Three ‘E’ challenges for siRNA drug development

Theoretically, siRNA has the ability to target any gene of interest, potentially addressing disease targets that are 'undruggable' for small molecules and proteins.Currently, there are six siRNA therapeutics that have been approved for clinical use, and approximately 20 additional candidates have progressed to late stages of clinical investigation.Targeted accumulation and cellular uptake (entry), endolysosomal escape (escape), and in vivo pharmaceutical performance (efficacy) (three 'E' challenges) are the most critical bottlenecks in siRNA drug development.Ligand-conjugated siRNAs are promising platforms that have made a breakthrough in robust extrahepatic delivery.Sophisticated and appropriate chemical modification may bring astounding breakthroughs in the stability and long-term efficacy of siRNA modalities. siRNA therapeutics have gained extensive attention, and to date six siRNAs are approved for clinical use. Despite being investigated for the treatment of metabolic, cardiovascular, infectious, and rare genetic diseases, cancer, and central nervous system (CNS) disorders, there exist several druggability challenges. Here, we provide insightful discussions concerning these challenges, comprising targeted accumulation and cellular uptake ('entry'), endolysosomal escape ('escape'), and in vivo pharmaceutical performance ('efficacy') – the three 'E' challenges – while also shedding light on siRNA drug development. Moreover, we propose several promising strategies that hold great potential in facilitating the clinical translation of siRNA therapeutics, including the exploration of diverse ligand-siRNA conjugates, expansion of potential disease targets, and excavation of novel modification geometries, as well as the development of combination therapies. siRNA therapeutics have gained extensive attention, and to date six siRNAs are approved for clinical use. Despite being investigated for the treatment of metabolic, cardiovascular, infectious, and rare genetic diseases, cancer, and central nervous system (CNS) disorders, there exist several druggability challenges. Here, we provide insightful discussions concerning these challenges, comprising targeted accumulation and cellular uptake ('entry'), endolysosomal escape ('escape'), and in vivo pharmaceutical performance ('efficacy') – the three 'E' challenges – while also shedding light on siRNA drug development. Moreover, we propose several promising strategies that hold great potential in facilitating the clinical translation of siRNA therapeutics, including the exploration of diverse ligand-siRNA conjugates, expansion of potential disease targets, and excavation of novel modification geometries, as well as the development of combination therapies. Viewed through the prism of pharmaceutical history, small molecules have enjoyed over a century of use as the earliest developed and applied therapeutic modality, while proteins and antibodies emerged relatively late and have been investigated for almost half a century. Although nucleic acid molecules, as a novel therapeutic approach, have had a shorter developmental timeline (20–30 years), they have already captured significant global attention from the pharmaceutical industry, emerging as the third most prominent modality [1.Opalinska J.B. Gewirtz A.M. Nucleic-acid therapeutics: basic principles and recent applications.Nat. Rev. Drug Discov. 2002; 1: 503-514Crossref PubMed Scopus (504) Google Scholar]. Nucleic acid drugs are still undergoing rapid exploration and development, particularly in the realm of RNAi, where their broad and profound therapeutic potential is increasingly manifest. With this in mind, we believe that the coming period will be a pivotal era for nucleic acids, both expanding the scope of treatment options and offering new possibilities in the field. Compared with traditional small molecules and antibodies, siRNA (see Glossary) has the advantage of abundant disease targets, high development success rate, short development time, robust and long-lasting efficacy, and outstanding attributes of platform-based modalities [2.Hopkins A.L. Groom C.R. The druggable genome.Nat. Rev. Drug Discov. 2002; 1: 727-730Crossref PubMed Scopus (2776) Google Scholar, 3.Wu S.Y. et al.RNAi therapies: drugging the undruggable.Sci. Transl. Med. 2014; 6240ps7Crossref Scopus (216) Google Scholar, 4.Finan C. et al.The druggable genome and support for target identification and validation in drug development.Sci. Transl. Med. 2017; 9eaag1166Crossref PubMed Scopus (312) Google Scholar]. Currently, six siRNA drugs (patisiran, givosiran, lumasiran, inclisiran, vutrisiran, and Rivfloza) have been successfully commercialized [5.Guo S. et al.Membrane-destabilizing ionizable lipid empowered imaging-guided siRNA delivery and cancer treatment.Exploration. 2021; 1: 35-49Crossref Scopus (103) Google Scholar, 6.Hu B. et al.Therapeutic siRNA: state of the art.Signal Transduct. Target. Ther. 2020; 5: 101Crossref PubMed Scopus (617) Google Scholar, 7.Zhang M. Huang Y. siRNA modification and delivery for drug development.Trends Mol. Med. 2022; 28: 892-893Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar]. Despite the broad application prospects of siRNA drugs in clinical practice, their development faces pivotal challenges, including targeted accumulation and cellular uptake (entry), endolysosomal escape (escape), and in vivo pharmaceutical performance (efficacy) (three 'E' challenges) (Figure 1). In this opinion article, we elaborate on the current status and future prospects of siRNA therapeutics, summarize the pivotal challenges encountered in this field, and propose a series of circumventing strategies. By offering extensive insights and inspiration, this opinion article seeks to provide valuable guidance to the scientific and pharmaceutical communities alike. In recent years, siRNA therapy has shown immense potential in the development of numerous candidate drugs for preclinical and clinical research [8.Zogg H. et al.Current advances in RNA therapeutics for human diseases.Int. J. Mol. Sci. 2022; 23: 2736Crossref PubMed Scopus (65) Google Scholar,9.Forgham H. et al.Keeping up with the COVID's – could siRNA-based antivirals be a part of the answer?.Exploration. 2022; 220220012Crossref Scopus (4) Google Scholar]. As of August 2023, there are globally 15 investigational siRNA drugs in clinical Phase 2 or later stages (Table 1), covering a wide range of treatment areas including rare diseases and genetic diseases and extending to common diseases. Leading pharmaceutical companies have expanded their research focus to encompass popular disorders such as metabolic diseases, cardiovascular disease, hepatitis B, and cancer. For instance, ALN-AGT (NCT04936035i, NCT05103332ii, randomized) is currently in development for the treatment of hypertension and has progressed to Phase 2 trials [10.Huang S.A. et al.Safety and tolerability of ALN-AGT, an RNA interference therapeutic targeting hepatic angiotensinogen synthesis, in hypertensive patients during sodium depletion or irbesartan coadministration.Circulation. 2021; 144A11276Google Scholar]. Olpasiran (NCT05581303iii, randomized) is intended to treat atherosclerotic plaques and is undergoing Phase 3 study [11.Malick W.A. et al.Clinical trial design for lipoprotein(a)-lowering therapies: JACC Focus Seminar 2/3.J. Am. Coll. Cardiol. 2023; 81: 1633-1645Crossref PubMed Scopus (9) Google Scholar]. SLN360 (NCT05537571iv, randomized), a lipid-lowering siRNA, has progressed to Phase 2 investigation. RBD1016 (NCT05961098v, randomized), a N-acetylgalactosamine (GalNAc)-conjugated siRNA for the treatment of hepatitis B, will start Phase 2 trials in the Europe. STP705 and STP707 are made of two siRNAs that target transforming growth factor beta 1 (TGF-β1) and cyclooxygenase 2 (COX-2) and are formulated in peptide nanoparticles (PNPs). STP705 was locally administered to diseased tissue and investigated for the treatment of in situ squamous cell carcinoma (isSCC) (NCT04844983vi, Phase 2, randomized) and basal cell carcinoma (BCC) (NCT04669808vii, Phase 2, non-randomized), while STP707 (NCT05037149viii, Phase 1, non-randomized) was intravenously injected into the body for the treatment of several solid tumors and fibrotic liver diseases such as primary sclerosing cholangitis (PSC).Table 1Selected commercialized or late-stage investigated siRNA therapeuticsDrug nameTarget geneDelivery technologyIndicationSponsorPhase and NCT numberAdministration routeaAbbreviations: i.d., intradermal injection; i.t., intratracheal administration; ita, intratumoral administration; i.v., intravenous injection; o.a., ophthalmic administration; s.c., subcutaneous injection.PatisiranTransthyretin (TTR)L NPsPolyneuropathy of hereditary TTR-mediated amyloidosis (hATTR)AlnylamApprovedi.v.GivosiranAminolevulinate synthase 1 (ALAS1)GalNAc-siRNA conjugateAcute hepatic porphyria (AHP)AlnylamApproveds.c.LumasiranHydroxyacid oxidase 1 (HAO1)GalNAc-siRNA conjugatePrimary hyperoxaluria type 1 (PH1)AlnylamApproveds.c.InclisiranProprotein convertase subtilisin/kexin type 9 (PCSK9)GalNAc-siRNA conjugateHypercholesterolemiaAlnylam, The Medicine Company, NovartisApproveds.c.VutrisiranTTRGalNAc-siRNA conjugatePolyneuropathy of hATTR amyloidosisAlnylamApproveds.c.RivflozaLactate dehydrogenase A (LDHA)GalXC™ RNAi platformPH1Novo NordiskApproveds.c.Olpasiran, AMG 890, ARO-LPAApolipoprotein (APO) A1 (APOA1), Lp(a)GalNAc-siRNA conjugateCardiovascular disease, atherosclerotic cardiovascular diseaseAmgen, ArrowheadPhase 2, NCT04270760xviiiPhase 3, NCT05581303iiis.c.ARO-APOC3APOC3GalNAc-siRNA conjugateType I hyperlipoproteinemia, hypertriglyceridemia, congenital lipid metabolism disordersArrowheadPhase 3, NCT05089084xixs.c.Tivanisiran, SYL1001Transient receptor potential cation channel subfamily V member 1 (TRPV1)None (unmodified, carrier-free)Dry eye disease, Sjögren's syndromeSylentisPhase 3, NCT03108664xii NCT04819269xiiio.a.AOC 1020Double homeobox 4 (DUX4)Antibody-siRNA conjugateFSHDAvidity BiosciencesPhase 2, NCT05747924xxi.v.SLN360APOA1, Lp(a)GalNAc-siRNA conjugateCardiovascular diseases, atherosclerosis, Lp(a)SilencePhase 2, NCT05537571ivs.c.SLN-124Transmembrane serine protease 6 (TMPRSS6)GalNAc-siRNA conjugatePolycythemia veraSilencePhase 1/2, NCT05499013xxis.c.Zilebesiran, ALN-AGTAngiotensinogen (AGT)GalNAc-siRNA conjugateHypertensionAlnylamPhase 2, NCT04936035i, NCT05103332iis.c.ALN-HSDHydroxysteroid 17-beta dehydrogenase 13 (HSD17B13)GalNAc-siRNA conjugateNASHAlnylam, RegeneronPhase 2, NCT05519475xxiis.c.OLX10010Connective tissue growth factor (CTGF)Cell-penetrating asymmetric siRNA (cp-asiRNA)Hypertrophic scarringOlix, Alira HealthPhase 2, NCT04877756xxiiii.d.XalnesiranHBV geneGalNAc-siRNA conjugateHepatitis B virus (HBV)Dicerna, Novo NordiskPhase 2, NCT04225715xxivs.c.RBD1016HBV geneGalNAc-siRNA conjugateHBVRibo Life Science LtdPhase 2, NCT05961098vs.c.SYL1801NOTCH regulated ankyrin repeat protein (NRARP)NoneWet macular degeneration, neovascular age-related macular degeneration, macular degenerationSylentisPhase 2, NCT05637255xxvo.a.SYL040012Adrenoceptor beta 2 (ADRB2)NoneOpen-angle glaucomaSylentisPhase 2, NCT02250612xxvi, NCT01739244 xxviio.a.STP705COX-2, TGF-β1PNPsBCC, intraepidermal SCC, skin SCC in situ (isSCC, keloid), keloidSirnaomicsPhase 2, NCT04669808vii, NCT04844983vi, NCT04844840xxviiis.c., i.d., itasiG12D-LODERKRAS proto-oncogene, GTPase (KRAS)LODER®Pancreatic ductal adenocarcinomaSilenseedPhase 2, NCT01676259xxixitaa Abbreviations: i.d., intradermal injection; i.t., intratracheal administration; ita, intratumoral administration; i.v., intravenous injection; o.a., ophthalmic administration; s.c., subcutaneous injection. Open table in a new tab From a product pipeline perspective, a notable breakthrough in siRNA therapy lies in its expansion into extrahepatic diseases [12.Weng Y. et al.RNAi therapeutic and its innovative biotechnological evolution.Biotechnol. Adv. 2019; 37: 801-825Crossref PubMed Scopus (185) Google Scholar, 13.Lu M. et al.Photoactivatable silencing extracellular vesicle (PASEV) sensitizes cancer immunotherapy.Adv. Mater. 2022; 34e2204765Crossref Scopus (22) Google Scholar, 14.Zhang M. et al.Conscription of immune cells by light-activatable silencing NK-derived exosome (LASNEO) for synergetic tumor eradication.Adv. Sci. (Weinh.). 2022; 9e2201135Google Scholar, 15.Guo S. et al.A novel polyethyleneimine-decorated FeOOH nanoparticle for efficient siRNA delivery.Chin. Chem. Lett. 2021; 32: 102-106Crossref Scopus (19) Google Scholar, 16.Hu B. et al.Lipid-conjugated siRNA hitchhikes endogenous albumin for tumor immunotherapy.Chin. Chem. 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Nanotechnol. 2011; 6: 658-667Crossref PubMed Scopus (372) Google Scholar, 22.Zheng Z. et al.Folate-displaying exosome mediated cytosolic delivery of siRNA avoiding endosome trapping.J. Control. Release. 2019; 311–312: 43-49Crossref PubMed Scopus (85) Google Scholar], including realms that have remained elusive for small molecules and antibody drugs, such as CNS disorders. ALN-APP (NCT05231785ix, Phase 1, randomized) is an intrathecally administered siRNA targeting amyloid precursor proteins (APPs) for the treatment of Alzheimer's disease (AD) [23.Mishra N. et al.Role of siRNA-based nanocarriers for the treatment of neurodegenerative diseases.Drug Discov. Today. 2022; 27: 1431-1440Crossref PubMed Scopus (14) Google Scholar] and cerebral amyloid angiopathy (CAA) [24.Nat. Biotechnol. 2022; 40: 1439-1440Crossref PubMed Scopus (2) Google Scholar]. Recently, the ongoing Phase 1 study of ALN-APP has attained positive mid-term results in the single-drug dose escalation trialx. ARO-SOD1 (NCT05949294xi, Phase 1, randomized) is an investigational siRNA targeting superoxide dismutase 1 (SOD1) in the CNS for potential treatment of amyotrophic lateral sclerosis (ALS) caused by SOD1 mutations, which is undergoing Phase 1 study. In addition, clinically developed RNAi therapies are progressing towards the delivery of siRNAs to other tissues, such as eye, muscle, lung, and fat. Tivanisiran (SYL1001) (NCT03108664xii, NCT04819269xiii, randomized) is currently in a Phase 3 clinical study for the treatment of dry eye disease. ARO-DUX4xiv (Phase 1/2) for the treatment of facioscapulohumeral muscular dystrophy (FSHD) has been submitted for clinical trials. ARO-MUC5AC (NCT05292950xv, Phase 1, randomized), ARO-RAGE (NCT05276570xvi, Phase 1, randomized), and ARO-MMP7 (NCT05537025xvii, Phase 1/2a, randomized) are investigated for the treatment of pulmonary disorders. It is noteworthy that the administration frequency of siRNA has achieved a historic breakthrough. The enhanced stabilization modification of siRNA enables durable gene repression and treatment effect in vivo while avoiding potential sequence-dependent off-target effects. As an example, Leqvio requires administration only twice in the first 3 months, followed by treatments every 6 months, to effectively manage primary hypercholesterolemia or mixed dyslipidemia. There are currently over 100 companies worldwide engaged in the siRNA field, with approximately 30 of them specifically focusing on siRNA drug development. As Informa Pharma Intelligence's Biomedtracker recorded, there are currently approximately 200 siRNA/RNAi-based drugs undergoing preclinical and clinical investigation. Since 2016, a total of 14 siRNAs and antisense oligonucleotides (ASOs) have been approved for commercialization. Additionally, the field of oligonucleotide therapeutics has witnessed significant activity in terms of mergers and acquisitions. There have been several notable licensing agreements in recent years in fields such as cardiovascular and metabolic diseases, neurological disorders, and hepatitis B. Representative siRNA delivery platforms include lipid nanoparticle (LNP), GalNAc-siRNA conjugates (GalAheadTM, PDoV-GalNAc, etc.), GEMINI™, TRiM™, PNPs, RIBO-GalSTAR®, and RIBO-OncoSTAR [25.Gao S. My 20 years together with Journal of Oral Pathology and Medicine.J. Oral Pathol. Med. 2023; 52: 324-327Crossref PubMed Scopus (1) Google Scholar], while IKARIA™ was established to develop long-acting siRNA. Despite the significant progress that has been made in siRNA drug research, some critical challenges remain that should be overcome. Specifically, the three 'E' challenges (entry, escape, efficacy) are the three pivotal issues that limit the clinical translation and application of siRNA. The first challenge is to achieve efficient enrichment of siRNAs in target organs/tissues and effective internalization into the target cells (Figure 1A). Due to their large size and anionic charge, unmodified naked siRNAs display low bioavailability, with a half-life as short as several minutes [26.Gao S. et al.The effect of chemical modification and nanoparticle formulation on stability and biodistribution of siRNA in mice.Mol. Ther. 2009; 17: 1225-1233Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar]. Nanocarrier-encapsulated siRNAs are typically bound by serum proteins, leading to uptake by the reticuloendothelial system (RES) and phagocytic clearance [27.Blanco E. et al.Principles of nanoparticle design for overcoming biological barriers to drug delivery.Nat. Biotechnol. 2015; 33: 941-951Crossref PubMed Scopus (4580) Google Scholar]. Moreover, siRNA can be rapidly degraded by nucleases or phosphatase present in plasma, tissues, and cytoplasm. After systemic clearance, siRNAs must cross the endothelium of capillaries to enter the tissue, which is particularly challenging due to extensive adhesion and tight junctions. Although siRNA may passively accumulate in porous sites such as liver or tumor tissues, delivering these therapeutic agents to other parts of the body beyond the organs that preferentially absorb these molecules, as well as the efficient crossing of barriers such as the blood–brain barrier (BBB) and blood–retinal barrier, still faces great challenges [28.Pecot C.V. et al.RNA interference in the clinic: challenges and future directions.Nat. Rev. Cancer. 2011; 11: 59-67Crossref PubMed Scopus (701) Google Scholar]. The second challenge is how to achieve efficient endosomal and lysosomal escape. Although siRNA can enter cells through endocytosis, less than 1% of siRNAs can escape from the endosome, with a passive siRNA escape rate of less than 0.01% [29.Dowdy S.F. Overcoming cellular barriers for RNA therapeutics.Nat. Biotechnol. 2017; 35: 222-229Crossref PubMed Scopus (706) Google Scholar]. The asialoglycoprotein receptor (ASGPR) is a notable exception, with liver cell expression levels of approximately 500 000 or higher and a recycle time of less than 20 min [30.Fakhr E. et al.Precise and efficient siRNA design: a key point in competent gene silencing.Cancer Gene Ther. 2016; 23: 73-82Crossref PubMed Scopus (113) Google Scholar]. Sufficient GalNAc-siRNA conjugates can accumulate in the cytoplasm of hepatocytes to achieve therapeutic levels during treatment. While this provides hope for future RNAi-based targeting of liver therapies, siRNA escape remains an unresolved issue for other types of cells. The expression range of most surface receptors is 10 000–100 000 or less, and receptor recycle times are approximately or longer than 90 min [31.Juliano R.L. The delivery of therapeutic oligonucleotides.Nucleic Acids Res. 2016; 44: 6518-6548Crossref PubMed Scopus (600) Google Scholar] (Figure 1B). As a result of the degradation of siRNA in both the cytoplasm and the endosome, it was observed that only a minuscule fraction of endocytosed GalNAc-siRNA conjugate is present in the cytoplasm in vivo at any given moment [32.Brown C.R. et al.Investigating the pharmacodynamic durability of GalNAc-siRNA conjugates.Nucleic Acids Res. 2020; 48: 11827-11844Crossref PubMed Scopus (116) Google Scholar]. Remarkably, while endosomally entrapped RNA therapeutics serve as a depot, thereby sustaining a long single-dose response duration, this advantage is offset by the substantial proportion of endocytosed RNA therapeutics that fail to penetrate the cytoplasm. Consequently, while the release from endosomes is indeed the primary barrier that inhibits broader application of RNA therapeutics in the treatment of human diseases, it is noteworthy that there needs to be a counterbalance to maintain a depot effect to some extent, ensuring sustained responses over an extended period. To date, attempts to enhance endosomal escape using modified pH sensitivity, ion-penetrating agents, chloroquine-like lysosomotropic agents, pore-forming peptides such as melittin [33.Hou K.K. et al.A role for peptides in overcoming endosomal entrapment in siRNA delivery – a focus on melittin.Biotechnol. Adv. 2015; 33: 931-940Crossref PubMed Scopus (60) Google Scholar], dodecylphosphocholine (DPC), and/or GalNAc-conjugated melittin-like peptide (NAG-MLP) have not fully resolved the relationship between cytotoxicity and increased endosomal escape. The third challenge is the requirement for good in vivo stability, long-lasting effects, and safety. The use of viral vectors for in vivo nucleic acid delivery has some toxic side effects [34.Deyle D.R. et al.A genome-wide map of adeno-associated virus-mediated human gene targeting.Nat. Struct. Mol. Biol. 2014; 21: 969-975Crossref PubMed Scopus (11) Google Scholar,35.Zhu H. et al.Nanoparticle-mediated corneal neovascularization treatments: toward new generation of drug delivery systems.Chin. Chem. Lett. 2023; 34107648Crossref Scopus (5) Google Scholar] and is currently mainly limited to preclinical studies. Chemically synthesized carrier systems such as cationic lipids [36.Meraz I.M. et al.Adjuvant cationic liposomes presenting MPL and IL-12 induce cell death, suppress tumor growth, and alter the cellular phenotype of tumors in a murine model of breast cancer.Mol. Pharm. 2014; 11: 3484-3491Crossref PubMed Scopus (20) Google Scholar] and most inorganic nanoparticles [37.Mohammapdour R. Ghandehari H. Mechanisms of immune response to inorganic nanoparticles and their degradation products.Adv. Drug Deliv. Rev. 2022; 180114022Crossref PubMed Scopus (31) Google Scholar] may induce apoptosis and inflammation in vivo. The delivery systems must also ensure ease of production, quality control, and transport to achieve large-scale clinical applications [38.Humphreys S.C. et al.Considerations and recommendations for assessment of plasma protein binding and drug–drug interactions for siRNA therapeutics.Nucleic Acids Res. 2022; 50: 6020-6037Crossref PubMed Scopus (18) Google Scholar]. In addition, the widely used mouse model in current preclinical studies of RNA drugs is not a toxicity evaluation model, as the RNA dose–response relationship obtained from mouse models cannot be directly applied to human beings. Non-primate models often lack sufficient overlap of genomic sequences with humans to predict pharmacodynamic effects, so it is necessary to expand the use of non-human primate (NHP) models or, as a potential choice, disease-related organoids [39.Pauli C. et al.Personalized in vitro and in vivo cancer models to guide precision medicine.Cancer Discov. 2017; 7: 462-477Crossref PubMed Scopus (651) Google Scholar]. Oligonucleotides without modification normally are unstable in vivo and are easily degraded by nucleases in the bloodstream. Moreover, exogenous oligonucleotides may display immunogenicity and cause immune reactions in the body. With technological breakthroughs, chemical modifications [e.g., modifications on the phosphorothioate (PS) backbone, ribose, and the end of the strand] have been widely used to enhance siRNA stability and reduce/erase off-target effects and immunogenicity [40.Khvorova A. Watts J.K. The chemical evolution of oligonucleotide therapies of clinical utility.Nat. Biotechnol. 2017; 35: 238-248Crossref PubMed Scopus (731) Google Scholar, 41.Ge Q. et al.Effects of chemical modification on the potency, serum stability, and immunostimulatory properties of short shRNAs.RNA. 2010; 16: 118-130Crossref PubMed Scopus (57) Google Scholar, 42.Robbins M. et al.2′-O-methyl-modified RNAs act as TLR7 antagonists.Mol. Ther. 2007; 15: 1663-1669Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar], thereby improving the 'efficacy' of siRNAs [43.Ray K.K. et al.Two Phase 3 trials of inclisiran elevated LDL cholesterol.N. Engl. J. Med. 2020; 382: 1507-1519Crossref PubMed Scopus (702) Google Scholar,44.Desai A.S. et al.Zilebesiran, an RNA interference therapeutic agent for hypertension.N. Engl. J. Med. 2023; 389: 228-238Crossref PubMed Scopus (15) Google Scholar] (Figure 1C). Through sophisticated modification, siRNA has successfully achieved 99% gene silencing and persistent existence in the body, allowing low-dose quarterly, semiannual, or even annual dosing [45.Fitzgerald K. et al.A highly durable RNAi therapeutic inhibitor of PCSK9.N. Engl. J. Med. 2016; 376: 41-51Crossref PubMed Scopus (290) Google Scholar]. The evolutionary history of chemical modifications and their effects on siRNA efficacy is a fascinating area of research that deserves further comprehensive exploration. However, despite these promising achievements, some challenges remain. For example, modification-induced stability and specificity enhancement may reduce the silencing activity or cause unexpected adverse effects [46.Jackson A.L. Linsley P.S. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application.Nat. Rev. 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WO2013074974 describes a dsRNA duplex with motifs comprising three identical modifications on three consecutive nucleotides in one or both strands, particularly near the cleavage site. Additionally, WO2018185241 focuses on modification strategies for nucleotides at positions 2 and 14 from the 5′ end of the antisense strand as well as the nucleotides on the sense strand, which correspond to position 11, 13, 11 and 13, or 11–13 of the antisense strand. These patents pose significant barriers to siRNA drug development, necessitating the establishment of unique technologies by other entities in the field. To address these challenges and advance the development of siRNA therapeutics, several promising strategies or approaches are worth exploration. Optimizing chemical modifications is an important direction to improve siRNA stability, specificity, safety, and bioavailability. This includes the development of novel chemical modification monomers, modification patterns, and RNAi trigger structures (Figure 2A–C ). Traditional siRNA modifications mainly involve 2′-O-methylation (2′-OMe), 2′-fluoro-deoxyribonucleotide (2′-F), and PS, while the development of novel modification monomers and modification patterns will further refine the pharmacokinetic and safety profiles of siRNAs. For example, novel monomers such as glycol nucleic acid (GNA) and 5′-(E)-vinylphosphonate [5′-(E)-VP] (Figure 2B), novel modification patterns such as enhanced stabilization chemistry (ESC) plus (ESC+) (Figure 2A), as well as novel RNAi trigger structures such as small circular interfering RNAs (sciRNAs) [48.Egli M. Manoharan M. Chemistry, structure and function of approved oligonucleotide therapeutics.Nucleic Acids Res. 2023; 51: 2529-2573Crossref PubMed Scopus (65) Google Scholar,49.Jahns H. et al.Small circular interfering RNAs (sciRNAs) as a potent therapeutic platform for gene-silencing.Nucleic Acids Res. 2021; 49: 10250-10264Crossref PubMed Scopus (7) Google Scholar], asymmetric siRNAs [50.Khvorova A. et al.Fully Stabilized Asymmetric siRNA. University of Massachusetts, 2016Google Scholar,51.Turanov A.A. et al.RNAi modulation of placental sFLT1 for the treatment of preeclampsia.Nat. Biotechnol. 2018; 36: 1164-1173Crossref Scopus (119) Google Scholar], and divalent siRNA scaffold [52.Alterman J.F. et al.A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system.Nat. Biotechnol. 2019; 37: 884-894Crossref PubMed Scopus (104) Google Scholar] have been developed (Figure 2C). In addition, siRNA design and modification can now be achieved using algorithms. For example, Alnylam has developed several generations of siRNA designs, including