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Review ArticleReview Article
Open Access

RNA Drugs and RNA Targets for Small Molecules: Principles, Progress, and Challenges

Ai-Ming Yu, Young Hee Choi and Mei-Juan Tu
RHIAN M. TOUYZ, ASSOCIATE EDITOR
Pharmacological Reviews October 2020, 72 (4) 862-898; DOI: https://doi.org/10.1124/pr.120.019554
Ai-Ming Yu
Department of Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, California (A.-M.Y., Y.H.C., M.-J.T.) and College of Pharmacy and Integrated Research Institute for Drug Development, Dongguk University-Seoul, Goyang-si, Gyonggi-do, Republic of Korea (Y.H.C.)
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Young Hee Choi
Department of Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, California (A.-M.Y., Y.H.C., M.-J.T.) and College of Pharmacy and Integrated Research Institute for Drug Development, Dongguk University-Seoul, Goyang-si, Gyonggi-do, Republic of Korea (Y.H.C.)
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Mei-Juan Tu
Department of Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, California (A.-M.Y., Y.H.C., M.-J.T.) and College of Pharmacy and Integrated Research Institute for Drug Development, Dongguk University-Seoul, Goyang-si, Gyonggi-do, Republic of Korea (Y.H.C.)
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RHIAN M. TOUYZ
Roles: ASSOCIATE EDITOR
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    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.

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    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.

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    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.

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    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.

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    Fig. 5.

    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.

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    Fig. 6.

    Chemical structures of some small-molecule compounds under clinical or preclinical studies that act on various types of RNAs for the treatment of different diseases. (A) Ataluren targets rRNA to promote insertion of near-cognate tRNAs at the site of the dystrophin gene toward nonsense suppression for the treatment of Duchenne muscular dystrophy. (B) Netilmicin and anthraquinone derivatives targeting HIV TAR RNA motif; benzimidazole derivative Isis-11 and diaminopiperidine analog acting on HIV IRES; MTDB as an inhibitor of SAR-CoV RNA pseudoknot; and DPQ identified to attenuate influenza A promoter RNA for the control of virus infections. (C) Synthetic ribocils and natural roseoflavin mimic FMN riboswitch ligand to modulate target gene expression to exert antimicrobial activities. (D) Risdiplam and branaplam, interacting with SMN2 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.

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    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.

Tables

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    TABLE 1

    Characteristics of inorganic and small-molecule organic compound drugs, as well as macromolecule protein and nucleic acid therapeutics

    PropertiesInorganic Compound DrugsSmall-Molecule Organic Compound DrugsProtein TherapeuticsRNA Therapeutics
    ChemistryTypical mol. wt. < 200 Da; ionicTypical mol. wt. < 500 Da; hydrophobicTypical mol. wt. > 100 kDa; positive/negative/neutralTypical mol. wt. > 7 kDa; negative charge
    DosingPrimarily oral; often dailyPrimarily oral; often dailyMainly intravenous and subcutaneous; weekly to monthlyIntravenous, subcutaneous, intrathecal, intravitreal (various); weekly to once every 3–6 mo
    ADME/PK propertiesOrally bioavailable;Orally bioavailable;Not orally bioavailable;Not orally bioavailable;
    distributed to all organs and tissues, cell permeable;distributed to all organs and tissues, cell permeable;distributed mainly in plasma or extracellular fluids, cell impermeable;Distributed extensively to kidney and liver, cell impermeable;
    usually not metabolized;metabolized by phase I and II enzymes;catabolized extensively to peptides or amino acids;catabolized extensively by nucleases to (oligo)nucleotides;
    excreted primarily in urineexcreted mainly in bile and urinelimited excretionlimited excretion
    Molecular targetsProteinsMainly proteinsProteinsMainly RNAs, besides proteins and DNAs
    Site of action and PDExtra-/intracellular;Extra-/intracellular;Extracellular/membrane;Primarily intracellular;
    direct or indirect relationship to blood PKDirect or indirect relationship to blood PKdirect or indirect models linked to blood PKmore relevant to tissue PK, whereas PD can be linked to blood PK
    Safety/toxicityRisk of off-target effectsRisk of off-target effectsRisk of immunogenicityRisk of immunogenicity
    • ADME, absorption, metabolism, distribution, and excretion.

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    TABLE 2

    RNA therapeutics approved by the US Food and Drug Administration for the treatment of human diseases

    Note that two PMO drugs are included because the nucleobase thymine (T) is also known as 5-methyluracil, or m5U.

    RNA DrugChemistryDosage RegimenMechanisms of ActionDiseaseYear ApprovedCurrent StatusReferences
    Pegaptanib (aptamer)28-nt aptamer; pegylated, all PO, 2′-F, and 2′-OMe; G and A methylated; mol. wt. ∼50 kDa0.3 mg every 6 wk, intravitreal injectionSelective VEGF (165 isoform) antagonist; antiangiogenesis in the eyeNeovascular AMD2004PrescriptionGragoudas et al., 2004; Gryziewicz, 2005
    Mipomersen (ASO)20-mer gapmer; all PS, 2ʹ-MOE, and 2′-deoxy; C and U methylated; mol. wt. ∼7.6 kDa200 mg once weekly, s.c.Selectively binds to ApoB-100 mRNA to inhibit the translation of synthesis of ApoB in liverHoFH2013Discontinued in 2018Crooke and Geary, 2013; Morrow, 2013
    Eteplirsen (ASO)30-mer PMO; m5U; mol. wt. ∼10.3 kDa30 mg/kg once weekly, i.v. infusionSelectively binds to exon 51 of dystrophin pre-mRNA to alter splicing, leading to production of functional muscle protein dystrophinDMD2016PrescriptionCirak et al., 2011; Mendell et al., 2016; Stein, 2016; Syed, 2016
    Nusinersen (ASO)18-mer; all PS, fully 2ʹ-MOE; m5U; m5C; mol. wt. ∼7.5 kDaLoading: 12 mg every 2 wk for three doses, then 12 mg for 30 days, i.t. Maintenance: 12 mg once every 4 mo, i.t.Selectively binds to SMN2 mRNA to alter splicing, leading to the production of full-length SMN proteinSMA2016PrescriptionAartsma-Rus, 2017; Ottesen, 2017
    Patisiran (siRNA)21-bp double-stranded siRNA; all PO, and 2ʹ-OMe; lipid nanoparticle mol. wt. ∼14.3 kDa0.3 mg/kg (b.wt. < 100 kg) or 30 mg (b.wt. ≥ 100 kg), every 3 wk, i.v. infusionSelectively binds to TTR mRNA to decrease hepatic production of TTR proteinhATTR amyloidosis2018PrescriptionAdams et al., 2018; Wood, 2018; Zhang et al., 2020b
    Inotersen (ASO)20-mer gapmer; all PS, 2ʹ-MOE, and 2′-deoxy; C and U methylated; mol. wt. ∼7.2 kDa284 mg once weekly, s.c.Selectively binds to TTR mRNA to cause mRNA degradation and reduce protein productionhATTR amyloidosis2018PrescriptionBenson et al., 2018; Keam, 2018
    Givosiran (siRNA)Double-stranded siRNA; PO and PS, 2-F′, 2′-O-Me, and triantennary GalNAc; mol. wt. ∼16.3 kDa2.5 mg/kg once monthly, s.c.Selectively binds to hepatic ALAS1 mRNA, leading to ALAS1 mRNA degradation through RNA interferenceAHP2019PrescriptionSardh et al., 2019; de Paula Brandao et al., 2020; Scott, 2020
    Golodirsen25-mer PMO; m5U; mol. wt. ∼8.6 kDa30 mg/kg once weekly, i.v. infusionSelectively binds to exon 53 of dystrophin pre-mRNA to alter splicing, leading to production of functional muscle protein dystrophin in patients with genetic mutations that are amenable to exon 53 skippingDMD2019PrescriptionHeo, 2020
    • 2′-F, 2′-fluoro; 2′-OMe, 2′-methoxy.

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    TABLE 3

    Analytical methods for oligonucleotides or RNAs

    MethodAnalytesAdvantagesDisadvantages
    qPCR (amplification)Unmodified oligonucleotides (chemical modifications may affect the assay)High sensitivity, broad dynamic range (4–8 orders of magnitudes), medium to high throughputExtensive sample preparation
    Depends on primers and fluorophore
    Low resolution, accuracy, and precision
    Limited specificity and potential matrix effects
    May not distinguish metabolites from the analyte
    Hybridization-based assays or ELISA (no amplification)Modified or unmodified oligonucleotidesHigh specificity (sequence dependent)Depends on the reliability of probe, detection marker, or antibody
    Wide dynamic rangePossible matrix effects
    Good sensitivityMay not distinguish metabolites from the analyte
    Reasonable accuracy and reproducibility
    LC-UV (or FL)Modified or unmodified oligonucleotidesHigh specificity after separationExtensive sample preparation
    Good accuracyLow sensitivity
    Broad dynamic rangeRequires good separation
    Low to medium throughput(Sensitive FL detection depends on the fluorophore-labeled probe to be hybridized with the analyte)
    LC-MS/MSSmall oligonucleotides (e.g., <25 nt)High specificity (for modified RNAs), wide dynamic range, good sensitivity (for modified RNAs), medium to high throughput, good accuracy and reproducibilityCostly instrument
    Limited specificity and sensitivity, particularly for unmodified RNAs
    • LC-MS/MS, LC tandem mass spectrometry.

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    TABLE 4

    Small-molecule drugs approved for clinical practice or under development that act on RNA targets

    CompoundMolecular Targets and Mechanisms of ActionClinical ApplicationCurrent StatusReferences
    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 synthesisAntibiotics to treat infectionsApproved for medical useFourmy 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 synthesisAntibioticsApproved for medical useBrodersen 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 synthesisAntibioticsApproved for medical useVannuffel 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 synthesisAntibioticsApproved for medical useIppolito 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 suppressionTreatment of patients with DMD with nonsense mutationApproved 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 levelsTreatment of patients with SMAPhase II/III trial (NCT02913482)Ratni et al., 2016, 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 proteinTreatment of patients with SMAPhase I/II trial (NCT02268552)Palacino et al., 2015; Cheung et al., 2018; Dangouloff and Servais, 2019
    AnthraquinonesBinds to HIV TAR element to inhibit viral replicationTreatment of HIV infectionPreclinical developmentGanser et al., 2018
    Benzimidazoles (e.g., Isis-11)Binds to HCV IRES to inhibit viral replicationTreatment of hepatitis CPreclinical developmentSeth et al., 2005; Paulsen et al., 2010; Dibrov et al., 2012
    RibocilsBinds to FMN riboswitch selectively to suppress riboswitch-targeted gene expression and thus inhibit bacterial growthAntibioticsPreclinical developmentHowe et al., 2015, 2016; Wang et al., 2017; Rizvi et al., 2018
    Targaprimir-96 and its bleomycin A5 conjugateBinds to pri-miR-96 to inhibit Drosha-mediated processing or induce cleavage of pri-miR-96AnticancerPreclinical developmentVelagapudi et al., 2016; Li and Disney, 2018
    Targapremir-210 and its conjugateBinds to pre-miR-210 to inhibit Dicer-mediated processing or recruit RNase L for cleavageAnticancerPreclinical developmentCostales et al., 2017, 2019b
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Review ArticleReview Article

RNA-Based Therapies

Ai-Ming Yu, Young Hee Choi and Mei-Juan Tu
Pharmacological Reviews October 1, 2020, 72 (4) 862-898; DOI: https://doi.org/10.1124/pr.120.019554

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RNA-Based Therapies

Ai-Ming Yu, Young Hee Choi and Mei-Juan Tu
Pharmacological Reviews October 1, 2020, 72 (4) 862-898; DOI: https://doi.org/10.1124/pr.120.019554
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    • I. Introduction
    • II. Classification and General Features of RNA-Based Therapeutics
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