RNA therapeutics in targeting G protein-coupled receptors: Recent advances and challenges.

Introduction

G protein-coupled receptors (GPCRs) are transmembrane proteins that transduce signals from the extracellular environment to modulate cellular responses by regulating the intracellular signaling pathways. 1 The human genome encodes more than 800 GPCRs that respond to a wide variety of signals. 2 GPCRs play significant roles in almost all physiological and pathophysiological processes. Therefore, members of the GPCR superfamily are promising therapeutic targets. However, drugs have been developed successfully for only 110 of the 826 knowns human GPCRs, including ∼400 olfactory receptors. 3 , 4 The major obstacle in developing conventional drugs such as chemical agonists or antagonists for specific GPCRs is the existence of various subtypes and splice variants for each of the known GPCRs and the high structural similarity between members of the GPCR family. 5 Therefore, novel strategies are urgently needed for the development of effective GPCR-targeting drugs with high specificity.

RNA-based therapy has shown significant clinical potential in the last two decades. It is based on the delivery of exogenous therapeutic RNA into cells to correct or modify defective biological pathways that cause diseased conditions. 6 RNA-based drugs show high specificity and potency, low toxicity, and can be synthesized rapidly on a large scale. 7 , 8 Therefore, the RNA-based drug approach is a promising strategy for developing novel drugs for difficult targets, including the GPCRs. The global RNA-based therapeutics market was valued at USUS$6.16 billion in 2022 and is predicted to achieve an exponential compound annual growth rate (CAGR) of 19.13% between 2022 and 2030. 9

GPCRs are major drug targets for many human diseases, and RNA-based therapy has shown great promise in targeting GPCRs with high specificity. 10 Therefore, it is expected that GPCR-targeting RNA drugs will elicit successful clinical outcomes. In this review, we summarize and discuss the status and recent advances in the field of GPCR-targeting RNA therapeutics, including mechanistic details, clinical applications, and RNA drug delivery systems.

Current GPCR-Targeting Drugs

GPCR is a tractable class of drug targets

GPCRs are activated by a variety of ligands, including ions, hormones, neurotransmitters, proteins, and protons. 3 , 11 The human GPCR family has been classified into four subfamilies: class A (rhodopsin), B (secretin and adhesion), C (glutamate), and F (Frizzled), which are significantly associated with diabetes, 12 cardiovascular diseases, 13 immunological disorders, 14 infectious diseases, 15 cancer, 16 and neurodegenerative diseases, including Alzheimer’s disease, 17 Parkinson’s disease, 18 and Huntington’s disease. 3 , 19 , 20 The extracellular domain of the GPCRs consists of a deep cleft as the orthosteric binding site, which is well suited for the design of small synthetic molecules that inhibit (antagonists) or activate (agonists) the receptors. 21 Therefore, the receptor-binding site for the drugs is easily accessible and does not require membrane permeability, which is an essential criterion for the drugs that target intracellular molecules. Furthermore, GPCRs have highly dynamic structures during activation. When the receptor is activated, the transmembrane helices rearrangement enables binding of the C terminus of the G protein α subunit and stabilizes the activated GPCR. 21 , 22 The binding of ligands to the GPCR triggers the associated G proteins and/or β-arrestins to stimulate downstream signaling events. 3 , 23 Amplification of the downstream signaling pathways further enhances the effect of the ligands or the drugs that interact with the GPCRs. Therefore, GPCR-targeted drugs elicit an effective drug response by significantly altering the cellular functions.

Current GPCR drug market

Currently, almost 60% of drugs in the developmental stages and 36% of approved drugs target GPCRs. 24 In 2020, the global market for GPCRs was estimated at US$2.6 billion and is expected to reach US$3.9 billion by 2027, with a CAGR of 5.7% between 2020 and 2027. 25 North America is currently the largest market for GPCR-targeting drugs. In 2021, the market for GPCR-related drugs in the USA was estimated at US$974.6 million. The GPCR drug market has also grown robustly in the Asia-Pacific region because of the increasing population, cancer incidence rates, and research activities. China is expected to reach a market size of US$318.7 million by 2027 for GPCR drugs. The forecast for Japan and Canada is to grow at 4.9% and 5.7%, respectively, for the GPCR drugs. The CAGR is expected to be 5.4% between 2020 and 2027 in the European market for GPCR drugs. 25 In general, the demand from the global market indicated the huge potential market for the drugging of the currently “undruggable” GPCRs.

The increased adoption of GPCR-targeting drugs for the treatment of various human diseases is expected to fuel the growth of the global market for the GPCR-related drugs. 26 As shown in Figure 1 A, the US Food and Drug Administration (FDA) has approved 104 GPCR-targeting drugs during the last 10 years (2014–2023), from which 79% drugs were targeted class A, 14% were class B, 5% were class C. However, only 2% of GPCR drugs were class F ( Figure 1 B). In addition, of these approved GPCR drugs, 24% were used for treatment of endocrine diseases, while 14% were nervous system diseases and 10% were mental diseases ( Figure 1 C). Table 1 summarizes the FDA-approved GPCR drugs in the last 7 years. However, in recent years, there has been no significant increase of new drugs launched in the market, and only 25 new GPCR-targeting drugs were approved between 2021 and 2023. More than 90% of the agents that enter phase I clinical trials did not obtain FDA approval because of efficacy or safety concerns, unknown prevalence, and impact of genetic variations. Therefore, targeted drugs are available for only less than 13% of the known GPCRs, and there is a great scope for discovering effective and safe drugs for the remaining GPCRs. 21 Figure 1 Landscape of FDA-approved GPCR drugs from 2014 to 2023 The trials were further analyzed based on (A) FDA-approved drug numbers, (B) GPCR classes, and (C) disease types. GPCR drugs approved in 2014–2016 were obtained from Hauser et al. 24 Approved GPCR drug numbers from 2017 to 2023 were analyzed based on the data shown in Table 1 . Table 1 FDA-approved drugs targeting GPCRs in the 2017 to August 2023 Substance Brand name Targets GPCR class Indications Approval year Abaloparatide Tymlos PTHR1 B, parathyroid hormone osteoporosis 2017 Etelcalcetide Parsabiv CaSR C, calcium-sensing hyperparathyroidism 2017 Naldemedine Symproic OPRM A, opioid opioid-induced constipation 2017 Angiotensin II Giapreza (La Jolla Pharma) AGTR1 A, angiotensin septic or other distributive shock 2017 Exenatide Bydureon BCise GLP-1 B, glucagon type 2 diabetes 2017 Oxymetazoline hydrochloride Rhofade α 1 -AR A, adrenaline rosacea 2017 Fluticasone propionate and salmeterol AirDuo RespiClick β 2 -AR A, adrenaline asthma 2017 Macimorelin Macrilen (Novo Nordisk) GHSR A, ghrelin adult growth hormone deficiency 2017 Semaglutide (injection) Ozempic (Novo Nordisk) GLP-1R B, glucagon type 2 diabetes, cardiovascular risk reduction 2017 Latanoprostene bunod Vyzulta (Bausch and Lomb) PTGFR A, prostanoid glaucoma or ocular hypertension 2017 Cetirizine hydrochloride Zerviate H1R A, histamine conjunctivitis, allergic 2017 Epinephrine Symjepi α/β-AR A, adrenaline allergic reactions 2017 Triptorelin Triptodur GnRH A, gonadotrophin-releasing hormone precocious puberty 2017 Buprenorphine Sublocade μ-opioid A, opioid opioid use disorder 2017 Brimonidine tartrate Lumify α 2 -AR A, adrenaline ocular redness 2017 Gilteritinib Xospata (Astellas) Serotonin receptors A, 5-hydroxytryptamine relapsed or refractory acute myeloid leukemia 2018 Revefenacin Yupelri (Mylan Ireland) CHRM1-CHRM5 A, acetylcholine chronic obstructive pulmonary disease 2018 Tolvaptan Jynarque V2R A, vasopressin and oxytocin adults at risk of rapidly progressing autosomal dominant polycystic kidney disease 2018 Lofexidine hydrochloride Lucemyra α 2 -AR A, adrenaline opiate withdrawal 2018 Lutetium 177 dotatate Lutathera (AAA USA) SSTR2 A, somatostatin gastroenteropancreatic neuroendocrine tumors 2018 Fosnetupitant/palonosetron Akynzeo (Helsinn Healthcare) NK1R/5-HT3R A, tachykinin chemotherapy-associated nausea and vomiting prevention 2018 Elagolix Orilissa (Abbvie) GNRHR A, gonadotropin endometriosis-associated moderate-to-severe pain 2018 Mogamulizumab-kpkc Poteligeo (Kyowa Kirin) CXCR4 A, chemokine non-Hodgkin lymphoma 2018 Cannabidiol Epidioloex (GW Research) CNR1 A, cannabinoid spasticity related to multiple sclerosis and epilepsy 2018 Prucalopride Motegrity 5-HT4R A, hydroxytryptamine chronic idiopathic constipation 2018 Albuterol sulfate ProAir Digihaler β 2 -AR A, adrenaline asthma and COPD 2018 Galcanezumab-gnlm Emgality CGRP B, calcitonin migraine prevention, cluster headaches 2018 Fostamatinib Tavalisse (Rigel Pharma) Multiple targets, including ADORA3 A, adenosine chronic immune thrombocytopenia 2018 Mogamulizumab-kpkc Poteligeo CCR4 A, chemokine refractory mycosis fungoides, or Sézary syndrome 2018 Glasdegib Daurismo SMO F, smoothened acute myeloid leukemia 2018 Erenumab-aooe Aimovig (Amgen) CALCRL B, calcitonin migraine (prevention) 2018 Semaglutide (oral) Rybelsus (Novo Nordisk) GLP-1R B, glucagon type 2 diabetes mellitus 2019 Sumatriptan Tosymra 5-HT1B/1D A, hydroxytryptamine migraine 2019 Brexanolone Zulresso GABA C, GABA postpartum depression 2019 Aclidinium bromide and formoterol fumarate Duaklir Pressair muscarinic acetylcholine receptor (mAChR)/β 2 -AR A, acetylcholine/adrenaline COPD 2019 Lasmiditan Reyvow (Eli Lilly) HTR1F A, 5-hydroxytryptamine migraine 2019 Pitolisant Wakix (Harmony) HRH3 A, histamine narcolepsy, excessive daytime sleepiness 2019 Lumateperone Caplyta (Intra-Cellular) HTR2A, DRD1, DRD2 A, dopamine, 5-hydroxytryptamine schizophrenia 2019 Ubrogepant Ubrelvy CGRP B, calcitonin migraine 2019 Bremelanotide Vyleesi MC1R A, melanocortin sexual arousal disorder 2019 Afamelanotide Scenesse MC1R A, melanocortin prevention of phototoxicity in erythropoietic protoporphyria 2019 Lemborexant Dayvigo (Eisai) HCRTR1 A, orexin insomnia 2019 Cetirizine hydrochloride Quzyttir H1R A, histamine urticaria 2019 Gallium 68 dotatoc NA (UIHC-PET Imaging Center) SSTR2 A, somatostatin diagnostic agent for neuroendocrine tumors 2019 Phenylephrine hydrochloride Biorphen α 1 -AR A, adrenaline hypotension 2019 Siponimod Mayzent (Novartis) S1PR1, S1PR5 A, lysophospholipid relapsing forms of multiple sclerosis 2019 Istradefylline Nourianz (Kyowa Kirin) ADORA2A A, adenosine Parkinson’s disease 2019 Cysteamine Procysbi (Horizon Pharma) NPY2R A, neuropeptide radiation sickness 2020 Ozanimod Zeposia (Celgene) S1PR1, S1PR5 A, lysophospholipid relapsing forms of multiple sclerosis 2020 Oliceridine Olinvyk OPRM1 A, opioid moderate-to-severe acute pain 2020 Rimegepant Nurtec ODT (Biohaven Pharm) CALCRL B, calcitonin migraine 2020 Setmelanotide Imcivree mACh4R A, acetylcholine weight loss (obesity/overweight) 2020 Eptinezumab-jjmr Vyepti (Lundbeck) CALCRL B, calcitonin migraine (prevention) 2020 Relugolix Orgovyx GnRH A, gonadotrophin-releasing hormone prostate cancer 2020 Amisulpride Barhemsys D2R A, dopamine nausea/vomiting, postoperative 2020 Clascoterone Winlevi mAChR A, acetylcholine acne 2020 Tramadol hydrochloride Qdolo μ-opioid A, opioid pain 2020 ozanimod Zeposia S1P A, lysophospholipid multiple sclerosis, ulcerative colitis 2020 Difelikefalin Korsuva κ-opioid receptor A, opioid pain following abdominal surgery 2021 Avacopan Tavneos C5aR A, complement peptide severe active anti-neutrophil cytoplasmic autoantibody-associated vasculitis 2021 Ponesimod Ponvory S1P A, lysophospholipid multiple sclerosis 2021 Varenicline solution Tyrvaya mAChR A, acetylcholine dry eye disease 2021 pilocarpine ophthalmic Vuity mAChR A, acetylcholine presbyopia 2021 naloxone hydrochloride Zimhi opioid A, opioid opioid emergency 2021 Atogepant Qulipta CGRP B, calcitonin prevent migraine headache 2021 Relugolix Myfembree GnRH A, gonadotrophin-releasing hormone premenopausal women to control heavy menstrual bleeding due to uterine fibroids 2021 Leuprolide Camcevi GnRH A, gonadotrophin-releasing hormone adult patients with advanced prostate cancer 2021 Baclofen Lyvispah GABBR1 C, GABA spasticity 2021 Daridorexant Quviviq OXR A, orexin insomnia 2022 Ganaxolone ZTALMY GABA C, GABA CDKL5 deficiency disorder 2022 Dexmedetomidine Igalmi α 2 -AR A, adrenaline agitation 2022 Tirzepatide Mounjaro GLP-1 B, glucagon type 2 diabetes in adults to decrease blood sugar levels 2022 Terlipressin Terlivaz V1R/V2R A, vasopressin and oxytocin hepatorenal syndrome 2022 Omidenepag isopropyl Omlonti EP2 A, prostanoid glaucoma and ocular hypertension 2022 Naloxone RiVive μ-opioid receptor A, opioid the reversal of an opioid overdose or suspected opioid overdose 2023 Zuranolone Zurzuvae GABAA C, GABA postpartum depression 2023 Albuterol and budesonide Airsupra β 2 -AR A, adrenaline asthma 2023 Sparsentan Filspari AT1R/ETAR A, angiotensin/endothelin primary immunoglobulin A nephropathy 2023 Zavegepant Zavzpret CGRP B, calcitonin migraine 2023 Rizatriptan RizaFilm 5-HT1B/1DR A, hydroxytryptamine migraine 2023 Phenylephrine hydrochloride and tropicamide Mydcombi α 1 -AR A, adrenaline pupillary dilation 2023 Fezolinetant Veozah NK3R A, tachykinin menopausal disorders, hot flashes 2023 Nalmefene hydrochloride Opvee μ-opioid receptor A, opioid opioid overdose 2023 COPD, chronic obstructive pulmonary disease.

Drugs that target different classes of GPCRs

According to Yang et al. 3 and the Drugs@FDA database ( accessdata.fda.gov ), FDA has approved more than 500 drugs that target class A GPCRs, including 75% for the aminergic receptors and 10% for the peptide ligand receptors. These drugs are used as analgesics or in the treatment of allergies, cardiovascular diseases, hypertension, pulmonary diseases, and cancer-related fatigue. 3 , 27

Class B GPCRs are classified into two subfamilies—secretin (B1, with 15 receptors) and adhesion (B2, 33 receptors)—and are therapeutic targets for several human diseases, including obesity, type 2 diabetes mellitus (T2DM), osteoporosis, migraine, depression, and anxiety. 3 , 28 , 29 Currently, the FDA has approved 12 drugs that specifically target class B GPCRs. 30 For example, multiple receptor agonists have been developed for the glucagon-like peptide 1 (GLP-1) through selective amino acid substitutions, blocking enzyme cleavage, and/or conjugation to increase the entity bound to plasma proteins. 3 These methods slow down renal clearance of the peptides and extend the half-life of the drug. For example, semaglutide, approved for T2DM in 2017, is the fifth approved GLP-1 receptor agonist, and it has a longer half-life (168 h) than native GLP-1 (1–2 min). 28 , 31

Class C GPCRs include 22 receptors that are subdivided into five subfamilies, including eight orphan receptors, one calcium-sensing receptor (CaSR), two gamma-aminobutyric acid (GABA) type B receptors, three taste 1 sensory receptor (TS1R1–3), and eight metabotropic glutamate receptors (mGluR1–8). 3 , 32 To date, 16 drugs that target the class C GPCRs have been approved by the FDA. At present, 15 drug candidates targeting the metabotropic glutamate receptor subtypes (mGluRs) are undergoing clinical trials for pain, migraine, and Parkinson’s disease. 3 For example, acamprosate is an antagonist of mGluR5 and was approved by FDA in 2004 as an anti-neoplastic agent. 33 Furthermore, allosteric modulators of class C have also attracted significant drug development efforts. For example, cinacalcet is a small-molecule positive allosteric modulator (PAM) of the CaSR and a calcimimetic that was approved by the FDA in 2004 for hyperparathyroidism. 34 Class F GPCRs include 11 members and are characterized as coiled-coil receptors (FZD1–10) that mediate Wnt signaling and are essential for the embryonic development. 3 Only one of the class F GPCRs (smoothened receptor [SMO]) has been validated as a drug target. 3 In 2012, the FDA approved the small-molecule antagonist of SMO, glasdegib, for the treatment of patients with acute myeloid leukemia (AML). 35

Biased ligands

Recent studies on biased GPCR signaling have transformed the understanding of GPCR signaling and opened a new area in GPCR-targeted drug development. “Biased signaling” occurs when a selective ligand preferentially activates one signaling pathway over others that use the same receptor in a single cellular system. 23 , 36 The biased GPCR ligands offer the potential for highly targeted GPCR therapeutics while avoiding side effects. 36 , 37 Biased GPCR signaling involves angiotensin II receptor type 2 (AT2R), μ-opioid receptor (OR), κ-OR, β-adrenergic receptors (βARs), dopamine receptor D2 (DRD2), calcitonin receptor (CTR), chemokine receptors (CCRs), and adenosine receptors (ARs). In 2020, the FDA approved oliceridine (Olinvyk), a μ-OR-biased ligand, for restricted use to manage moderate to severe acute pain in adults. 38 Oliceridine acts as a "biased agonist" of the μ-OR and preferentially activates the G-protein-dependent pathway with minimal receptor phosphorylation to trigger the b-arrestin dependent (or G-protein-independent) pathway. Compared to the traditional opioids, oliceridine shows significantly reduced risk of opioid-related adverse effects, such as constipation and respiratory depression. 38 Additionally, a recent study has shown that MIPS521, a PAM of the adenosine A1 receptor (A1R), exhibits analgesic efficacy in animal neuropathic pain model through the stabilization of the adenosine-receptor-G-protein complex bound to its endogenous agonist. This study elucidates the A1R is a potential agent of non-opioid analgesia on neuropathic pain management. 39

GPCR dimerization

The signaling function of GPCRs involves formation of homo- or heterodimers and oligomers. 40 Therefore, therapeutic strategies involve modulation of receptor dimerization and/or oligomerization. 41 For example, pre-eclampsia is associated with altered GPCR heterodimerization. In patients with pre-eclampsia, significant increase in the formation of the angiotensin II receptor type I (AGT1R)/bradykinin B2 receptor (B2R) heterodimers promotes angiotensin II (Ang II)-stimulated activation of Gαq/11 on the membranes and upregulates Ang II-related hypersensitivity. 42 However, there are limited approaches for targeting GPCR dimerization. Previous studies have demonstrated that transmembrane peptides can be used to inhibit GPCR dimerization. For example, in an animal model, drinking behavior was affected by inhibiting dimerization of the secretin receptor and AGTR1 using a peptide from transmembrane domain 4 (TM4). 43 The TM5/TM6 cannabinoid receptor (CB1R) peptide inhibits dimerization of the CB 1 R and serotonin receptor (5HT2AR) and is an ideal candidate for cannabis-based pain management. 44

Peptide drugs

FDA had approved ∼50 GPCR peptide drugs up to 2021 and more than 10 potential peptide therapeutics are in the pipeline. 45 A majority of the GPCR-targeting peptide drugs functionally mimic the structure of endogenous peptides and target class A or class B GPCRs. Peptide drugs targeting various class A GPCRs, such as μ-OR and κ-OR, and vasopressin receptors are used to relieve pain, induce labor, and treat cardiovascular diseases; these peptides are, however, highly unstable and demonstrate low plasma protein binding. 31 Therefore, chemical modifications are required to enhance pipette circulation time and to improve their biological activity, selectivity, and stability for reducing blood clearance. For example, selepressin is a novel selective vasopressin V1a receptor agonist with a longer plasma half-life because of the introduction of non-natural amino acids at the proteolysis site. 46 , 47 Semaglutide is a GLP-1 receptor agonist with a half-life of 168 h that is approved for the treatment of T2DM. Semaglutide contains a free fatty acid linker that allows the molecule to form a non-covalent reversible interaction with serum albumin, which significantly enhances its half-life. 31 Furthermore, tirzepatide (Mounjaro), a dual GIP/GLP-1, is a key regulator of insulin secretion and has also been used for T2DM treatments in adults. 48

Monoclonal antibodies

GPCR-targeted monoclonal antibodies (mAbs) are another significant area of drug development because of the high specificity, affinity, and ease of purification. 49 GPCR-targeted mAbs have been developed for the treatment of cancers, inflammation, and metabolic disorders. 3 Currently, three GPCR-targeting mAbs have been approved by the FDA. Erenumab (Aimovig), a calcitonin gene-related peptide receptor (CGRP-R) antagonist, was the first GPCR-targeted mAb approved by the FDA in 2018 for the treatment of migraine. 50 Mogamulizumab (Potilegeo), a chemokine receptor CCR4 binder, was approved by the FDA in 2018 for the treatment of mycosis fungoides or Sézary syndrome and acts by targeting the CCR4-positive T cell lymphomas. 51 In 2020, the FDA approved eptinezumab (Vyepti), an anti-CGRP monoclonal antibody for the preventive treatment of migraine in adults. 52 To date, 57 GPCR-targeting monoclonal antibodies are in preclinical and clinical trials. 53 In addition to mAbs, bispecific antibodies, nanobodies, antibody-drug conjugates, and antibody-peptide conjugates are potential variants for future drug development. 54

Photopharmacology

Photopharmacology is another new approach to targeting GPCRs without genetic manipulation, wherein GPCR activity is modulated by small molecules that are regulated by light. 55 Photopharmacology has great therapeutic potential because it allows precise spatiotemporal control of receptor activation. For example, Taura et al. developed a caged light-sensitive A2A adenosine receptor (A2AR) antagonist, MRS7145, to treat movement disorders. The active antagonist is locally released upon illumination by violet light (405 nm) and activates the A2AR. MRS7145 induces accumulation of cyclic AMP (cAMP) mediated by A2AR in a light- and dose-dependent manner. 55 , 56

RNA therapeutics

Advances in RNA-based therapy has shown great promise in the drug development industry because they can theoretically target genetic components based on Watson-Crick base pairing, and demonstrate high precision, relatively predictable performance, high thermostability, and lower cost of synthesis compared to the protein- or peptide-based drugs. 57 , 58 At present, there are six distinct categories of RNA therapeutics: (1) antisense oligonucleotides (ASOs), (2) aptamers, (3) small interfering RNAs (siRNAs), (4) small activating RNAs (saRNAs), (5) microRNAs (miRNAs), and (6) messenger RNAs (mRNAs). The RNA-based drugs approved by the FDA in the six categories are listed in Table 2 and Figure 2 , and those undergoing clinical trials are listed in Table 3 . Currently, there are 21 FDA-approved RNA drugs that use ASO, siRNA, mRNA, and aptamer approaches. Moreover, there are significant basic research and preclinical publications that have demonstrated the potential of RNA therapeutics that are not yet in clinical trials. 59 The mechanisms and recent developments in each of these six categories are summarized in Figure 3 , and the details are discussed in the following sections. Table 2 FDA-approved RNA drugs RNA drugs Brand name Targets Diseases Company Approval year ASOs Fomivirsen Vitravene CMV mRNA CMV retinitis Ionis Pharmaceutical, Novartis 1998 Mipomersen Kynamro Apo-B-100 mRNA homozygous familial hypercholesterolemia Kastle Therapeutics, Ionis Pharmaceuticals, Genzyme 2013 Nusinersen Spinraza SMN2 pre-mRNA spinal muscular atrophy Ionis Pharmaceuticals, Biogen 2016 Eteplirsen Exondys 51 exon 51 of DMD DMD Sarepta Therapeutics 2016 Inotersen Tegsedi TTR mRNA familial amyloid polyneuropathy Ionis Pharmaceuticals 2018 Milasen N/A CLN7 Mila Makovec’s CLN7 gene associated with Batten disease Boston Children’s Hospital 2018 Golodirsen Vyondys 53 exon 53 of DMD DMD Sarepta Therapeutics 2019 Viltolarsen Viltepso exon 53 of DMD DMD NS Pharma 2020 Casimersen Amondys 45 exon 45 of DMD DMD Sarepta Therapeutics 2021 Tofersen Qalsody SOD-1 amyotrophic lateral sclerosis Biogen 2023 Aptamers Pegaptanib Macugen heparin- binding domain of VEGF-165 neovascular age-related macular degeneration OSI Pharmaceuticals 2004 Defibrotide Defitelio adenosine A1/A2receptor veno-occlusive disease in liver Jazz Pharmaceuticals 2016 siRNAs Patisiran Onpattro TTR mRNA polyneuropathy caused by hATTR amyloidosis Alnylam 2018 Givosiran Givlaari ALS1 mRNA acute hepatic porphyria Alnylam 2020 Lumasiran Oxlumo HAO1 mRNA primary hyperoxaluria type 1 Alnylam 2020 Inclisiran Leqvio PCSK9 atherosclerotic cardiovascular disease Novartis 2021 Vutrisiran Amvuttra TTR mRNA polyneuropathy caused by hATTR amyloidosis Alnylam 2022 Nedosiran Rivfloza LDH mRNA primary hyperoxaluria type 1 Novo Nordisk 2023 mRNAs BNT162b2 Comirnaty SARS-CoV-2 S antigens COVID-19 vaccine Pfizer- BioNTech 2020 mRNA-1273 Spikevax SARS-CoV-2 S antigens COVID-19 vaccine Moderna 2020 Figure 2 Summary of FDA-approved RNA drugs Number of RNA drugs was analyzed based on data in Table 2 . Table 3 RNA therapeutics in clinical development RNA therapeutics Target(s) Disease Company Identifier and status ASOs 1018 ISS (CpG-OND-1018) TLR9 non-Hodgkin’s lymphoma Dana-Farber Cancer Institute, Brigham and Women’s Hospital, Massachusetts General Hospital, University of Rochester NCT00251394 (phase II) Apatorsen (OGX-427) HSP27 urologic, bladder, prostate, urothelial, and non-small-cell lung cancer Achieve Life Sciences PRA Health Sciences NCT00487786 , NCT01454089 (phase I/II) Cenersen (EL625) TP53 acute myelogenous leukemia and lymphoma Eleos NCT00074737 (phase II) ARRx (AZD5312) AR prostate cancer AstraZeneca NCT02144051 , (phase I/II) Custirsen (OGX-011) ApoJ prostate, breast, and non-small-cell lung cancer Clinical Trials Group Achieve Life Sciences NCT00054106 , NCT00138658 , (phase I/II) Aptamers NOX-A12 CXCL12 pancreatic, colorectal cancer, and multiple myeloma NOXXON Pharma Merck Sharp & Dohme NCT01521533 , NCT01521533 , NCT03168139 (phase I/II) NOX-E36 CCL2 diabetic nephropathy and renal impairment NOXXON Pharma NCT01372124 , NCT01547897 (phase I/II) Zimura C5 geographic atrophy macular degeneration IVERIC bio NCT02686658 (phase II/III) Pegcetacoplan C3 transplant-associated thrombotic microangiopathy Swedish Orphan Biovitrum Apellis Pharmaceuticals NCT05148299 (phase II) E10030 PDGF age-related macular degeneration National Eye Institute NCT02859441 (phase II) siRNAs TKM-080301 PLK1 cancer with hepatic metastases, and hepatocellular cancer National Cancer Institute Arbutus Biopharma Corporation NCT01437007 , NCT02191878 (phase I/II) AGN211745 VEGFR1 choroidal neovascularization-age-related macular degeneration (CNV-AMD) Allergan NCT00363714 (phase I/II) PF-04523655 VEGFR1 CNV-AMD Quark Pharmaceuticals NCT00713518 (phase II) Atu027 Protein kinase N3 advanced solid tumors and pancreatic cancer Silence Therapeutics NCT00938574 , NCT01808638 (phase I/II) Cosdosiran (QPI-1002) p53 acute kidney injury and delayed graft function Quark Pharmaceuticals NCT02610283 , NCT02610296 (phase I/I) TD101 KRTGA pachyonychia congenita Huntsman Cancer Institute NCT00716014 (phase I) siG12D LODER KRASG12D pancreatic cancer Silenseed NCT01676259 , NCT01188785 (phase I/II) ARO-HIF2 HIF2A renal cell carcinoma Arrowhead Pharmaceuticals NCT04169711 (phase I) APN401 CBLB brain cancer, melanoma, pancreatic cancer, and renal cell cancer Wake Forest University Health Sciences National Cancer Institute NCT03087591 , NCT02166255 (phase I) STP705 TGF-β1 and COX-2 basal cell carcinoma, keloid, hypertrophic scar, hepatocellular carcinoma, liver metastases, and cholangiocarcinoma Sirnaomics NCT04669808 , NCT05421013 , NCT04844840 , NCT02956317 , NCT04676633 (phase I/II) Cosdosiran (QPI-1007) Caspase-2 nonarteritic anterior ischemic optic neuropathy Quark Pharmaceuticals NCT01965106 (phase III) Tivanisiran (SYL1001) TRPV1 dry eye disease Sylentis NCT03108664 , NCT04819269 (phase III) Nedosiran (DCR-PHXC) HAO1 primary hyperoxaluria Dicerna Pharmaceuticals NCT04042402 (phase III) Cemdisiran (ALN-CC5) C5 immunoglobulin A nephropathy Alnylam Pharmaceuticals NCT03841448 (phase II) Fitusiran (ALN-AT3SC) antithrombin hemophilia A or B Genzyme NCT03417102 (phase III) saRNAs MTL-CEBPA CEBPA liver cancer, solid tumor MiNA Therapeutics NCT02716012 , NCT04710641 , NCT04105335 (phase I/II) MTL-STING STING malignant solid tumor MiNA Therapeutics Preclinical trial (Enter phase 1 evaluation in 2023) miRNAs TargomiRs miR-16 malignant pleural mesothelioma, non-small cell lung cancer EnGeneIC NCT02369198 (phase I) MRG-110 miR-92a wound healing miRagen NCT03603431 (phase I) RG-125 (AZD4076) miR-103/107 NASH, T2D with NAFLD AstraZeneca NCT02612662 , NCT02826525 (phase I) RGLS4326 miR-17 autosomal dominant polycystic kidney disease Regulus Therapeutics NCT04536688 (phase I) Miravirsen miR-122 HCV Roche/Santaris NCT01200420 (phase II) CDR132L miR-132 heart failure, acute myocardial infarction Cardior Pharmaceuticals NCT05350969 (phase II) MRG-201 (Remlarsen) miR-29 keloids miRagen Therapeutics NCT03601052 (phase II) RG-012 (Lademirsen) miR-21 Alport syndrome Sanofi NCT03373786 (phase II) MRG-106 (Cobomarsen) miR-155 cutaneous T cell lymphoma/mycosis fungoides Fungoides miRagen Discontinued RG-101 miR-122 chronic hepatitis C Regulus Therapeutics Discontinued pSil-miR200c and PMIS miR-200a/c miR200a tooth extraction University of Iowa Discontinued MRX34 miR-34a melanoma; primary liver cancer; hematologic malignancies Mirna Therapeutics Discontinued mRNAs CVnCoV SARS-CoV-2 S antigens COVID-19 CureVac NCT04652102 (phase III) CV7202 RABV-G rabies CureVac NCT03713086 (phase I) AZD8601 VEGF-A ischemic heart disease AstraZeneca NCT03370887 (phase II) MRT5005 CFTR CF Translate Bio NCT03375047 (phase I/II) mRNA-3704 MUT methylmalonic aciduria Moderna NCT03810690 (phase I/II) BNT111 mutated TAAs (NY-ESO-1, MAGEA3, tyrosinase, and TPTE) advanced melanoma BioNTech NCT02410733 (phase I) mRNA-1273 SARS-CoV-2 S antigens COVID-19 ModernaTX NCT05584202 (phase II) Figure 3 Schematic illustrating different classes of RNA therapeutics ASO, antisense oligonucleotide; RNA, ribonucleic acid; siRNA, small interfering RNA; saRNA, small activating RNA; miRNA, microRNA; mRNA, messenger RNA; RISC, RNA-induced silencing complex; Ago2, Argonaute 2; RNase H, ribonuclease H; RNAP II, RNA polymerase II. Graph was created with BioRender ( https://app.biorender.com ).

Antisense oligonucleotides

Antisense oligonucleotides (ASOs) are short, single-stranded oligonucleotides that downregulate the synthesis of their target proteins. ASOs were first reported in 1978 60 and are widely used for studying target gene functions and as a viable therapeutic approach for specific human diseases. 61 ASOs silence gene expressions through several mechanisms: (1) inhibition of 5′ cap formation, (2) alteration of the splicing process, (3) ribonuclease H (RNase H)-dependent activation, and (4) RNase H-independent ASO activation (steric blocker). 62 , 63 ASO targeting to the 5′ UTR sequence could inhibit 5′ cap formation, thus leading to gene silencing. 63 In addition, pre-mRNAs splice noncoding introns and exons to form mature mRNA. This slicing process could be altered by ASO for disease treatments. For example, ASOs are utilized to delete certain sequences in Duchenne muscular dystrophy (DMD)/dystrophin pre-mRNA that can repair RNA by facilitating splicing, thus effectively treating DMD. 63 , 64 ASO could also work through RNase H-dependent and -independent (steric-blocker) actions. RNase H-dependent ASOs form a duplex with their target RNAs. Subsequently, RNase H recognizes the central region of the RNA-DNA hybrid and cleaves the corresponding RNA strand, thus decreasing target gene transcript levels. 65 The binding of the RNase H-independent ASOs to the target RNA generates a steric block that prevents pre-mRNA maturation or mRNA translation without RNA degradation. The steric-blocker ASOs impede protein-RNA binding interactions between the splicing machinery components and the pre-mRNAs. Therefore, steric-blocker ASOs can be used to regulate the target protein levels by interfering with the splicing of the target RNAs (pre-mRNAs or mRNAs). 66 Typically, steric-blocking ASOs could prevent recognition and cleavage by RNase H with sugar modification, including 2′-O-2-methoxyethyl (2′-O-MOE), 2′-O-methyl (2′-O-Me), 2′-fluoro (2′-F), and locked nucleic acid (LNA). 61 , 67 , 68

In 1998, the FDA approved fomivirsen (Vitravene), the first drug with phosphorothioate (PS) linkages within the ASO backbone, for the treatment of cytomegalovirus retinitis (CMV). Fomivirsen blocked the translation of a key CMV protein, UL123, by binding to the complementary sequence of its mRNA. 69 However, this drug was removed from the market in 2006 because of the success of anti-retroviral therapy, which significantly reduced the probability of opportunistic infections in subjects with HIV. Since then, several ASO therapeutics have been successfully marketed in the US, including mipomersen (Kynamro) and inotersen (Tegsedi). These are also known as ASO gapmers because they contain chemically modified RNA bases flanking both sides of a central 8- to 10-base DNA “gap.” 70 Mipomersen (Kynamro) is a 20-mer gapmer and is approved by the FDA for the treatment of homozygous familial hypercholesterolemia (HoFH) because it significantly reduces the levels of apolipoprotein B ( ApoB ) mRNAs and low-density lipoprotein (LDL). 71 , 72 Inotersen (Tegsedi) is approved for the treatment of adult subjects with hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis) and acts by selectively suppressing the transthyretin (TTR) mRNA levels. 73 Nusinersen is a 2′-MOE-modified 18-mer ASO that modulates alternative splicing of SMN2 and was approved by the FDA in 2016 for the treatment of spinal muscular atrophy (SMA). 74 Milasen is a personalized ASO that was specifically designed for a 6-year-old child with a rare Batten disease, which was caused by premature translational termination of the ceroid lipofuscinosis 7 ( CLN7 ) pre-mRNA because of improper exon splicing. 59 Milasen restores normal exon 6–7 splicing of the CLN7 pre-mRNA. 61 Eteplirsen (Exondys 51), golodirsen (Vyondys 53), viltolarsen (Viltepso), and casimersen (Amondys 45) are ASO drugs with phosphorodiamidate morpholino oligomer (PMO) modifications that were approved by the FDA in 2016, 75 2019, 76 2020, 77 and 2021, 78 respectively, for the treatment of DMD, a lethal neuromuscular disorder that is caused by genetic mutations that alter the reading frame of the X-linked dystrophin gene. 73 Additionally, tofersen (Qalsody) is an ASO that targets the production SOD1 for the treatment of amyotrophic lateral sclerosis (ALS). It was approved by FDA in April 2023. 79 Currently, several new ASOs are undergoing clinical trials ( https://www.clinicaltrials.gov/ .) for the treatment of specific human diseases ( Table 3 ).

RNA aptamers

Aptamers were first developed by the Gold and Szostak groups in 1990. They are short, synthetic, single-stranded RNA or DNA oligonucleotides or their modified analogs that form complex three-dimensional (3D) structures by specifically binding to their target molecules. 80 Aptamers are selected from large libraries of random oligonucleotide libraries (>1,015 random sequences) using the systematic evolution of ligands by exponential enrichment (SELEX) method. 81 These aptamers show high affinity and specificity and interact with a wide variety of specific molecular targets, such as proteins, DNA, RNA, small molecules, and ions. 82 RNA aptamer functions like a nucleic acid antibody or a chemical inhibitor and modulates target protein functions. 73 , 83 RNA aptamer also serves as a targeting ligand to modify and bind nanocarriers that improve the specificity of target drug delivery in clinical therapy. 84 The metabolic stability and pharmacokinetic properties of the RNA aptamers can be improved through chemical modifications, as described for the ASOs. 72

Pegaptanib (Macugen) was the first FDA-approved aptamer in 2004 for the treatment of neovascular age-related macular degeneration. 85 Pegaptanib binds to vascular endothelial growth factor (VEGF) with high affinity and prevents its binding with the VEGF receptor. 86 In 2016, defibrotide (Defitelio) was approved by the FDA for the treatment of hepatic veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS). Defibrotide protected the endothelium lining of the blood vessels during chemotherapy with fludarabine by increasing the levels of prostaglandins and prostacyclin, altering the activity of platelets, and decreasing the activity of the plasminogen activator inhibitor-1 ( PAI-1 ). 87 However, the molecular mechanism of defibrotide is poorly understood. 87 , 88 Both pegaptanib and defibrotide show minimal side effects and highlight the potential of aptamer-based therapies. Currently, several aptamers are being evaluated in clinical trials. For example, NOX-A12 is an anti- C-X-C motif chemokine ligand 12 ( CXCL12 ) aptamer that increases the number of circulating tumor-infiltrating T cells by neutralizing CXCL12 and is proposed for the treatment of pancreatic cancer ( NCT01521533 ), colorectal cancer ( NCT01521533 ), and multiple myeloma ( NCT03168139 ). 89 , 90 , 91 Zimur is an anti-C5 aptamer that is undergoing a clinical trial for the treatment of geographic atrophy secondary to age-related macular degeneration ( NCT02686658 ). 92

siRNA

siRNAs regulate gene expression in eukaryotic cells through a phenomenon known as RNA interference (RNAi). The first step of RNA interference occurs in the cytoplasm, wherein Dicer, an endoribonuclease, produces mature 21- to 23-base siRNAs by cleaving long double-stranded RNAs or short hairpin RNAs. 93 After processing, mature siRNAs are incorporated into the RNA-induced silencing complex (RISC), which then interacts with the Argonaute 2 (Ago2) protein; subsequently, the RNA duplex is unwound, and the passenger strand is degraded. 72 The antisense strand then guides the RISC to the complementary mRNA for subsequent endonucleolytic mRNA cleavage, thereby suppressing protein synthesis.

In the field of biomedicine, siRNAs are a popular tool for downregulating genes of interest. For example, in cancer research, siRNA-targeted downregulation of sphingosine-1-phosphate lyase 1 ( S1P lyase ) decreased invasiveness of cancer cells by upregulating E-cadherin, 94 siRNA-mediated silencing of remodeling and spacing factor-1 ( RSF-1 ) induced apoptosis and cell-cycle arrest of the cervical cancer cells, 95 and PD-L1 -specific siRNAs significantly improved the efficacy of immunotherapy against melanoma and colon cancer cells. 96 siRNA-based therapy has also been applied to neurodegenerative diseases. Zhou et al. developed a glycosylated “triple-interaction” stabilized polymeric siRNA nanomedicine (Gal-NP@siRNA) to target the β-site amyloid precursor protein (APP) cleavage enzyme 1 ( BACE1 ) in the APP/PS1 transgenic mouse model of Alzheimer’s disease. Gal-NP@siRNA efficiently penetrated the blood-brain barrier through glycemia-controlled GLUT1-mediated transport, thereby inducing siRNA-mediated downregulation of BACE1 expression to alter related signaling pathways without significant side effects. 97

To date, the FDA has approved six siRNA agents ( Table 2 ): patisiran (Onpattro), givosiran (Givlaari), lumasiran (Oxlumo), inclisiran (Leqvio), vutrisiran (Amvuttra), and nedosiran (Rivfloza). Patisiran was the first siRNA agent that received FDA approval in 2018. Patisiran targets mutated transthyretin ( TTR ) mRNA and significantly reduces TTR deposition caused by hATTR amyloidosis in patients with polyneuropathy. 93 A lipid nanoparticle formulation was used to shield and stabilize the novel siRNA (ALN-18328) in patisiran from nuclease degradation. 98 In 2019, the FDA approved givosiran for the treatment of acute hepatic porphyria (AHP), which is caused by defective heme biosynthesis resulting in a toxic buildup of porphobilinogen (PBG) and delta-aminolevulinic acid (ALA). 59 Givosiran targets the mRNA of ALA synthase 1 ( ALAS1 ) in the liver and reduces the serum levels of neurotoxic intermediates such as aminolevulinic acid and porphobilinogen. 99 Lumasiran was approved by the FDA in 2020 for the treatment of primary hyperoxaluria type 1 (PH1) in pediatrics and adults by decreasing the levels of oxalate in the urine and blood. Lumasiran targets hydroxyacid oxidase 1 ( HAO1 ) mRNA and reduces the levels of the glycolate oxidase. This reduces the levels of glyoxylate, a substrate for the synthesis of oxalate. 100 Inclisiran injection was approved by the FDA in 2021. It acts by targeting the proprotein convertase subtilsin/kexin type 9 ( PCSK9 ) mRNAs to reduce the levels of LDL. PCSK9 is a key player in lipid metabolism and participates in the regulation of cholesterol levels. Inclisiran injection was approved by the FDA for use in combination with statin therapy for the treatment of heterozygous familial hypercholesterolemia (HeFH) and clinical atherosclerotic cardiovascular disease (ASCVD). 101 Furthermore, vutrisiran was approved in June 2022 by the FDA for the treatment of polyneuropathy caused by hATTR amyloidosis in adults through degradation of the variant and wild-type TTR mRNAs. 102 In September 2023, the FDA approved nedosiran, an siRNA drug, to treat primary hyperoxaluria 1 (PH1) once monthly by inhibiting the expression of LDH enzyme. 103 Givosiran, lumasiran, inclisiran, vutrisiran, and nedosiran are all composed of 2′-O-Me, 2′-F, and PS chemical modifications that enhance stability and reduce immunogenicity. 104 These five drugs are also conjugated to the GalNAc ligand, which was developed to overcome the biological barriers and facilitate targeted delivery. 105 When the bound siRNAs are internalized into hepatocytes through ASGPR-mediated endocytosis, GalNAc siRNAs are rapidly cleaved from the target moiety within acidic endosomes. Subsequently, these siRNAs silence the activity of the targeted protein in the liver. 106 , 107 Except as mentioned above, several siRNA-based therapies are currently being evaluated in clinical trials and are shown in Table 3 .

saRNA

The clinical research on siRNA has led to the use of small RNAs to inhibit the transcription of disease-related genes. However, RNA therapeutics are not yet available for specifically rescuing the transcription of silenced functional genes in specific diseases. RNA activation involves specific upregulation of the target gene expression using small RNA oligos, which are called saRNAs. Multiple studies have shown that saRNAs activate a wide variety of genes in several mammalian species. 108 , 109 , 110 The saRNAs are double-stranded noncoding RNAs that are 21 nucleotides long with a two-nucleotide overhang at the 3′ ends. 111 The saRNAs form a complex consisting of guide RNA, heterogeneous nuclear ribonucleoproteins (hnRNPs), and Ago2 in the cytoplasm. Subsequently, this complex translocates into the nucleus, binds to the corresponding DNA sequence, facilitates assembly of an RNA-induced transcriptional activation (RITA) complex, and recruits RNA polymerase II to initiate transcription and productive elongation of the target mRNA. 112 , 113 Thus, saRNAs expose the target gene promoter and facilitate the binding of RNA polymerase II (RNAPII) at the transcription start site and assembly of the transcription pre-initiation complex. 112 In some cases, saRNAs alter the target mRNAs by binding to the promoter-associated transcripts or long noncoding RNAs instead of the complementary DNA sequences. 114

MTL-CCAAT/enhancer binding protein alpha (CEBPA) is a first-in-class saRNA-based therapy that has recently progressed in the clinical trials. MTL-CEBPA uses liposomal nanoparticles called SMARTICLES (which are composed of different mixtures of 1-palmi- toyl-2-oleoyl-glycero-3-phosphocholine (POPC), DOTAP, DMGSucc, and cholesterol) to encapsulate a modified 2 'O-Me-conjugated saRNA (21-mer), which activates transcription of the tumor suppressor CEBPA gene for the treatment of hepatocellular carcinoma (HCC). 111 , 115 In the phase I and Ib study (OUTREACH-2; NCT02716012 ), MTL-CEBPA was evaluated as a monotherapy or in combination with sorafenib in 51 HCC patients with cirrhosis that resulted from non-alcoholic steatohepatitis or liver metastases. This clinical trial is estimated to be completed in January 2023. The preliminary results from this clinical trial show that MTL-CEBPA mediated RNA activation and reduced immune suppression biomarkers in HCC patients; moreover, MTL-CEBPA in combination with sorafenib showed significant tumor suppression and a good safety profile. 116 Therefore, in January 2022, MiNA Therapeutics announced the global phase II clinical trial (OUTREACH-2; NCT04710641 ) in advanced HCC patients for MTL-CEBPA in combination with sorafenib. 117 Furthermore, MTL-CEBPA in combination with pembrolizumab (PD-L1 inhibitor) is undergoing phase 1 clinical trial ( NCT04105335 ) in patients with advanced solid tumors.

Recently, MiNA Therapeutics announced the proof-of-mechanism data for MTL- Stimulator of interferon genes (STING) as a second drug candidate for RNA therapeutics. STING is a master regulatory protein for the identification of cancer cells by the immune system. The downregulation of STING is a key immune evasion mechanism in the cancer patients and is also the main mechanism for inactivating the cGAS/cGAMP/STING pathway in the innate immune response. 118 MTL-STING is initially being developed as a combination treatment for solid tumor malignancies and is expected to enter phase I evaluation in 2023. 119

miRNAs

miRNAs were first identified in Caenorhabditis elegans and represent an abundant class of noncoding single-stranded RNAs that are 22–61 nucleotides in length. 120 The miRNA coding genes are initially transcribed as primary miRNA transcripts (pri-miRNAs) and are subsequently processed into shorter precursor miRNAs (pre-miRNAs) by the RNase III enzyme, Drosha. 72 , 120 Some pre-miRNAs are directly excised from the introns of protein-coding genes. 121 The pre-miRNAs are then exported from the nucleus into the cytoplasm by Exportin-5 and converted to double-stranded miRNA molecules by the cytoplasmic endoribonuclease, Dicer. Subsequently, miRNAs are loaded into miRNA-induced silencing complexes (miRISCs) and repress protein translation through selective base pairing between the single-stranded guide sequence of the miRNA and the target mRNA. 122 , 123 miRNAs have been the focus of several clinical studies for the treatment of human diseases such as cancers, viral infections, and inflammatory diseases. 124 , 125 , 126 Furthermore, miRNA mimics or miRNA antagonists (anti-miRs) have been proposed as potential agents for the restoration of miRNA expression levels. 59 , 127

Currently, the FDA has not approved any miRNA or anti-miR drugs. However, several studies are ongoing regarding the use of miRNA and anti-miR therapeutics. So far, 12 miRNA drugs have been evaluated in clinical trials. Among these, four are in phase I trials, three are in phase II trials, and five have been terminated ( Table 3 ). Currently, none of the potential miRNA therapeutics are in phase III trials, and more than 40% of the tested miRNA drugs have been terminated in clinical trials, which indicates significant obstacles for the miRNA therapeutics. Due to the off-target effects, the miRNAs show a flexible complementary ratio with the target sequence (range: 20%–90%), and none of the miRNAs are 100% complementary. Therefore, most of the miRNAs target 30–1,000 genes, through mRNA degradation or translation blockage, thus leading to gene-silencing effects. 128 In comparison, the siRNA mechanism requires 100% target sequence specificity. siRNA drugs downregulate target genes via mRNA cleavage. 128 Therefore, siRNAs show significantly higher specificity than the miRNAs. 129 However, even though the flexible complementary miRNAs allow the regulation of multiple target genes, the off-target effects are one of the major challenges of miRNA therapeutics.

MesomiR-1 is a phase1 study ( NCT02369198 ) aimed at delivering miR-16 mimics packaged in EDV nanocells targeted with EGFR antibodies (TargomiRs) for the treatment of malignant pleural mesothelioma and non-small cell lung cancer (NSCLC). The phase 1 trial of TargomiRs ( NCT02369198 ) concluded successfully and is expected to continue to phase II despite some complications. 130 RG-125 (AZD4076) is a GalNAc-conjugated anti-miR targeting miR-103/107 for the treatment of non-alcoholic steatohepatitis (NASH) in patients with type 2 diabetes or pre-diabetes through reduction of fasting glucose and insulin levels. 131 MRG-110 has completed phase I clinical trials to test the safety and efficacy of the miR-92a inhibitor in healthy subjects ( NCT03603431 ). MRG-110 shows potential for treating impaired wound healing in conditions such as diabetes because of the pro-angiogenic effects of miR-92a inhibition. 59 , 132 CDR132L is a noncoding RNA inhibitor that targets miR-132, a central regulator of the pathological cardiac remodeling process in patients with cardiac diseases such as heart failure. Since miR-132 levels are elevated in the cardiac tissues of patients with heart failure, it is hypothesized that the inhibition of miR-132 by CDR132L would potentially restore normal cardiac muscle function by correcting the aberrant signaling pathways. 133 MRG-201 (Remlarsen) is a synthetic miRNA agonist of miR-29 for reversing fibrosis because miR29 decreases synthesis of collagen and other related proteins involved in fibrosis. 134

mRNAs

The mRNAs are excellent candidates for the treatment of diseases with a known genetic component because of their high translation efficiency and immunostimulatory properties. 135 However, mRNA drugs can be associated with toxicity because mRNA expression at nontarget sites results in unwanted protein expression, 136 the off-target effects of mRNA accumulation in the liver and spleen requiring a higher dose of mRNA treatment, 137 and instability related to the relatively long mRNA sequence. 136 These concerns have hampered the use of mRNAs for in vivo clinical applications. Furthermore, the field of mRNA therapeutics has received a major boost due to the development of sophisticated regulatory systems for mRNA expression and the increased stability of mRNAs through chemical modifications, as well as the advancement of delivery platforms. 138

The mRNA therapeutics are used in protein replacement therapy, including delivery of VEGF-A to sites of myocardial infarction, 139 production of vaccines for infectious diseases, 135 and the in vivo production of programmed cell death protein 1 (PD-1) human mAbs. 140 , 141 Furthermore, during the recent onset of the worldwide COVID-19 pandemic, clinical trials of mRNA-based vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) progressed rapidly and resulted in the approval of two mRNA vaccines for emergency use by the World Health Organization (WHO). 72 The first mRNA vaccine approved by the FDA was BNT162b2 (Comirnaty), which was developed by Pfizer-BioNTech and consisted of the coding sequence for the full-length membrane-anchored spike (S) glycoprotein of SARS-CoV-2 with two minor mutations (K986P and V987P) to increase conformational stability. The second FDA-approved mRNA vaccine was mRNA-1273 (Spikevax), which was developed by Moderna and contained the coding sequence for the S glycoprotein of SARS-CoV-2, which was stabilized with two proline substitutions (K986P and V987P) and an intact S1–S2 cleavage site. 142 ( Table 2 ). Both vaccines were encapsulated in lipid nanoparticles, including 1-methyl-pseudouridine, to escape the innate immune-sensing mechanisms and increase translational capacity and mRNA stability. 59 , 138 Furthermore, two additional mRNA vaccine candidates are currently in clinical trials ( Table 3 ). CVnCoV is a COVID-19 vaccine candidate that is produced by CureVac and is currently in phase III clinical trials. CVnCoV is a chemically unmodified mRNA that encodes the full-length S glycoprotein of SARS-CoV-2. 143 CV7202 is another mRNA vaccine candidate in phase I clinical trials for rabies prevention. CV7202 is composed of the rabies virus glycoprotein mRNA to induce a rabies-neutralizing antibody response. 144

There are several mRNA drug candidates in clinical trials for various human diseases. AZD8601 is a candidate VEGF-A drug manufactured by AstraZeneca for ischemic heart disease and is currently being evaluated in phase II clinical trials. 145 MRT5005 is a candidate drug for producing the cystic fibrosis transmembrane conductance regulator protein (CFTR). MRT5005 is manufactured by Translate Bio and is currently in phase I/II clinical trials for the potential treatment of cystic fibrosis (CF) lung disease. 146 Moderna is currently conducting phase I/II clinical trials for mRNA-3704, which is an mRNA therapeutic for restoring function of the mitochondrial enzyme methylmalonic-CoA mutase (MUT) in rare cases of methylmalonic acidemia. 62 FixVac (BNT111) is an intravenously administered liposomal RNA (RNA-LPX) vaccine that targets four highly prevalent tumor-associated antigens in melanoma, including New York esophageal squamous cell carcinoma 1 (NY-ESO-1), melanoma-associated antigen A3 (MAGE-A3), tyrosinase, and transmembrane phosphatase with tensin homology (TPTE). This drug is currently in phase I dose escalation clinical trials for the treatment of advanced melanoma and is manufactured by BioNTech. 147

Challenges for the RNA therapeutics

Research in the last two decades has established that RNA-based therapeutics is a promising area of drug development in various human diseases, especially those that are undruggable. However, several factors have limited their clinical use, including issues regarding RNA stability, penetration efficiency, endosomal escape, immunogenetic problems, and off-target effects. The main obstacles are discussed in this section.

RNA stability

In vivo stability of the RNA molecules is an important criterion for RNA-based therapeutics. The naked RNA oligonucleotides have a very short half-life of 6 min in the plasma and are rapidly cleared by the RNases in the systemic circulation after intravenous injections. 148 Furthermore, there are other biological barriers that prevent naked oligonucleotides from reaching the target cells and tissues. For example, glomerular filtration in the kidney eliminates small molecules that are <50 kDa. These small molecules cannot cross the cell membrane and return into circulation. Therefore, it is estimated that the renal system eliminates intravenously injected naked oligonucleotides into urine within 1 h. 149 The liver also contributes to the clearance of oligonucleotides from the human body. In the reticuloendothelial system (RES), circulating naked oligonucleotides are eliminated from the liver by the Kupffer cells and from the spleen by the macrophages. 150 Therefore, prolonged in vivo stability of the RNA therapeutics in circulation requires specialized or modified delivery systems, which are discussed in later sections.

Penetration efficiency and endosomal escape

Another critical factor for the efficiency of RNA drugs is the ability to penetrate the cell membrane. The lipid bilayer prevents the passage of large amounts of negatively charged molecules, including RNA. 59 , 151 Furthermore, the intracellular route of nonviral oligonucleotide delivery occurs via endocytosis. RNA molecules in the endosomal vesicles are targeted to the lysosomes for degradation. 152 Therefore, endosomal escape is a significant challenge for the efficient delivery of RNA-based therapeutics. Effective strategies are required for the oligonucleotides to escape the endosomes and lysosomal degradation. Currently, this has been achieved by using lipid and dendrimer nanoparticles, as well as ligand conjugation, which results in the release of the loaded therapeutic cargo into the cytoplasm. 153

Immunogenicity

The endogenous immune system considers the injected small RNA molecules as pathogens. Therefore, injection of foreign RNA stimulates the innate immune response and results in undesirable adverse effects and poor therapeutic effects because of drug elimination. 62 Systemic administration of the RNA duplexes stimulates excessive production of inflammatory cytokines and type I interferons (IFNs) through the Toll-like receptors 7/8 (TLR7/8) and represents a key challenge for RNA therapy. 154 The immune response also depends on the length and the nature of the injected RNAs. Longer dsRNAs induce a stronger immune response with high inflammatory cytokine levels. 155 The immune effects can be partially overcome through chemical modifications of the RNA molecules, which are discussed in detail later.

Off-target effects

RNA therapeutics can cause off-target effects and compromise specificity and safety. These off-target effects occur when small RNA molecules are partially complementary to one or more cellular mRNAs besides the target gene or mRNA. 72 The imperfectly matched oligonucleotide binds non-specifically to these nontarget mRNAs and induces silencing through miRNAs binding to their 3′-UTR sequences. 156 Additionally, due to the lack of effective delivery system, RNA drugs could not reach the on-target cells, tissues, and organs, which also lead to off-target effects, thereby resulting in toxicity and reducing the efficacy of the RNA therapeutics. 157

Low exposure

Low exposure to oligonucleotides is another major challenge in the clinical practice of RNA-based therapeutics. 148 Poor enzymatic stability is one of the reasons for this low exposure, as the phosphodiester bond of oligonucleotides is extremely susceptible to RNases and phosphatases. 106 Once the RNA molecule enters the circulation, it is rapidly degraded into segments by endonucleases or exonuclease enzymes, preventing the accumulation of entire RNA molecules in vivo . 106 On the other hand, because the RNA molecule is relatively small, has a short half-life, and lacks the ability to strongly bind to plasma proteins, it is easily cleared during glomerular filtration in the kidneys and eliminated into urine, 158 which also leads to low exposure to RNA drugs.

Chemical modifications of RNA-based drugs

Efficient outcomes of the systemic administration of RNA therapeutics depend on the ability to overcome the physiological challenges mentioned above. Therefore, an optimal RNA drug should also be biocompatible, biodegradable, and nonimmunogenic. 159 Chemical modification of RNA molecules can enhance their stability and protect against the potential immunogenic barrier. Modified nucleotide bases can increase stability, binding affinity, and specificity of the potential RNA drugs to the target sequence. 106 , 160 The most common chemical modifications of the nucleotide bases are 5′-methylcytosine (5′-mC) and 5-propynyl pyrimidine. 161 However, these modifications are associated with steric hindrance, which affects the function of RNA drugs by interfering with the interaction between siRNAs and the RISC. 162

The deoxyribose sugar moiety in the DNA and the ribose sugar moiety in the RNA can be modified to increase the stability of the oligos against nuclease degradation and reduce the adverse immune responses. For example, 2′ modified nucleosides of short RNA, such as 2′-O-methyl (2′- OMe) or 2′-fluoro-2′-deoxy (2′-F) nucleoside, have a prolonged half-life because of increased resistance to endonucleases. 163 , 164 These modifications are incorporated by connecting the 2′-O to the C4′ with a methylene linkage and have been used effectively in the construction of siRNAs, gapmers, splice-switching, and antagomirs. 161 Modification of the phosphorothioate (PS) backbone can also improve the stability of the RNA therapeutics by suppressing exonuclease-mediated degradation and clearance. However, this modification can decrease binding affinity with the target RNA and induce non-specific protein binding. To overcome this problem, PS chemistry is often combined with the base and/or sugar modifications, such as the PMOs, which are short single-stranded DNA analogs that consist of a backbone of morpholine rings connected by the phosphorodiamidate linkages. 161 , 165 The FDA has recently approved two PMO-based ASO drugs, eteplirsen and golodirsen, to resolve the skipping of DMD exons 51 and 53. Both drugs bind to the dystrophin pre-mRNA, alter the exon splicing of the RNA, and enhance the production of full-length dystrophin. The progression of DMD can be prevented or suppressed by increasing the quantity of the full-length dystrophin protein through these modified RNA therapeutics. The uncharged nature of the PMO also protects them against biological degradation. 166 , 167 Another method is the covalent attachment of the hepatocyte asialoglycoprotein receptor-binding N-acetylgalactosamine (GalNAc) to the ASOs, siRNAs, miRNAs, and aptamers to achieve gene silencing. 168 , 169 PS modifications can improve the target-binding affinity of the aptamers. Several strategies have been employed to improve the stability of the in vitro -transcribed (IVT) mRNAs, with greater focus on the structural modification of the nucleic acid. It is well known that the length of the poly(A) tail, incorporation of 5′ and 3′ UTR, 136 and inclusion of the regulatory sequences are essential in determining the intracellular stability of the mRNAs. 164

In vivo delivery of RNA-based drugs

Currently, there is an urgent requirement for innovative delivery systems to improve the in vivo efficacy of RNA therapeutics. In recent years, nanoparticle-based (NP) delivery systems have been developed to provide effective delivery of the loaded RNA cargo with improved protection. NP delivery systems also reduce drug-associated toxicity by minimizing drug accumulation in the off-target tissues and organs. 170 Furthermore, recent efforts have been directed toward developing NP systems with stimuli-responsive drug release, including those triggered by light, redox reactions, temperature, or pH. 171 , 172 , 173

Lipid-based nanoparticles

Liposomes were the earliest lipid-based nano-delivery systems that were used for drug delivery. Hence, they are the most thoroughly studied and successfully developed nano-delivery systems. The advantages of liposomes for the delivery of genes or chemical drugs include the ease of preparation, low cost, and low toxicity. Liposomes are formed by the dispersion of polar lipids such as phospholipids (which contain polar head groups and nonpolar tails) in an aqueous phase. Surface-modified liposome-based particles are advantageous in delivering a variety of therapeutics. Doxil 174 is a liposome-encapsulated formulation of doxorubicin for the treatment of cancer with reduced cardiac toxicity. This breakthrough demonstrated the efficacy of nanoparticle-based drug delivery with altered biodistribution of the drug and increased safety. 175 Lipid-based nanoparticles (LNPs) approved by the FDA contain variations of the following four basic components: (1) cationic or ionizable lipids for electrostatic interactions with the RNA, (2) cholesterol for improving cell entry, (3) helper lipids such as phospholipids, and (4) polyethylene glycol (PEG) for improving stability and circulation time by preventing serum protein binding. 176 Scientists have investigated the structure of lipid-based nucleic acid delivery systems and demonstrated that the lipid structure alters the interaction of the LNPs with cells, protects them from degradation, and prolongs their time of circulation. Onpattro (Patisiran) is an FDA-approved siRNA drug (2018, Alnylam Pharmaceuticals) that is encapsulated inside four lipid components, namely DSPC, cholesterol, DLin-MC3-DMA, and PEG2000-C-DMG. 98

Recently, Alnylam, Moderna, and Pfizer/BioNTech/Acuitas developed LNPs for delivering mRNAs with four components: (1) cationic or ionizable lipids such as DLin-MC3-DMA (Alnylam), SM-102 (Moderna), or ALC-0315 (Pfizer/BioNTech/Acuitas); (2) cholesterol; (3) PEG-lipids such as PEG-2000-C-DMG (Alnylam), PEG-2000-DMG (Moderna), or ALC-0159 (Pfizer/BioNTech/Acuitas); and (4) DSPC. 177 Although most preclinical studies have shown that the structure of the cationic or ionizable lipid is the most important criterion for drug delivery, the other three components also influence drug delivery. 178 For example, an LNP-based siRNA delivery to the pulmonary and cardiovascular endothelial cells in mice was re-targeted to deliver siRNAs to the bone marrow by altering the cholesterol and PEG-lipid components. 179 The interaction of PEG and lipid components of the LNPs with the aqueous component in blood determined the interaction of LNPs with the cells. 180 Luo et al. incorporated the leukocyte membrane proteins into the surface of the liposomes to generate leukosomes, which could localize to the sites of inflammation, reduce the expression levels of pro-inflammatory genes (interleukin [IL]-6, IL-1b, and tumor necrosis factor [TNF]-α), and increase the expression levels of the anti-inflammatory genes (IL-10 and transforming growth factor [TGF]-β). 181 This is a promising strategy to generate novel mRNA therapeutics that selectively target sites of inflammation, but further investigations are necessary. To further enhance the capability of drug delivery at targeted sites, many ligands conjugating with lipid-based nanoparticles have also been employed, including small compounds, carbohydrates, peptides, proteins, and antibodies. 182 , 183 For example, Sato et al. used vitamin A-conjugated liposomes to deliver siRNA against heat-shock protein 47 ( HSP47 ) for reversing human liver cirrhosis. 184 Another study has developed human epidermal growth factor (hEGF) or anti-HER2 Affibody as targeting moieties on the surface of liposomes exhibit better antitumor outcomes. 185

Exosomes

Exosomes are nanosized extracellular vesicles with a diameter of 30–150 nm that are composed of DNA, RNA, lipids, cytosolic metabolites, and cell surface proteins. 186 , 187 Since exosomes originate in cellular secretion, they possess a natural ability in intercellular cargo delivery. 187 , 188 Once targeted to recipient cells, exosomes can transfer their genetic materials and molecules by endocytosis, receptor interaction, or cell membrane fusion, thus regulating cellular functions. 187 , 189 Similar to a lipid-based nanoparticle, exosomes have a special lipid bilayer structure with an aqueous core and a lipophilic shell. 189 Exosomes have high stability and longer circulation time in vivo , efficient cellular uptake, lower immunogenicity, and lower toxicity. 189 Therefore, the unique structure and physicochemical characteristics make the exosome a highly efficient and promising natural carrier for RNA delivery. 189 Li et al. applied arrowtail RNA nanoparticles to ginger-derived exosome-like nanovesicles (GDENs) with folic acid (FA) ligands on the surface for siRNA delivery, this system successfully inhibited tumor growth through intravenous administration. 190 Another recent study has established exosomes for RNA loading by constructing a fusion protein, CD9-HuR, which successfully delivers miR-155 to recipient cells. 191 Furthermore, the exosome can be applied to mRNA delivery as well. Tsai et al. demonstrated that exosomes can efficiently deliver mRNAs encoding immunogenic forms of the SARS-CoV-2 Spike and nucleocapsid proteins in vitro and in vivo , further supporting the development of this system as a novel vaccine and therapeutic approach. 192

Polymer-based nanoparticles

Natural and artificial polymers can be used to construct nanoparticles. The polymer-based nanoparticles show high structural stability, drug encapsulation efficiency, cellular uptake, drug release rate, and ease of modification for the multifunctional and targeted delivery of RNA therapeutics. 193 The polymer properties, including charge, degradability, and molecular weight, can be varied to alter the RNA delivery into the cells. 177

Polyethyleneimine and poly(L-lysine)

Cationic polymers with amine groups such as polyethyleneimine (PEI) and poly(L-lysine) (PLL) can complex with RNAs through electrostatic interactions and can be used to deliver drugs into cells. 177 , 194 However, chemical modifications of PEI and PLL are required to enhance their in vivo efficacy and tolerability. For example, nanoparticles generated with PEG-grafted PEI and cyclodextrin-PEI conjugates have been used for the in vivo delivery of mRNAs. 195 , 196

Poly(beta-amino ester)s

Poly(beta-amino ester)s (PBAEs) are another class of cationic polymers that are synthesized by conjugating amine monomers to the diacrylates. PBAEs with cationic amines and ester bonds show better biodegradation and cytocompatibility compared to PEI and PLL. 197 PBAEs have been used for the intranasal delivery of mRNAs and for the siRNA delivery in a mouse orthotopic glioblastoma tumor model. 198 Furthermore, studies on lipid-polymer hybrids have shown that the serum stability and delivery are enhanced by the addition of lipids to PBAE. 199

Dendrimers

Dendrimers are highly branched polymeric molecules with a 3D structure that is composed of multiple perfect branches radially emanating from a central core and includes multiple functional groups on the surface. 170 Organic and inorganic dendrimer-based nanoparticles have been widely studied and exhibit great potential in the targeted delivery of cargo, including drugs or chemicals, to specific cells. 170 The dendrimer structure has also been modified for protecting the nucleic acids from enzymatic degradation and improving endosomal escape. There are many distinct types of dendrimers that have been used for drug delivery. Here, we focus on the functions and applications of poly(amidoamine) (PAMAM), poly(propylene imine) (PPI), and peptide dendrimers.

PAMAM dendrimers

PAMAM dendrimer is the most thoroughly studied dendrimer type with a 3D spherical structure. 200 PAMAM dendrimer consists of ammonia or ethylenediamine (EDA) core and multiple extended branch points. However, advances in synthetic chemistry have resulted in triethanolamine being frequently used as the core because it offers better loading of monomer molecules. 170 PAMAM dendrimers regulate drug release through a combination of complexation or conjugation. Complexation refers to the loading of drugs into the internal voids of the dendrimers or the gaps surrounded by multiple dendrimers through electrostatic interactions, hydrogen bonding, or hydrophobic interactions. Conjugation involves covalent binding of the drug molecules to the surface of the dendrimer through chemical bonds. 201 Modifications on the surface of the PAMAM dendrimers could improve drug targeting and reduce immunotoxicity. Lai et al. reported that the PAMAM-amide-DOX conjugate for cancer treatment showed slower drug release and reduced cytotoxicity compared to the free drug control at pH = 4.5. 201 Luong et al. reported the use of Fe 3 O 4 nanoparticles decorated with folic acid-PAMAM for delivering an anticancer drug, 3,4-difluorobenzylidene-curcumin, in the treatment of ovarian and cervical cancers. 202 Amreddy et al. reported the use of FA and PEI-conjugated PAMAM nanocarriers loaded with cis -diamine platinum drugs and siRNAs for lung cancer treatment. 203

Amphiphilic dendrimers (ADs) are a new type of PAMAM-based carriers with an affinity for the cell membrane lipids and with improved ability to bind to DNA in the cationic dendrimers. 204 Since the structure of the ADs is more flexible, the amino terminal and carbon chain of ADs can be easily modified. Therefore, modified ADs are widely used in the field of biomedicine. In the nucleic acid delivery process, the positively charged amino terminal of the ADs interacts with the negatively charged nucleic acid molecules through electrostatic interactions. 205 Yu et al. demonstrated that ADs synthesized with different alkyl tail lengths efficiently delivered siRNAs and produced significant gene-silencing effect. 204

PPI dendrimers

PPI dendrimers were first synthesized by Buhleier and Vogtle. 206 PPI dendrimers are synthesized using a divergent approach that involved a two-reaction sequence consisting of repeated Michael addition reaction and amidation from the core to the propylenimine branching unites. 207 Typically, 1,4-butane diamine is used as the core of PPI dendrimers and complexing with the nucleic acid molecules reduces the cytotoxicity of the PPI dendrimers. 208 In order to improve the ability of drug targeting, the numerous groups on the surface of PPI can also be modified. 209 PPI is also used in the construction of nucleic acid biosensors. 210

Peptide dendrimers

Peptide dendrimers are radial or wedge-like branched macromolecules that consist of a peptidyl branching core and/or covalently attached surface functional units. 211 Peptide dendrimers are widely used as biomedical diagnostic reagents, protein mimetics, anticancer and antiviral agents, vaccines, as well as drug and gene delivery vehicles. 212 Zhang et al. generated a mPEGylated peptide dendrimer-doxorubicin conjugate-based nanoparticle using an enzyme-responsive tetrapeptide linker sequence (Gly-Phe-Leu-Gly) as a drug delivery carrier and showed efficient inhibition of breast cancer growth in a mouse model. 213

Cell-penetrating peptide

Cell-penetrating peptides (CPPs), consisting of 5–30 amino acid residues, 214 are another effective tool for the delivery of membrane-impermeable nucleic acids in vitro and in vivo . 215 Due to its cationic or amphipathic nature, it can transport nucleic acid into the cytoplasm via electrostatic interactions, resulting in the self-assembly of peptides with nucleic acids and the formation of nanosized complexes. 169 , 214 CPP delivery facilitates by direct cell membrane penetration or improves cellular uptake. 216 For example, Yang et al. successfully synthesized graphene oxide (GO)/PEI/PEG/CPP/siRNA targeting the Rictor system, which significantly suppressed tumorigenicity in triple-negative breast cancer (TNBC) cells. 217 Another study has described Lp-PPRP, which contains a cationic polymer and a palmitic acid-modified CPP (R8-PA) for a novel ASO delivery tool in tumor therapy. 218 Dastpeyman et al. found that a CPP, HA2-ApoE, used in combination with Nusinersen, an FDA-approved ASO drug for the treatment of SMA, exhibits efficient endosomal escape and cytoplasmic delivery capabilities and significantly improved blood-brain-barrier permeability and central-nervous-system activity of transgenic mouse models. 219

Silica nanoparticles

Silica nanoparticles have gained considerable attention in drug-delivery applications because of good biocompatibility, ease of synthesis, and surface modifications. 220 There are three main types of silica nanoparticles, namely solid, nonporous, and mesoporous. The amorphous particle size, stability, porosity, and surface of the silica nanoparticles depend on the synthesis parameters. 221 , 222 Mesoporous silica nanoparticles consist of many empty pores or compartments like a honeycomb. Therefore, the surface of mesoporous silica nanoparticles can be modified by positively charged moieties to transport substantial amounts of negatively charged RNA. Silica nanoparticles are suitable for the controlled release of therapeutic agents because of their large surface area, pore volume, and high stability. 222 , 223 However, particle aggregation causes safety issues. Lee et al. developed a positively charged structure with a large pore to load anionic siRNAs against Bcl-2 and a negatively charged structure with a small pore to load the anticancer drug doxorubicin; these dual-pore hybrid silica nanoparticles were generated to deliver a combination of genetic and chemotherapeutic drugs simultaneously and their efficacy was demonstrated in the HeLa cells in vitro . 224

Carbon and gold nanomaterials

Gold nanoparticles, quantum dots, nanographene oxide, and carbon nanotubes are synthesized nanostructures that can harbor RNA molecules, protect them from degradation, and deliver them to the targeted site. 62 Jayasekara et al. demonstrated the potential of AuNPs-AR agonist/antagonist conjugates as GPCR therapeutic targets and were associated with biological properties similar to their monomeric counterparts. 225 Xue et al. developed a novel siRNA delivery nanosystem with encapsulated siRNA and aptamer-incorporated core/shell (CS) gold nanoparticle (siRNA/Ap-CS). 226 This system exhibited high serum stability and long circulation time, and it significantly inhibited the growth of malignant tumor. 226 He et al. showed that siRNA conjugated with quantum dots (QDs) can be used to target genes by overcoming the cellular bilayer and deliver the QD-siRNA complex into cells. 227

Administration routes for RNA-based drugs

Due to the different physicochemical properties of RNA, the routes and sites of administration have a significant impact on the clinical efficacy and biodistribution to the RNA drugs. 128 , 228 Currently, there are two main clinical administration routes for RNA therapy: (1) local administered by intramuscular, intrathecal, intravitreal, or intradermal injections, etc.; or (2) systemic administration by intravenous, subcutaneous, or intraperitoneal injections. 228 , 229

Local administration

RNA-based drugs treated by local administration normally exhibit therapeutic effect at the specific sites and could reduce the problems in terms of pharmacokinetic and RNA stability. 228 Intrathecal injection of RNA agents has been explored for the central nervous system (CNS) 230 ; for example, Nusinersen is used for the treatment of SMA by intrathecal injection every 4–6 months. 231 Local administration has also become the preferred method of administration for ophthalmic treatment due to the complexity of the ocular surface. 228 For example, fomivirsen and pegaptanib are used to treat ocular diseases via direct intravitreal injections. 231 Furthermore, naked mRNA has been used to heal various skin diseases and to improve wound healing in the skin by subcutaneous or intradermal injection. 232 Local administration can also be used for siRNA drugs with liposome carriers. 106 For example, intratracheal administration of siRNA targeting Plekhf1 with liposome carrier could effectively inhibit Plekhf1 expression levels in the lungs, and this strategy can be used to protect against pulmonary fibrosis disease. 233

However, the effectiveness of local administration is limited by the low bioavailability of RNA therapeutics and the increased absorption time. 231 Therefore, systemic administration is the preferred route of administration for the treatment of many diseases, especially cancer, because it allows the drugs to reach both local and metastatic sites. 234

Systemic administration

Systemic RNA delivery is more advanced and complicated compared to local administration. 228 Typically, a carrier is required to systematically deliver RNA drugs to prolong the half-life of the drugs. Currently, 50% of siRNA clinical trials have been conducted via systemic delivery, and all these siRNA-based therapies involve nanoparticle delivery systems. 234 , 235 siRNA-lipid nanoparticles are easily selectively concentrated in the liver due to the high apolipoprotein E (ApoE) content and resulting in significant hepatic specificity drug responses. 228 For example, Patisiran is used to inhibit hepatocyte-derived transthyretin by intravenous infusions every 3 weeks. 235 Melamed et al. demonstrated that intraperitoneal administration of mRNA delivered by lipid nanoparticles containing cationic helper lipids facilitate protein expression in pancreatic islets. 228 , 229 Furthermore, givosiran, lumasiran, inclisiran, and vutrisiran represent approved GalNAc-siRNA conjugate drugs, which are administered subcutaneously for acute liver porphyria, primary hyperoxaluria type 1, ASCVD, and transthyretin-mediated amyloidosis, respectively. 104 However, the development of naked RNA drug for systemic administration is hampered by RNA stability and rapid renal clearance considerations. 115 The only exception is QPI-1002, a naked siRNA targeting p53 to treat acute kidney injury, 106 in which intravenous administration of QPI-1002 is used to target tubule cells with high renal siRNA concentration. 104

Application of the RNA therapeutic platform in targeting GPCRs

GPCRs are the most intensively studied therapeutic targets for a plethora of human diseases. However, it is difficult to develop drugs targeted to specific GPCRs because of their complex structure, presence of multiple subtypes, and common activation mechanisms between related GPCRs. RNA therapeutics has opened a new avenue of developing specific drugs targeting GPCRs for various human diseases.

The targeting of GPCRs is mainly focused on either directly modulating GPCR expression level or the downstream signaling molecules. The most widely used strategy for targeting GPCRs is siRNA therapy. Candidate oncogenes are highly expressed in tumors and play critical roles in tumor growth, metastasis, angiogenesis, and drug resistance. Cobalt (III) oxide nanoparticles (Co 3 O 4 NPs) were used to deliver β-arrestin1 siRNAs into the 1321N1 cells; MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay showed that Co 3 O 4 NPs were non-toxic in this cell line. 236 G protein-coupled receptor 1 (OGR1 or GPR68) mediates contraction of the airway smooth muscle signaling in response to pH changes and contributes to asthma pathology; therefore, siRNAs against OGR1 represent valuable therapeutics in obstructive lung diseases. 237 , 238 Ku et al. reported that Gpr27 was a positive regulator of insulin production by using an MIN6-based siRNA screening system with four independent siRNAs targeting mouse-selected GPCR-related genes that were transfected into a reporter cell line. 239 This suggested that siRNA-based targeting of a specific GPCR can be used to modulate insulin production and represents a new avenue for treating diabetes.

Currently, there is only one report on the activation of GPCRs using saRNA. Xiong et al. reported that saRNAs delivered using an amphiphilic dendrimer vector significantly increased the expression of the MAS receptor ( MAS1 ), a GPCR, and inhibited tumorigenesis of multiple cancers by suppressing the classical angiotensin II pathway. 240 This study was the first to use saRNAs to modulate GPCR signaling in cancer therapy and presented a new methodology for effectively targeting GPCRs.

Chemokine receptor 4 coupled with the G protein (CXCR4) is specifically associated with cancer metastasis and HIV-1 infection. The CXCR4-CXCL12 axis plays a key role in orchestrating the recruitment of immune and stromal cells within the tumor microenvironment (TME), thereby influencing tumor cell growth and progression. 241 Therefore, many studies have focused on using RNA therapeutics to target the CXCR4-CXCL12 axis. For example, Dong et al. showed that COL1A1-014 sponges miR-1273 h-5p and increases the levels of the CXCL12 and CXCR4 proteins, thereby promoting the proliferation and metastasis of gastric cancer cells. 242 , 243 Furthermore, overexpression of lncNORAD reduces the growth of NSCLC cells by suppressing the expression levels of CXCR4 and CXCL12. 244 Moreover, overexpression of miR-193a-5p reduces colorectal cancer (CRC) proliferation by suppressing CXCR4 expression levels. Therefore, miR-193a-5pin combination with 5-fluorouracil (5-FU) and oxaliplatin is postulated to be an effective therapy for CRC. 243 , 245 LncRNA FEZF1 antisense RNA 1 (FEZF1-AS1) induces proliferation of osteosarcoma cells and their resistance to apoptosis by inhibiting miR-144, which directly targets CXCR4. 243 , 246 NOX-A12 is a PEGylated 45-nucleotide L-form RNA aptamer (L-RNA aptamer; Spiegelmer is the trade name) and a new RNA drug under phase 2 clinical trial. It is designed to selectively target CXCL-12 with high affinity. It has shown efficacy in preventing the binding of CXCL-12 to its receptors, CXCR4/7, thereby inhibiting angiogenesis and metastasis and improving other anticancer therapies. 83 , 247

Noncoding RNAs (ncRNAs) have been widely used to modulate GPCRs. CD97 is a pro-metastatic GPCR and a direct target of miR-126, and it has been shown to promote breast cancer cell invasion, migration, and angiogenesis. 248 MiR-138-5p enhanced gefitinib sensitivity of the NSCLC cells by regulating GPR124/ADGRA2. 249 EPI-2010 is a respirable ASO (RASON) that is used to target the adenosine A1 receptor and inhibit asthma. Low doses of RASONs can be administered to target respiratory tissues and have been shown to be safe and long acting. 250 Many RNA aptamers have been shown to bind and stabilize the β 2 -adrenoceptor (β 2 AR), which is a non-peptide ligand GPCR with inactive or ligand-specific conformation. 251 Aptamers function like neutralizing antibodies and block the interactions between ligands and their receptors. Anti-PD-L1 aptamers reduce tumor growth and improve immune surveillance by blocking the PD-1/PD-L1 signaling axis. 41

Outlook and future perspectives

RNA molecules are highly versatile therapeutics and have great potential for a wide range of medical conditions. The success of COVID-19 mRNA vaccines also demonstrated the immense potential of RNA-based technologies in the field of infectious disease prevention and in RNA therapeutics for a wide range of applications. More importantly, RNA drugs offer a promising solution for targeting “untagged GPCR,” particularly orphan receptors. However, the challenge lies in our limited understanding of these orphan receptors. Hence, a concerted effort is needed to elucidate the physiological functions of these receptors before translating the RNA drug approach into therapeutic applications.

Precision targeting of specific GPCRs within subfamilies poses difficulties due to structural similarities among multiple subtypes. Classic small-molecule drugs raise concerns about cross-reactivity, as seen in chemokine and β-adrenergic receptor families. RNA drugs therefore present an excellent alternative, although achieving broad inhibition of multiple targets may prove challenging. However, controlling common downstream signaling molecules, if feasible, could overcome this obstacle.

The success of oliceridine, the first GPCR-biased agonist, highlights the potential to reduce side effects by preferentially activating the G protein pathway over the β-arrestin pathway GPCR. 252 , 253 This achievement poses a challenge for RNA drugs, mainly because most approaches involve regulating receptor expression and translation. But recent research on using the A62 agonist aptamer in activating insulin receptor 7 suggests the potential of using aptamer for selectively activating receptor-biased signaling of GPCRs.

RNA drugs, based on the delivery systems, could provide organ- or tissue-specific drug effects, crucial for cancer therapy. In addition, nanoparticle delivery systems enable RNA drugs to synergize with other therapeutic approaches. Combining multiple cell-killing strategies, such as chemotherapy and immunotherapy, 254 not only enhances drug responses but also mitigates potential drug resistance in cancer cells.

In addition, since genetic variations affect an individual’s response to drugs, combining pharmacogenomics analysis and RNA therapeutics may provide innovative and personalized treatments to maximize efficacy and minimize potential adverse effects. In the future, by addressing patient-specific variability, RNA therapeutics could lead to more informed, precise, and tailored treatments that ultimately could optimize outcomes and improve quality of life for patients.

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Acknowledgments

This work was funded by The 10.13039/501100003009 Science and Technology Development Fund, Macau SAR (file no. 0002/2023/RIA1) and 10.13039/501100004733 University of Macau (file nos. and MYRG2022-00240-FHS and MYRG-GRG2023-00093-FHS).

Author contributions

Conceptualization/design, W.Y. and L.L.; original draft preparing and writing, W.Y.; preparation of tables and figures, W.Y.; manuscript reviewing, editing, and supervision, X.S. and L.L. All authors read and approved the final manuscript.

Declaration of interests

The authors declare no competing interests.