (Kulinski et al., 2003) , binds to the Tat protein to form a Tat/TAR complex that stimulates transcription and HIV-1 replication (Le Grice, 2015; Connelly et al., 2016) . Efforts have been made to interfere with Tat/TAR interactions to control HIV-1 infections, including the identification and development of TAR-targeted small molecules (Hermann, 2016 ; Di Giorgio and Duca, 2019). Interactions of representative small-molecule antibiotics with specific nucleotide residues of individual rRNAs within ribosomal subunits. (A) Paromomycin binds to 16S rRNA in the decoding region A-site on the 30S subunit. Specifically, ring I stacks against the G1491 and interacts with A1408 through hydrogen bonding. Ring I also penetrates into the RNA helix and facilitates the flipping out of A1493. Ring II forms tight interactions with both nucleobases and the backbone of the RNA, whereas ring III just makes weak contacts. (B) Linezolid binds to 23S rRNA in the A-site cleft near PTC of the 50S subunit to interfere with tRNA accommodation. In particular, the O4 of ring-C interacts with U2585 via hydrogen bonding. Although G2576 stacks directly onto G2505, other residues clustered around U2504 form the binding pocket linezolid; among them, C2452 interacts with U2504 through the indicated hydrogen bonds. (C) Telithromycin binds to 23S rRNA in the NPET of the 50S subunit through its 3-keto group and hydrophobic interactions.

For example, through viral screening of TAR dynamic structures against .50,000 compounds and fluorescencebased experimental assays, one study identified and validated six small molecules that were able to bind to TAR (Kd = 55 nM to 22 mM) and inhibit its interaction with Tat (Ki = 710 nM to 169 mM) (Stelzer et al., 2011) . Among them, netilmicin (Fig. 6) , a semisynthetic aminoglycoside antibiotic drug, exhibited high selectivity for TAR in the presence of tRNA. The antiviral activity of netilmicin (100 mM) was further demonstrated in mammalian cell lines infected with an HIV-1 clone (Stelzer et al., 2011) . Using a Tat peptide displacement assaybased high-throughput screening, the same group of investigators identified more TAR binders, including five anthraquinone derivatives ( Fig. 6 ; Table 4 ) that showed selectivity relevant to tRNA (Ganser et al., 2018) . Nevertheless, the efficacy and safety of TAR-targeted small molecules in animal disease models remain obscure.

Another well studied viral RNA target is the internal ribosome entry site (IRES) element located in the 59UTR of the hepatitis C virus (HCV) viral genome (Dibrov et al., 2014; Hermann, 2016; Di Giorgio and Duca, 2019) . Being a highly structured RNA motif, HCV IRES functions as an RNA-based initiation factor to directly recruit host cell ribosomes with only a subset of host cell initiation factors and thus mediates cap-independent translation initiation (Lukavsky, 2009) . Through MS-based high-throughput screening against a 29-nt HCV IRES subdomain IIa, imidazoles were identified as lead binders (Seth et al., 2005) . Further structure-activity relationship studies afforded a number of new analogs, e.g., Table 4) , showing comparable binding affinity (Kd = 1.7 mM) and antireplication activity (EC 50 = 1.5 mM) (Seth et al., 2005) . Separate X-ray crystallography and NMR spectroscopy studies characterized the binding complex between HCV pre-mRNAs to switch splicing and thus elevate the expression of functional SMN protein for the treatment of spinal muscular atrophy amenable to SMN deficiency. (E) Targarprimir-96 and targarpremir-210 that block the biogenesis of oncogenic miRNAs through direct binding to pri-miR-96 and pre-miR-210, respectively, to elicit antitumoral activities.

RNA-Based Therapies 885 IRES subdomain IIa and Isis-11 (Paulsen et al., 2010; Dibrov et al., 2012) . As another example, a synthetic diaminopiperidine derivative (Fig. 6 ) was identified to bind with HCV IRES subdomain IIa using a fluorescence-based ligand binding assay (Carnevali et al., 2010) , which likely competes with the magnesium ions, locks the IIa RNA switch in the bent conformation, and prevents ribosome release to inhibit IRES function (Dibrov et al., 2014) . Although the HCV IRES arises as a promising target for the treatment of HCV infections, no smallmolecule inhibitors have entered clinical trials yet.

Efforts were also made to explore other viral RNA motifs as targets for small molecules. The SARS-CoV genome consists of an atypical three-stemmed RNA pseudoknot that stimulates 21 programmed ribosomal frameshifting to initiate translation of viral proteins essential for replication (Plant et al., 2005; Su et al., 2005) . Through virtual screening of about 80,000 compounds against a computationally constructed, three-dimensional SARS-CoV pseudoknot structure and experimental studies on a number of high-ranked compounds, a 1,4-diazepine analog, 2-[[4-(2-methylthiazol-4-ylmethyl) - (1, 4) diazepane-1-carbonyl]amino]benzoic acid ethyl ester (MTDB) (Fig. 6) , was identified to inhibit the 21 ribosomal frameshifting of SARS-CoV with an IC 50 of 0.45 mM (Park et al., 2011) . Indeed, MTDB directly interacts with SARS-CoV pseudoknot to block the formation of alternate conformers, as revealed by singlemolecule force spectroscopy studies . These findings support the concept of developing smallmolecule inhibitors against SARS-CoV RNA pseudoknot for the treatment of pandemic severe acute respiratory syndrome, including the ongoing SARS-CoV-2, in which the pseudoknot is well conserved.

Among most human influenza A virus variants, there is a highly conserved promoter RNA element comprising 13 nt at the 59 end and 12 nt at the 39 end of each viral RNA segment, which are folded together to form a partial duplex called a panhandle-like structure (Bae et al., 2001 ) that is recognized specifically by the RNAdependent RNA polymerase (Pflug et al., 2014) to control transcription initiation and viral replication (Ferhadian et al., 2018) . Through an NMR spectroscopy-based screening, a drug-like quinazoline compound, 6,7dimethoxy-2-(1-piperazinyl)-4-quinazolinamine (DPQ) (Fig. 6) , was found to directly bind to the influenza A virus RNA promoter (Kd around 50 mM) and exhibit antiviral activity against influenza viruses (IC 50 = 70-300 mM) . Further screening and structure-activity relationship studies with cell-based anti-influenza activity assays identified a number of quinazoline derivatives that show around 10-fold higher activity against influenza A virus than the lead compound, DPQ (Bottini et al., 2015) . Similar to other viral RNA-targeted small molecules described above, the discovery of quinazolines against influenza A panhandle-like promoter RNA is promising but still in its early stage.

Riboswitches are a diverse group of natural RNA "receptors" commonly found in bacteria that directly control gene expression critical for survival (Tucker and Breaker, 2005; Hallberg et al., 2017; McCown et al., 2017) . Riboswitches are usually located in the 59UTR of particular mRNAs, and each consists of a ligand-sensing domain (or aptamer domain) and a regulatory domain (or expression platform). The specific binding of small-molecule ligand to an "aptamer" domain triggers conformational changes and stimulates the regulatory domain to govern target gene expression. Most bacterial riboswitches modulate gene expression through transcriptional or translational regulation, although some, including eukaryotic riboswitches, may provoke alternative splicing, and others can induce self-cleavage to cause mRNA degradation. In particular, various endogenous metabolite ligands, such as amino acids, vitamins, and FMN, present within cells above threshold concentrations can directly and specifically bind to the aptamer domains of riboswitches (Winkler et al., 2002b; Lee et al., 2009; Ren et al., 2015; Chauvier et al., 2017) . Entrapment of a ligand can stabilize the incorporation of the switching sequence into the aptamer domain, and thus the expression platform is induced to fold into a specific structure to modulate the expression of genes controlling the biosynthesis and transport of metabolites (Serganov and Nudler, 2013; Hallberg et al., 2017) .

Riboswitches have emerged as potential therapeutic targets because they are highly structured to form unique ligand binding pockets and function as gene regulatory factors, similar to the structural and functional selectivity of proteins. Many riboswitches are unique in bacteria, which upon binding to smallmolecule ligands, turn off or on the transcriptional or translational expression of genes essential for pathogen survival or virulence (Zhang et al., 2004b) . Therefore, small molecules may be discovered and developed to mimic or block the actions of natural ligands to interact with riboswitches with reasonable affinity and specificity for the control of infections.

Indeed, some small molecules have been identified to directly bind to targeted riboswitches, although most of them exhibit lower affinities toward the same riboswitch than natural ligand. For instance, L-lysine binds to lysine riboswitch with a Kd value around 0.36 mM, in contrast to currently identified lysine mimics, showing Kd values at 1-30 mM (Sudarsan et al., 2003; Blount et al., 2007) . Likewise, thiamine pyrophosphate (TPP) binds to TPP riboswitch with high affinity (Kd = 50 nM), whereas other small-molecule binders interact with the TPP riboswitch at high concentrations (Kd $ 50 mM), except the TPP analog pyrithiamine pyrophosphate (Kd = 160 nM) (Sudarsan et al., 2005; Warner et al., 2014) .

Although challenges remain in the discovery of highaffinity small molecules to target bacterial riboswitches, 886 recent efforts have led to the discovery of a few inhibitors with improved drug-like properties. Roseoflavin (Fig. 6) , a natural analog of riboflavin and FMN, was revealed to directly bind to the aptamer domain of bacterial FMN riboswitch with a Kd value of ;100 nM to exhibit antimicrobial activity, in comparison with that of FMN (5 nM) and riboflavin (3 mM) (Lee et al., 2009; Serganov et al., 2009) . Through phenotypic screening, a structurally distinct compound, ribocil (Table 4) , was discovered to mimic FMN actions to suppress FMN riboswitchmediated gene expression and inhibit bacterial growth via direct binding to the aptamer domain (Kd = 16 nM) (Howe et al., 2015) . Further derivatization and structure-activity relationship studies identified ribocil-C (Fig. 6 ) as the most potent and highly selectively inhibitor of FMN riboswitch, showing antimicrobial activities against different bacterial strains, including medically significant Gram positive bacteria such as methicillin-resistant Staphylococcus aureus and Enterococcus faecalis (Howe et al., 2016; Wang et al., 2017) . Very recently, an MS-based automated ligand detection system was established and employed for selective detection of small-molecule-ncRNA interactions, which not only verified the direct binding between ribocils and FMN riboswitch but also identified a few new binders that show greater antimicrobial activities (Rizvi et al., 2018) . Although these compounds are still under preclinical investigation, these findings shall enlighten the development of a novel class of riboswitch-targeted antibiotics.

4. Precursor Messenger RNAs. Orally bioavailable small molecules (Fig. 6 ) have also been identified to directly interact with pre-mRNAs to modulate splicing. This strategy seems more feasible for the treatment of genetic disorders, especially those diseases with verified RNA targets. For instance, the neuromuscular disorder SMA is amenable to SMN2 genetic variations that alter pre-mRNA splicing and thus deficiency of functional SMN protein (Aartsma-Rus, 2017; Ottesen, 2017) , and targeting SMN2 pre-mRNA splicing as an effective therapeutic strategy is proven by the approval of the ASO drug nusinersen (Table 2) . Indeed, a pyrimidin-4-one analog, SMN-C3, was first identified as an effective SMN2 splicing modifier through chemical screening and optimization (Naryshkin et al., 2014) . Being orally bioavailable, SMN-C3 was able to increase SMN protein levels in severe SMA disease D7 mouse models, improve motor function, and protect the neuromuscular circuit. An SMN-C3 derivative, RG7800, was further discovered and shown to be effective in two different mouse models of SMA (Ratni et al., 2016) . Although RG7800 entered clinical trials, it was terminated as a precautionary measure after the observation of retinal toxicity for chronic treatment (39 weeks) in cynomolgus monkeys . Additional chemical modifications and preclinical efficacy and safety studies led to clinical development of RG7916 (risdiplam) (Fig. 6 ; Table 4 ).

As risdiplam was well tolerated among healthy volunteers and exhibited effectiveness in shifting SMN2 splicing (Sturm et al., 2019) , phase II/III studies (NCT02913482) are underway to evaluate the safety and efficacy of risdiplam in patients with SMA.

Branaplam (LMI070 and NVS-SM1) (Fig. 6) represents another class of small-molecule pyridazine analogs identified to modulate SMN2 pre-mRNA splicing for the treatment of SMA caused by deficiency of SMN protein (Palacino et al., 2015; Cheung et al., 2018) . Branaplam was found to bind to the pre-mRNA of SMN2 and stabilize the transient dsRNA structure formed by the SMN2 pre-mRNA and U1 small nuclear RNA protein complex, a key component of the spliceosome, which enhances SMN2 pre-mRNA splicing and increases the expression of full-length SMN mRNA and functional SMN protein (Table 4 ) (Palacino et al., 2015) . With a favorable PK profile, branaplam was effective at improving survival of SMA D7 mice in a dose-dependent manner (Palacino et al., 2015; Cheung et al., 2018) . Branaplam has entered a first-in-human study on the safety, tolerability, PK, pharmacological actions, and efficacy of oral branaplam in infants with type 1 SMA (NCT02268552).

MicroRNAs. Efforts were also made to explore small-molecule inhibitors against the biogenesis of oncogenic miRNAs in comparison with antagonism with complementary oligonucleotides as well as oncolytic miRNA replacement therapy, which was discussed before. This may be achieved through the interference with Drosha-mediated processing of pri-miRNA within nucleus or Dicer-controlled cleavage of pre-miRNA in cytoplasm because both precursors are folded into hairpin structures available for ligand binding (Fig. 7) . Indeed, a computational approach has been taken to design and identify lead small molecules based on the predicted human miRNA hairpin precursor structures (Velagapudi et al., 2014) . Among 27 lead compounds, a benzimidazole analog was shown to selectively inhibit the processing of pri-miR-96 into oncogenic miR-96 and thus alter miR-96 target gene expression and induce apoptosis in cancer cells. The focus on the Drosha processing site of oncogenic pri-miR-96 and optimization of benzimidazole led to the discovery of a dimeric benzimidazole and bisbenzimide compound, targaprimir-96 ( Fig. 7; Table 4 ), which showed a favorable PK profile and was effective at releasing tumor burden in a triple-negative breast cancer xenograft mouse model . In addition, the conjugate of targaprimir-96 with bleomycin A5 resulted in over 100-fold higher selectivity toward pri-miR-96 than DNA (Li and Disney, 2018) . Very recently, another dimeric benzimidazole and bisbenzimide analog, targaprimir (TGP)-515, was identified to target pri-miR-515, leading to an induction of human epidermal growth factor receptor 2 expression levels in breast cancer cells and enhancement of the RNA-Based Therapies efficacy of human epidermal growth factor receptor 2 antibody therapeutics (Costales et al., 2019a) .

Likewise, a bisbenzimide (Hoechst 33342) analog called targarpremir-210, or TGP-210 ( Fig. 7 ; Table 4 ), was identified to bind to pre-miR-210, leading to the inhibition of Dicer-mediated processing to mature miR-210 in human carcinoma cells and the outgrowth of xenograft tumors in mice (Costales et al., 2017) . Direct binding of targarpremir-210 with pre-miR-210 was also validated by a chemical pull-down assay, and the blue fluorescent dye compound remains with a high affinity to DNA. The attachment of a nuclease (RNase L) recruitment module onto targarpremir-210 offered a conjugate, TGP-210-RL, which indeed was able to recruit RNase L onto pre-miR-210 to induce the degradation of pre-miR-210 (Costales et al., 2019b) . Interestingly, this TGP-210-RL conjugate exhibited high binding selectivity to the pre-miR-210 while lacking interactions with DNA as compared with TGP-210.

A small-molecule microarray-based approach was also developed for the identification of binders of RNA motifs (Velagapudi et al., 2018) . After screening against a library of 727 pharmacologically active compounds being used in the clinic or human trials, a number of DNA-intercalating agents (and topoisomerases inhibitors), such as doxorubicin, ellipticine, and mitoxantrone, as well as other entities, were revealed to bind to RNAs. Further computational and experimental studies demonstrated that mitoxantrone was able to directly bind to pre-miR-21 and subsequently inhibit Dicermediated biogenesis of oncogenic miR-21. Although these pharmacological agents commonly exhibit multiple levels of mechanisms of action and druggability of these pri-and pre-miRNA inhibitors ( Fig. 7 ; Table 4 ) awaits further investigations, these findings support the development of small molecules to target oncogenic ncRNAs as anticancer drugs.

6. Other RNA Targets. Some small molecules were also found to act on other types of disease-related RNAs or pathogenic RNA motifs. Many of them are for RNA repeats, such as the expanded GGGGCC repeat [r(GGGGCC) exp , or r(G4C2) exp , or G-quadruplex RNA], which causes frontotemporal dementia and amyotrophic lateral sclerosis (Su et al., 2014; Simone et al., 2018) ; the CGG repeat (rCGG exp ), which causes fragile X-associated tremor/ataxia syndrome Qurashi et al., 2012; Yang et al., 2015; Green et al., 2019) ; and the CUG repeat r(CUG) exp (Parkesh et al., 2012; Luu et al., 2016; Rzuczek et al., 2017; Li et al., 2018a; Angelbello et al., 2019) , which is associated with myotonic dystrophy type 1. Interestingly, there are several G-quadruplex motifs located on the 59UTR of oncogenic KRAS transcript that could be targeted by 4,11-bis(2-aminoethylamino)anthra[2, 3-b] furan-5,10-dione and 4,11-bis(2-aminoethylamino)anthra[2,3-b]thiophene-5,10-dione for the control of cancer cell proliferation and colony formation (Miglietta et al., 2017) . Another polyaromatic molecule, namely RGB-1, was found to stabilize RNA G-quadruplex, leading to the inhibition of RNA translation and suppression of proto-oncogene neuroblastoma RAS viral oncogene homolog expression in breast cancer cells (Katsuda et al., 2016) . In addition, a benzopyrrole analog named synucleozid was identified very recently as a binder to the structured iron-responsive element Fig. 7 . Pri-and pre-miRNAs as therapeutic targets for small-molecule compounds. (A) Small molecules (e.g., the dimeric benzimidazole and bisbenzimide compound targarprimir-96) may directly bind to pri-miRNA (pri-miR-96) to inhibit Drosha-mediated processing to pre-miRNA. (B) Small molecules (e.g., the bisbenzimide analog targarpremir-210) can be identified to interfere with pre-miRNA (pre-miR-210) processing by Dicer. In addition, the binding of miRNA to target transcripts would be disrupted by small molecules, which remains to be explored. 888 Yu et al. located on the 59UTR of a-synuclein mRNA, thus reducing a-synuclein protein levels in human cells (Zhang et al., 2020a) , which may provide insight into developing smallmolecule remedies for a-synuclein-caused Parkinson disease and other a-synucleinopathies.

With growing interests in developing small molecules to target RNAs for the management of human diseases (Costales et al., 2020) , it should be noted that RNAs are unique macromolecules that are distinguished from proteins and are closely related to DNAs. Currently, there are a limited number of verified druggable RNA targets, and validation of a new therapeutic target requires tedious and extensive investigations. A proven therapeutic target is valid until administrative approval of respective medication. Meanwhile, studies should be conducted to critically define the specificity of a small molecule in interacting with the target RNA. Most importantly, caution should be exercised to simply adopt conventional "rules" that are applicable to protein-ligand interactions for the discovery and development of RNA-targeted small-molecule drugs. This notion is also supported by the fact that the majority of RNA-targeted antibiotics ( Fig. 4 ; Table 4 ) do not follow Lipinski's rule of five, and many "RNA binders" initially designed and/or identified are known to later act on other targets (e.g., direct binding to DNAs). Therefore, structural optimization is necessary to improve the PK properties and selectivity toward a projected RNA target when developing novel efficacious and safe RNAtargeted drugs (Fig. 6 ).

1. Identification of Druggable RNA Targets. Two critical questions should be addressed for the identification of a druggable RNA target to control chosen disease. The first question is whether the RNA target can be accessed by small-molecule compounds. Beyond the intrinsic properties of small molecules, the accessibility of target RNA is dictated by RNA properties such as the abundance of target RNA in diseased cells, the availability of highly structured and functional sites for smallmolecule binding, and so on. Indeed, rRNAs accounting for the majority of cellular RNAs (e.g., .80%) are folded to form functional binding pockets and clefts that are accessible by small molecules to intervene specifically (Fig. 5) , leading to the disruption of protein synthesis and thus bacterial growth. As oncogenic miRNAs are commonly overexpressed in tumor tissues and carcinoma cells, the unique hairpin structures of miRNA precursors amenable to Drosha or Dicer processing (Fig. 7 ) make them promising targets for the development of smallmolecule inhibitors. Rather, it is necessary to verify selective binding of small molecules with the projected RNA target among complex cellular substances, such as other forms of RNAs, as well as DNAs and large quantities of proteins.

The second question is whether interactions between small molecules and RNA targets in cells and body can lead to an effective control of disease. Prior knowledge of RNA functions related to the pathogenesis and progression of a chosen disease shall be helpful, which could have already involved extensive epidemiologic, genetic, and biochemical studies. Starting from phenotype, specific RNA targets might be pinpointed. On the other hand, the appearance or apparent management of disease with the gain or loss of RNA coding gene or function will strongly support the role of target RNA. As such, pharmacological perturbation of RNA target with small molecules becomes possible, which awaits critical and comprehensive studies using phenotypic assays and animal disease models before entering clinical investigations.

2. Specificity in Targeting RNAs. Similar to the selective actions of a drug on corresponding protein target, new RNA-targeted small-molecule drugs should bind to RNAs with reasonable specificity to elicit ontarget therapeutic efficacy and avoid off-target adverse effects. This is actually a common principle for drug development, but it poses a big challenge for the discovery and development of novel RNA-targeted small-molecule modalities. As different RNA targets possess their intrinsic characteristics, the requirements for and strategies to achieve "selective" targeting may vary (Donlic and Hargrove, 2018) . Although structural studies have clearly elucidated the binding of small-molecule antibiotics with rRNAs ( Fig. 5 ; Table 2 ), it is inevitable that their interactions are based on the participation of essential proteins to assemble ribosomes (Wilson, 2014; Lin et al., 2018) . As another example, the small-molecule splicing modifier named AMN-C not only binds to an exonic splicing enhancer ESE2 of exon 7 but also interacts with an RNA helix formed by the 59ss intron 7 and the 59 terminus of the U1 small nuclear RNA bound with the U1 small nuclear RNA protein to selectively alter SMN2 pre-mRNA splicing over other transcripts (Sivaramakrishnan et al., 2017) .

The concern about specificity of RNA-targeted small molecules is worsened by the fact that RNAs and DNAs are both negatively charged nucleic acids and have common structural features, as well as the observation that the majority of small-molecule binders identified thus far for RNAs are either fluorescent dye derivatives (e.g., bisbenzimide or Hoechst 33342, commonly used to stain DNA), polyaromatic molecules, or highly basic or polar molecules. Many compounds themselves (e.g., doxorubicin and mitoxantrone) are well known as multifunctional DNA-intercalating agents (Wakelin, 1986) . Some approaches have developed and successfully improved the selectivity toward target RNA motifs over DNA (Li and Disney, 2018; Costales et al., 2019b) ; however, the resulting conjugate becomes a much bigger molecule, with a molecular mass over 1500 Da, whose druggability warrants further and more extensive RNA-Based Therapies investigations. In any cases, further structural modification and optimization of lead RNA binders are necessary to improve the selectivity toward RNA target for safe and effective control or eradication of particular diseases (Costales et al., 2020) .

Unparalleled opportunities have emerged to develop the next generation of RNA-based therapeutics, including a broad range of RNA molecules as medications and disease-related RNAs as therapeutic targets for small molecules. RNA-based therapies hold the promise to greatly expand the range of therapeutic targets for safe and effective control or eradication of potentially all types of diseases. Indeed, a number of RNA drugs with novel pharmacological actions, including RNA aptamers, ASOs, and siRNAs, have been approved for medical use, and many others, such as mRNA vaccines and gRNAmediated gene editing therapies, are undergoing active clinical trials. In addition, there is accumulating evidence supporting the mechanistic actions of existing and newly designed antibiotics on rRNAs for the control of infections, which motivates the identification of new viable RNA targets and development of novel RNA-targeted smallmolecule drugs. With growing interest in and enormous efforts devoted to the development of RNA-based therapies and emerging technologies, more and more RNAbased modalities are expected to enter into clinical trials and be approved for clinical practice in the coming decades.

Different from many small-molecule and protein drugs, natural RNA molecules are negatively charged and highly susceptible to ubiquitous RNases that impede them from access to intracellular targets. Application of particular chemical modifications or derivatizations, conjugation with targeting ligands, and utilization of lipid-based carriers have been proved as effective means to improve the stability of RNAs and deliver sufficient RNA molecules into cells to exert the desired pharmacological effects, which are evident in the FDA-approved RNA drugs. However, although chemical synthesis and purification can be automated, chemoengineered RNA drugs are among the most expensive medications on the market (Simoens and Huys, 2017; Burgart et al., 2018) , and the access to large quantities of synthetic RNAs required for animal studies, clinical investigations, and medical use among a large population remains a challenge. There were also concerns about inevitable effects of artificial modifications on RNA folding, activity, and safety as compared with natural RNAs produced and folded in living cells (Ho and Yu, 2016; Yu et al., 2019 Yu et al., , 2020 . Advances in RNA therapeutics will likely rely on the improvements of design and utilization of proper RNA molecules as well as delivery systems and targeting technologies to ensure efficacy and avoid or minimize off-target effects and immunogenicity.

There is also growing interest in developing orally bioavailable small-molecule inhibitors against therapeutic RNA targets beyond the use of RNA entities such as ASOs, siRNAs, and miRNAs. Because highly structured RNAs differ from proteins in many ways, including physicochemical properties and accessibility, the principles of RNA-small-molecule interactions may not necessarily follow the same rules derived from protein-targeted small-molecule drugs. By contrast, biological RNA macromolecules share common features with DNAs, although their cellular localizations can be very different. Therefore, it is necessary to verify the selectivity of small-molecule binder toward RNA target over DNAs, in addition to other types of RNAs and relevant proteins. An improved understanding of RNA structures and functions (especially those ncRNAs), identification of new pathogenic RNAs or RNA motifs as viable targets, and validation of their accessibilities for small molecules will greatly benefit future discovery and development of RNA-targeted small-molecule drugs.

Participated in research design: Yu, Choi, Tu. Performed data analysis: Yu, Choi, Tu. Wrote or contributed to the writing of the manuscript: Yu, Choi, Tu.

874 C. Challenges in the Development of RNA Drugs

IV. RNAs as Therapeutic Targets for Small Molecules

889 1. Identification of Druggable RNA Targets

FDA approval of nusinersen for spinal muscular atrophy makes 2016 the year of splice modulating oligonucleotides

FDA approves eteplirsen for Duchenne muscular dystrophy: the next chapter in the eteplirsen saga

Targeting noncoding RNAs in disease

Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis

A combinatorial library of lipidlike materials for delivery of RNAi therapeutics

Recognition of doublestranded RNA and activation of NF-kappaB by Toll-like receptor 3

A phase I/IIa immunotherapy trial of HIV-1-infected patients with Tat, Rev and Nef expressing dendritic cells followed by treatment interruption

The functions of animal microRNAs

Long-term efficacy and safety of mRNA therapy in two murine models of methylmalonic acidemia

Systemic messenger RNA therapy as a treatment for methylmalonic acidemia

Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation

Precise small-molecule cleavage of an r(CUG) repeat expansion in a myotonic dystrophy mouse model

Mapping of the second tetracycline binding site on the ribosomal small subunit of E.coli

Search-and-replace genome editing without double-strand breaks or donor DNA

2012) miR-34 -a microRNA replacement therapy is headed to the clinic

The promise of microRNA replacement therapy

Structural features of an influenza virus promoter and their implications for viral RNA synthesis

Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation

Preclinical and clinical demonstration of immunogenicity by mRNA vaccines against H10N8 and H7N9 influenza viruses

The complete atomic structure of the large ribosomal subunit at 2.4 A resolution

CRISPR provides acquired resistance against viruses in prokaryotes

Synthesis of RNA by in vitro transcription

Phase I study of MRX34, a liposomal miR-34a mimic, administered twice weekly in patients with advanced solid tumors

Therapeutic antisense oligonucleotides are coming of age

Inotersen treatment for patients with hereditary transthyretin amyloidosis

Structural insight into the antibiotic action of telithromycin against resistant mutants

Role for a bidentate ribonuclease in the initiation step of RNA interference

UTR seed matches, but not overall identity, are associated with RNAi off-targets

Antibacterial lysine analogs that target lysine riboswitches

Exportin 5 is a RanGTPdependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs

Targeting influenza A virus RNA promoter

Discovery and development of therapeutic aptamers

Application of aptamers for in vivo molecular imaging and theranostics

Safety, pharmacokinetics and pharmacodynamics of the anti-hepcidin Spiegelmer lexaptepid pegol in healthy subjects

Development of therapeutic-grade small interfering RNAs by chemical engineering

The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit

A system for stable expression of short interfering RNAs in mammalian cells

Genetic mapping of chronic childhood-onset spinal muscular atrophy to chromosome 5q11.2-13.3

Revisiting the structures of several antibiotics bound to the bacterial ribosome

Aptamers come of age -at last

Ethical challenges confronted when providing nusinersen treatment for spinal muscular atrophy

RNA-based therapeutics: current progress and future prospects

Ataluren treatment of patients with nonsense mutation dystrophinopathy

The molecular interactions that stabilize RNA tertiary structure: RNA motifs, patterns, and networks

Oligodeoxynucleoside phosphorothioate stability in subcellular extracts, culture media, sera and cerebrospinal fluid

The RNA modification database, RNAMDB: 2011 update

A modular approach to synthetic RNA binders of the hepatitis C virus internal ribosome entry site

Potent and sustained huntingtin lowering via AAV5 encoding miRNA preserves striatal volume and cognitive function in a humanized mouse model of Huntington disease

The promises and pitfalls of RNA-interferencebased therapeutics

Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation

A dicer-independent miRNA biogenesis pathway that requires Ago catalysis

Real-time quantification of microRNAs by stem-loop RT-PCR

Strategies for nonviral nanoparticle-based delivery of CRISPR/Cas9 therapeutics

A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications

Discovery of small molecule splicing modulators of survival motor neuron-2 (SMN2) for the treatment of spinal muscular atrophy (SMA)

Genomewide view of gene silencing by small interfering RNAs

A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity

Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, doseescalation study

Inotersen preserves or improves quality of life in hereditary transthyretin amyloidosis

Altered mRNA splicing and inhibition of human E-selectin expression by an antisense oligonucleotide in human umbilical vein endothelial cells

Multiplex genome engineering using CRISPR/Cas systems

The emerging role of RNA as a therapeutic target for small molecules

Non-viral delivery of zinc finger nuclease mRNA enables highly efficient in vivo genome editing of multiple therapeutic gene targets

A strategic approach to COVID-19 vaccine R&D

How we think about targeting RNA with small molecules

Small molecule inhibition of microRNA-210 reprograms an oncogenic hypoxic circuit

A designed small molecule inhibitor of a non-coding RNA sensitizes HER2 negative cancers to herceptin

Targeted degradation of a hypoxia-associated non-coding RNA enhances the selectivity of a small molecule interacting with RNA

Systemic microRNA-34a delivery induces apoptosis and abrogates growth of diffuse large B-cell lymphoma in vivo

Global transcriptional response to CRISPR/Cas9-AAV6-based genome editing in CD34 + hematopoietic stem and progenitor cells

Clinical pharmacological properties of mipomersen (Kynamro), a second generation antisense inhibitor of apolipoprotein B

RNA-targeted therapeutics

The dynamic landscapes of RNA architecture

In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight

RNA mediated Toll-like receptor stimulation in health and disease

RNA-Based Therapies 891

Clinical evidence supporting early treatment of patients with spinal muscular atrophy: current perspectives

Current progress on aptamer-targeted oligonucleotide therapeutics

Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles

Progress in the fight against multidrug-resistant bacteria? A review of U.S. Food and Drug Administration-approved antibiotics

iHIVARNA phase IIa, a randomized, placebocontrolled, double-blinded trial to evaluate the safety and immunogenicity of iHIVARNA-01 in chronically HIV-infected patients under stable combined antiretroviral therapy

A new understanding of the decoding principle on the ribosome

A structural basis for streptomycin-induced misreading of the genetic code

SDF-1 blockade enhances anti-VEGF therapy of glioblastoma and can be monitored by MRI

Processing of primary microRNAs by the Microprocessor complex

Leading RNA interference therapeutics part 2: silencing delta-aminolevulinic acid synthase 1, with a focus on givosiran

CRISPR/Cas9 b-globin gene targeting in human haematopoietic stem cells

A randomized, double-blind, placebocontrolled study of an RNAi-based therapy directed against respiratory syncytial virus

Structure of a hepatitis C virus RNA domain in complex with a translation inhibitor reveals a binding mode reminiscent of riboswitches

Hepatitis C virus translation inhibitors targeting the internal ribosomal entry site

Synthetic small-molecule RNA ligands: future prospects as therapeutic agents

A small molecule that targets r(CGG)(exp) and improves defects in fragile X-associated tremor ataxia syndrome

Inforna 2.0: a platform for the sequence-based design of small molecules targeting structured RNAs

Landscape of transcription in human cells

Targeting RNA in mammalian systems with small molecules

Overcoming cellular barriers for RNA therapeutics

Splice-modulating oligonucleotide QR-110 restores CEP290 mRNA and function in human c

Structures of the Escherichia coli ribosome with antibiotics bound near the peptidyl transferase center explain spectra of drug action

Inhibition of gene expression in plant cells by expression of antisense RNA

Nucleoside phosphorothioates

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

In vitro selection of RNA molecules that bind specific ligands

A transposon-derived small RNA regulates gene expression in Salmonella Typhimurium

A human snoRNA with microRNA-like functions

Non-coding RNAs in human disease

AAV5-miHTT gene therapy demonstrates broad distribution and strong human mutant huntingtin lowering in a Huntington's disease minipig model

Liposome-polyethylenimine complexes (DPPC-PEI lipopolyplexes) for therapeutic siRNA delivery in vivo

Storage stability of optimal liposome-polyethylenimine complexes (lipopolyplexes) for DNA or siRNA delivery

Quantification of oligonucleotides by LC-MS/MS: the challenges of quantifying a phosphorothioate oligonucleotide and multiple metabolites

Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection

Precise and efficient siRNA design: a key point in competent gene silencing

HIV-1 tat trans-activation requires the loop sequence within tar

Structural and functional motifs in influenza virus RNAs

Guide RNA modification as a way to improve CRISPR/Cas9-based genome-editing systems

Nusinersen versus sham control in infantile-onset spinal muscular atrophy

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind

A highly durable RNAi therapeutic inhibitor of PCSK9

RNA-based vaccines with dual activity induce balanced TLR-7 dependent adaptive immune responses and provide antitumor activity

Structure of the A site of Escherichia coli 16S ribosomal RNA complexed with an aminoglycoside antibiotic

Increased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy

Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases

Immunization of HIV-1-infected persons with autologous dendritic cells transfected with mRNA encoding HIV-1 Gag and Nef: results of a randomized, placebo-controlled clinical trial

High-performance virtual screening by targeting a high-resolution RNA dynamic ensemble

Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action

Identification of modifications in microbial, native tRNA that suppress immunostimulatory activity

Long terminal repeat CRISPR-CAR-coupled "universal" T cells mediate potent anti-leukemic effects

Knockout rats via embryo microinjection of zinc-finger nucleases

RNAi therapy targeting KRAS in combination with chemotherapy for locally advanced pancreatic cancer patients

Enhanced efficacy associated with early treatment of neovascular age-related macular degeneration with pegaptanib sodium: an exploratory analysis

Sequence determinants of innate immune activation by short interfering RNAs

Pegaptanib for neovascular age-related macular degeneration

High-throughput screening yields several smallmolecule inhibitors of repeat-associated non-AUG translation

The Microprocessor complex mediates the genesis of microRNAs

Regulatory aspects of drug approval for macular degeneration

Recent advances in developing small molecules targeting RNA

Creation of CRISPRbased germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD

European Medicines Agency review of ataluren for the treatment of ambulant patients aged 5 years and older with Duchenne muscular dystrophy resulting from a nonsense mutation in the dystrophin gene

TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing

Engineering and in vivo applications of riboswitches

An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells

Argo-naute2, a link between genetic and biochemical analyses of RNAi

The Drosha-DGCR8 complex in primary microRNA processing

The structures of four macrolide antibiotics bound to the large ribosomal subunit

Construction of Corynebacterium glutamicum cells as containers encapsulating dsRNA overexpressed for agricultural pest control

Overexpression system for recombinant RNA in Corynebacterium glutamicum using a strong promoter derived from corynephage BFK20

MicroRNAs in cancer: biomarkers, functions and therapy

Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach

Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors

Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells

A brave new world of RNAbinding proteins

Small molecules targeting viral RNA

Bioengineered noncoding RNAs selectively change cellular miRNome profiles for cancer therapy

Bioengineering of noncoding RNAs for research agents and therapeutics

In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Circular RNAs as therapeutic agents and targets

Phase 1 study of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumours

The many pathways of RNA degradation

Selective small-molecule inhibition of an RNA structural element

Atomic resolution mechanistic studies of ribocil: a highly selective unnatural ligand mimic of the E. coli FMN riboswitch

Disruption of the myostatin gene in porcine primary fibroblasts and embryos using zinc-finger nucleases

Age-related macular degeneration: experimental and emerging treatments

Plasma and liver protein binding of N-acetylgalactosamineconjugated small interfering RNA

Identification of GalNAc-conjugated antisense oligonucleotide metabolites using an untargeted and generic approach based on high resolution mass spectrometry

A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA

A microRNA in a multiple-turnover RNAi enzyme complex

Crystal structure of the oxazolidinone antibiotic linezolid bound to the 50S ribosomal subunit

Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons

Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons

Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis

Expression profiling reveals off-target gene regulation by RNAi

Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity

Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application

Selection of GalNAcconjugated siRNAs with limited off-target-driven rat hepatotoxicity

Identification of genes that are associated with DNA repeats in prokaryotes

Co-targeting of DNA, RNA, and protein molecules provides optimal outcomes for treating osteosarcoma and pulmonary metastasis in spontaneous and experimental metastasis mouse models

Effects of microRNA-34a on the pharmacokinetics of cytochrome P450 probe drugs in mice

Bioengineered Let-7c inhibits orthotopic hepatocellular carcinoma and improves overall survival with minimal immunogenicity

A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity

RNA quaternary structure and global symmetry

Advances in the delivery of RNA therapeutics: from concept to clinical reality

Delivery materials for siRNA therapeutics

A structural basis for the antibiotic resistance conferred by an N1-methylation of A1408 in 16S rRNA

Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA

Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability

Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development

2012) miRNA-34 prevents cancer initiation and progression in a therapeutically resistant K-ras and p53-induced mouse model of lung adenocarcinoma

A small molecule that represses translation of G-quadruplexcontaining mRNA

Efficacy and immunogenicity of unmodified and pseudouridine-modified mRNA delivered systemically with lipid nanoparticles in vivo

Aptamers in the therapeutics and diagnostics pipelines

Bioanalytical method validation: new FDA guidance vs. EMA guideline. Better or worse?

Inotersen: first global approval

Quantification of therapeutic miRNA mimics in whole blood from nonhuman primates

RNA-Based Therapies 893

AAV5-miHTT lowers Huntingtin mRNA and protein without off-target effects in patient-derived neuronal cultures and astrocytes

Eteplirsen treatment attenuates respiratory decline in ambulatory and non-ambulatory patients with Duchenne muscular dystrophy

Functional siRNAs and miRNAs exhibit strand bias

The chemical evolution of oligonucleotide therapies of clinical utility

Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy

CRISPR/Cpf1-mediated DNA-free plant genome editing

Sequence-and targetindependent angiogenesis suppression by siRNA via TLR3

Safety and pharmacokinetic study of RPI.4610 (ANGIOZYME), an anti-VEGFR-1 ribozyme, in combination with carboplatin and paclitaxel in patients with advanced solid tumors

Reconstitution of RNase P activity from inactive RNA and protein

RNA therapeutics: beyond RNA interference and antisense oligonucleotides

A structural basis for the antibiotic resistance conferred by an A1408G mutation in 16S rRNA and for the antiprotozoal activity of aminoglycosides

Delivering the messenger: advances in technologies for therapeutic mRNA delivery

miRBase: from microRNA sequences to function

miRBase: annotating high confidence microRNAs using deep sequencing data

MicroRNAs as clinical biomarkers and therapeutic tools in perioperative medicine

Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena

The apical loop of the HIV-1 TAR RNA hairpin is stabilized by a cross-loop base pair

tRNA fragments (tRFs) guide Ago to regulate gene expression post-transcriptionally in a Dicerindependent manner

Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery

Phase I clinical trial of an intranodally administered mRNA-based therapeutic vaccine against HIV-1 infection

Roseoflavin is a natural antibacterial compound that binds to FMN riboswitches and regulates gene expression

A novel small-molecule binds to the influenza A virus RNA promoter and inhibits viral replication

The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14

The nuclear RNase III Drosha initiates microRNA processing

MicroRNA maturation: stepwise processing and subcellular localization

MicroRNA genes are transcribed by RNA polymerase II

Identification and characterization of a spinal muscular atrophy-determining gene

Targeting the HIV RNA genome: high-hanging fruit only needs a longer ladder

Cellular localization of long non-coding RNAs affects silencing by RNAi more than by antisense oligonucleotides

Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses

Treating disease at the RNA level with oligonucleotides

A dimeric 2,9-diamino-1,10-phenanthroline derivative improves alternative splicing in myotonic dystrophy type 1 cell and mouse models

Chimeric microRNA-1291 biosynthesized efficiently in Escherichia coli is effective to reduce target gene expression in human carcinoma cells and improve chemosensitivity

Rapid production of novel pre-microRNA agent hsa-mir-27b in Escherichia coli using recombinant RNA technology for functional studies in mammalian cells

Bioengineered NRF2-siRNA is effective to interfere with NRF2 pathways and improve chemosensitivity of human cancer cells

Bioengineered miR-27b-3p and miR-328-3p modulate drug metabolism and disposition via the regulation of target ADME gene expression

Precise small molecule degradation of a noncoding RNA identifies cellular binding sites and modulates an oncogenic phenotype

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes

Tapping the RNA world for therapeutics

An investigational RNAi therapeutic targeting glycolate oxidase reduces oxalate production in models of primary hyperoxaluria

Post-transcriptional control of expression of the repA gene of plasmid R1 mediated by a small RNA molecule

Summary: the modified nucleosides of RNA

Ribosome-targeting antibiotics: modes of action, mechanisms of resistance, and implications for drug design

Understanding nucleic acid-ion interactions

Lead-and drug-like compounds: the rule-of-five revolution

The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44

Argonaute2 is the catalytic engine of mammalian RNAi

Oligonucleotide quantification and metabolite profiling by highresolution and accurate mass spectrometry

Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy

Lipid-like materials for low-dose, in vivo gene silencing

Spiegelmer, alone and with bortezomib-dexamethasone in relapsed/refractory multiple myeloma: a Phase IIa Study

Structure and function of HCV IRES domains

Nuclear export of microRNA precursors

A potent inhibitor of protein sequestration by expanded triplet (CUG) repeats that shows phenotypic improvements in a Drosophila model of myotonic dystrophy

Toxicity of cationic lipids and cationic polymers in gene delivery

Comparison of X-ray crystal structure of the 30S subunit-antibiotic complex with NMR structure of decoding site oligonucleotide-paromomycin complex

Structural origins of aminoglycoside specificity for prokaryotic ribosomes

An investigational RNAi therapeutic targeting antithrombin for the treatment of hemophilia A and B

A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action

RNA-guided human genome engineering via Cas9

Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activationresponse (TAR) element of human immunodeficiency virus

Singlestranded antisense siRNAs guide target RNA cleavage in RNAi

A new era for rare genetic diseases: messenger RNA therapy

Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA

Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes

RNA regulation: a new genetics

T cells expressing chimeric antigen receptors can cause anaphylaxis in humans

tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma

Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD

Riboswitch diversity and distribution

Ataluren in patients with nonsense mutation Duchenne muscular dystrophy (ACT DMD): a multicentre, randomised, double-blind, placebo-controlled

Chromatographic methods for the determination of therapeutic oligonucleotides

Ion exchange liquid chromatography method for the direct determination of small ribonucleic acids

Chemically modified Cpf1-CRISPR RNAs mediate efficient genome editing in mammalian cells

Discovery of selective, small-molecule inhibitors of RNA complexes--I. The Tat protein/TAR RNA complexes required for HIV-1 transcription

Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs

Eteplirsen Study Group and Telethon Foundation DMD Italian Network (2016) Longitudinal effect of eteplirsen versus historical control on ambulation in Duchenne muscular dystrophy

Eteplirsen for the treatment of Duchenne muscular dystrophy

Nusinersen versus sham control in later-onset spinal muscular atrophy

RNA G-quadruplexes in Kirsten Ras (KRAS) oncogene as targets for small molecules inhibiting translation

Developing therapies for age-related macular degeneration: the art and science of problem-solving: the

Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates

Design, characterization, and lead selection of therapeutic miRNAs targeting Huntingtin for development of gene therapy for Huntington's disease

AAV5-miHTT gene therapy demonstrates suppression of mutant huntingtin aggregation and neuronal dysfunction in a rat model of Huntington's disease

Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria

Discovery of key physicochemical, structural, and spatial properties of RNA-targeted bioactive ligands

Insights into the development of chemical probes for RNA

An open-label, phase 2 trial of RPI.4610 (Angiozyme) in the treatment of metastatic breast cancer

For patients who inherit homozygous familial hypercholesterolemia, 2 new treatments available

Pervasive regulatory functions of mRNA structure revealed by high-resolution SHAPE probing

Structure-function insights reveal the human ribosome as a cancer target for antibiotics

Genetic control by a metabolite binding mRNA

Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates

Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing

siRNA design software for a target gene-specific RNA interference

A brilliant disguise for self RNA: 59-end and internal modifications of primary transcripts suppress elements of innate immunity

GRK5-knockout mice generated by TALEN-mediated gene targeting

Motor neuron disease. SMN2 splicing modifiers improve motor function and longevity in mice with spinal muscular atrophy

An aptamer-siRNA chimera suppresses HIV-1 viral loads and protects from helper CD4(+) T cell decline in humanized mice

In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy

Rationally designed small molecules that target both the DNA and RNA causing myotonic dystrophy type 1

Post-transcriptional inducible gene regulation by natural antisense RNA

Recognition of cognate transfer RNA by the 30S ribosomal subunit

The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila

Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways

ISS-N1 makes the first FDA-approved drug for spinal muscular atrophy

Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells

SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice

Small nucleolar RNA-derived microRNA hsa-miR-1291 modulates cellular drug disposition through direct targeting of ABC transporter ABCC1

Phase Ib evaluation of a self-adjuvanted protamine formulated mRNA-based active cancer immunotherapy, BI1361849 (CV9202), combined with local radiation treatment in patients with stage IV nonsmall cell lung cancer

mRNA vaccines -a new era in vaccinology

Identification of RNA pseudoknot-binding ligand that inhibits the -1 ribosomal frameshifting of SARS-coronavirus by structure-based virtual screening

Design of a bioactive small molecule that targets the myotonic dystrophy type 1 RNA via an RNA motif-ligand database and chemical similarity searching

Targeting of antithrombin in hemophilia A or B with RNAi therapy

Induction of tumour immunity by targeted inhibition of nonsense-mediated mRNA decay

Inhibitor-induced structural change in the HCV IRES domain IIa RNA

Antitumor and antimetastatic activity of ribozymes targeting the messenger RNA of vascular endothelial growth factor receptors

Gene regulation by antisense transcription

Short RNAs repress translation after initiation in mammalian cells

Bioengineering of a single long noncoding RNA molecule that carries multiple small RNAs

MicroRNAs in non-small cell lung cancer: gene regulation, impact on cancer cellular processes, and therapeutic potential

RNA-Based Therapies 895

Structure of influenza A polymerase bound to the viral RNA promoter

Inhibition of translational initiation by Let-7 MicroRNA in human cells

A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal

MicroRNAs as biomarkers for clinical studies

Risdiplam distributes and increases SMN protein in both the central nervous system and peripheral organs

Multiplex genome-edited T-cell manufacturing platform for "off-the-shelf

A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold

Recombinant RNA technology: the tRNA scaffold

Comparative analysis of antisense oligonucleotide sequences targeting exon 53 of the human DMD gene: implications for future clinical trials

Metabolism and disposition of volanesorsen, a 29-O-(2 methoxyethyl) antisense oligonucleotide, across species

Bioavailable inhibitors of HIV-1 RNA biogenesis identified through a Rev-based screen

Restitution of tumor suppressor microRNAs using a systemic nanovector inhibits pancreatic cancer growth in mice

Chemical screen reveals small molecules suppressing fragile X premutation rCGG repeat-mediated neurodegeneration in Drosophila

Synthetic CRISPR RNA-Cas9-guided genome editing in human cells

Preclinical safety evaluation in rats of a polymeric matrix containing an siRNA drug used as a local and prolonged delivery system for pancreatic cancer therapy

Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity

Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation

Preclinical Justification of pbi-shRNA EWS/FLI1 Lipoplex (LPX) Treatment for Ewing's Sarcoma

Discovery of risdiplam, a selective survival of motor neuron-2 ( SMN2) gene splicing modifier for the treatment of spinal muscular atrophy (SMA)

Specific correction of alternative survival motor neuron 2 splicing by small molecules: discovery of a potential novel medicine to treat spinal muscular atrophy

Inclisiran in patients at high cardiovascular risk with elevated LDL cholesterol

Effect of 1 or 2 doses of inclisiran on low-density lipoprotein cholesterol levels: one-year follow-up of the ORION-1 randomized clinical trial

A stressresponsive RNA switch regulates VEGFA expression

Structural basis for molecular discrimination by a 39,39-cGAMP sensing riboswitch

Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition

Progress in the development of lipopolyplexes as efficient non-viral gene delivery systems

Pharmacology of a central nervous system delivered 29-Omethoxyethyl-modified survival of motor neuron splicing oligonucleotide in mice and nonhuman primates

Anti-frameshifting ligand reduces the conformational plasticity of the SARS virus pseudoknot

How drug-like are 'ugly' drugs: do drug-likeness metrics predict ADME behaviour in humans?

Intradermal vaccinations with RNA coding for TAA generate CD8+ and CD4+ immune responses and induce clinical benefit in vaccinated patients

Discovery of selective RNA-binding small molecules by affinity-selection mass spectrometry

29-O-methyl-modified RNAs act as TLR7 antagonists

Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA

Management of familial hypercholesterolemia: a review of the recommendations from the National Lipid Association Expert Panel on Familial Hypercholesterolemia

SDF-1 inhibition targets the bone marrow niche for cancer therapy

Pharmacokinetic and drug metabolism properties of novel therapeutic modalities

Fomivirsen approved for CMV retinitis

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

Intronic microRNA precursors that bypass Drosha processing

MicroRNA therapeutics: towards a new era for the management of cancer and other diseases

Ataluren: first global approval

Precise small-molecule recognition of a toxic CUG RNA repeat expansion

CRISPR/Cas system for genome editing: progress and prospects as a therapeutic tool

mRNA-based therapeutics--developing a new class of drugs

A genomewide search for ribozymes reveals an HDV-like sequence in the human CPEB3 gene

Circular RNAs: a novel class of functional RNA molecules with a therapeutic perspective

A comprehensive map of molecular drug targets

Phase 1 trial of an RNA interference therapy for acute intermittent porphyria

Liposome-polyethylenimine complexes for enhanced DNA and siRNA delivery

Structural characterization of an alternative mode of tigecycline binding to the bacterial ribosome

Approaches for the sequence-specific knockdown of mRNA

mRNA: a novel avenue to antibody therapy?

Adventures with RNA graphs

Asymmetry in the assembly of the RNAi enzyme complex

The effects of the anti-hepcidin Spiegelmer NOX-H94 on inflammation-induced anemia in cynomolgus monkeys

Givosiran: first approval

Specificity of short interfering RNA determined through gene expression signatures

Coenzyme recognition and gene regulation by a flavin mononucleotide riboswitch

A decade of riboswitches

SAR by MS: discovery of a new class of RNA-binding small molecules for the hepatitis C virus: internal ribosome entry site IIA subdomain

The current state and future directions of RNAi-based therapeutics

Market access of Spinraza (Nusinersen) for spinal muscular atrophy: intellectual property rights, pricing, value and coverage considerations

G-quadruplex-binding small molecules ameliorate C9orf72 FTD/ALS pathology in vitro and in vivo

Translational control of IS10 transposition

Binding to SMN2 pre-mRNA-protein complex elicits specificity for small molecule splicing modifiers

Bidirectional transcription and the regulation of Phage lambda repressor synthesis

Ribonuclease P: an enzyme with an essential RNA component

Eteplirsen approved for Duchenne muscular dystrophy: the FDA faces a difficult choice

FDA-approved oligonucleotide therapies in 2017

Apolipoprotein B synthesis inhibition with mipomersen in heterozygous familial hypercholesterolemia: results of a randomized, double-blind, placebo-controlled trial to assess efficacy and safety as add-on therapy in patients with coronary artery disease

A structural understanding of the dynamic ribosome machine

Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble

Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide

Olaptesed pegol (NOX-A12) with bendamustine and rituximab: a phase IIa study in patients with relapsed/refractory chronic lymphocytic leukemia

The regulation of HIV-1 transcription: molecular targets for chemotherapeutic intervention

The siRNA sequence and guide strand overhangs are determinants of in vivo duration of silencing

A phase 1 healthy male volunteer single escalating dose study of the pharmacokinetics and pharmacodynamics of risdiplam (RG7916, RO7034067), a SMN2 splicing modifier

An atypical RNA pseudoknot stimulator and an upstream attenuation signal for -1 ribosomal frameshifting of SARS coronavirus

Discovery of a biomarker and lead small molecules to target r(GGGGCC)-associated defects in c9FTD/ALS

Thiamine pyrophosphate riboswitches are targets for the antimicrobial compound pyrithiamine

An mRNA structure in bacteria that controls gene expression by binding lysine

Invention and early history of morpholinos: from pipe dream to practical products

N1-methyl-pseudouridine in mRNA enhances translation through eIF2a-dependent and independent mechanisms by increasing ribosome density

Eteplirsen: first global approval

Roadmap and strategy for overcoming infusion reactions to nanomedicines

Identification of biologically active, HIV TAR RNAbinding small molecules using small molecule microarrays

In vivo gene editing in dystrophic mouse muscle and muscle stem cells

Efficient nonmeiotic allele introgression in livestock using custom endonucleases

Toll-like receptor 8 senses degradation products of single-stranded RNA

Application of locked nucleic acid oligonucleotides for siRNA preclinical bioanalytics

Mipomersen, an apolipoprotein B synthesis inhibitor, reduces atherogenic lipoproteins in patients with severe hypercholesterolemia at high cardiovascular risk: a randomized, double-blind, placebo-controlled trial

Emerging frontiers in drug delivery

Structures of MLSBK antibiotics bound to mutated large ribosomal subunits provide a structural explanation for resistance

Bioengineered miRNA-1291 prodrug therapy in pancreatic cancer cells and patient-derived xenograft mouse models

Riboswitches as versatile gene control elements

Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase

Thermodynamic stability and Watson-Crick base pairing in the seed duplex are major determinants of the efficiency of the siRNA-based off-target effect

THPdb: database of FDA-approved peptide and protein therapeutics

Local dystrophin restoration with antisense oligonucleotide PRO051

) mRNA-based dendritic cell vaccination induces potent antiviral T-cell responses in HIV-1-infected patients

Do natural antisense transcripts make sense in eukaryotes

Mechanism of action of streptogramins and macrolides

Turning mirror-image oligonucleotides into drugs: the evolution of Spiegelmer(®) therapeutics

Design of a small molecule against an oncogenic noncoding RNA

Approved anti-cancer drugs target oncogenic non-coding RNAs

Sequence-based design of bioactive small molecules that target precursor microRNAs

Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis

Infusion reactions: diagnosis, assessment, and management

Preclinical mammalian safety studies of EPHARNA (DOPC nanoliposomal EphA2-targeted siRNA)

Polyfunctional DNA intercalating agents

Dual-targeting small-molecule inhibitors of the Staphylococcus aureus FMN riboswitch disrupt riboflavin homeostasis in an infectious setting

One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering

Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors

Advances in quantitative bioanalysis of oligonucleotide biomarkers and therapeutics

Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles

Bioengineering novel chimeric microRNA-34a for prodrug cancer therapy: high-yield expression and purification, and structural and functional characterization

Selection of hyperfunctional siRNAs with improved potency and specificity

Principles for targeting RNA with drug-like small molecules

Validating fragment-based drug discovery for biological RNAs: lead fragments bind and remodel the TPP riboswitch specifically

Comparative characterization of hepatic distribution and mRNA reduction of antisense oligonucleotides conjugated with triantennary N-acetyl galactosamine and lipophilic ligands targeting apolipoprotein B

Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA

Direct injection of protamine-protected mRNA: results of a phase 1/2 vaccination trial in metastatic melanoma patients

mRNA: fulfilling the promise of gene therapy

HIV gag mRNA transfection of dendritic cells (DC) delivers encoded antigen to MHC class I and II molecules, causes DC maturation, and induces a potent human in vitro primary immune response

RNA-Based Therapies 897

Targeting nuclear RNA for in vivo correction of myotonic dystrophy

Development of a lung cancer therapeutic based on the tumor suppressor microRNA-34

Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans

RNA as a source of transposase for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues

A phase IB study on intravenous synthetic mRNA electroporated dendritic cell immunotherapy in pretreated advanced melanoma patients

The A-Z of bacterial translation inhibitors

Ribosome-targeting antibiotics and mechanisms of bacterial resistance

The oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and effect tRNA positioning

Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression

An mRNA structure that controls gene expression by binding FMN

Direct gene transfer into mouse muscle in vivo

Targeted genome editing across species using ZFNs and TALENs

FDA approves patisiran to treat hereditary transthyretin amyloidosis

Effects of renal impairment on the pharmacokinetics, efficacy, and safety of inclisiran: an analysis of the ORION-7 and ORION-1 studies

Correction of a genetic disease in mouse via use of CRISPR-Cas9

Targeting of NLRP3 inflammasome with gene editing for the amelioration of inflammatory diseases

Creatine based polymer for codelivery of bioengineered MicroRNA and chemodrugs against breast cancer lung metastasis

Editing aberrant splice sites efficiently restores b-globin expression in b-thalassemia

CRISPR-mediated direct mutation of cancer genes in the mouse liver

Inhibition of non-ATG translational events in cells via covalent small molecules targeting RNA

mRNA vaccines: possible tools to combat SARS-CoV-2

Bioengineered miR-328-3p modulates GLUT1-mediated glucose uptake and metabolism to exert synergistic antiproliferative effects with chemotherapeutics

Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo

Targeting RNA: a transformative therapeutic strategy

Novel approaches for efficient in vivo fermentation production of noncoding RNAs

RNA therapy: are we using the right molecules?

Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide

RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals

Single processing center models for human Dicer and bacterial RNase III

Translation of the intrinsically disordered protein a-synuclein is inhibited by a small molecule targeting its structured mRNA

Lipidation of polyethylenimine-based polyplex increases serum stability of bioengineered RNAi agents and offers more consistent tumoral gene knockdown in vivo

DEG: a database of essential genes

Patisiran pharmacokinetics, pharmacodynamics, and exposure-response analyses in the phase 3 APOLLO trial in patients with hereditary transthyretin-mediated (hATTR) amyloidosis

Pharmacokinetics of patisiran, the first approved RNA interference therapy in patients with hereditary transthyretinmediated amyloidosis

Structures and stabilization of kinetoplastid-specific split rRNAs revealed by comparing leishmanial and human ribosomes

CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice

Genetically engineered pre-microRNA-34a prodrug suppresses orthotopic osteosarcoma xenograft tumor growth via the induction of apoptosis and cell cycle arrest

Combination therapy with bioengineered miR-34a prodrug and doxorubicin synergistically suppresses osteosarcoma growth

Mutant KRAS is a druggable target for pancreatic cancer

Clinical experiences with systemically administered siRNA-based therapeutics in cancer