Emerging concepts of miRNA therapeutics: from cells to clinic

Single microRNAs (miRNAs) regulate large subsets of mRNA targets. Although this property makes miRNAs potentially a powerful therapeutic tool, it also represents a major challenge in terms of controlling adverse effects that have been observed in clinical trials.Besides systemic applications via injection and infusion, advanced strategies emerge for miRNA-based drug administration via implantable 3D matrices, inhalation schemes, and intake via food.A combination of miRNA therapeutics with chemical modifications, biomolecule conjugation, or the use of carriers improves a site-directed and efficient cell targeting.A comprehensive risk assessment of miRNA therapeutics is required before any in vivo targeting to minimize off-target effects and to avoid overdosing of miRNAs. MicroRNAs (miRNAs) are very powerful genetic regulators, as evidenced by the fact that a single miRNA can direct entire cellular pathways via interacting with a broad spectrum of target genes. This property renders miRNAs as highly interesting therapeutic tools to restore cell functions that are altered as part of a disease phenotype. However, this strength of miRNAs is also a weakness because their cellular effects are so numerous that off-target effects can hardly be avoided. In this review, we point out the main challenges and the strategies to specifically address the problems that need to be surmounted in the push toward a therapeutic application of miRNAs. Particular emphasis is given to approaches that have already found their way into clinical studies. MicroRNAs (miRNAs) are very powerful genetic regulators, as evidenced by the fact that a single miRNA can direct entire cellular pathways via interacting with a broad spectrum of target genes. This property renders miRNAs as highly interesting therapeutic tools to restore cell functions that are altered as part of a disease phenotype. However, this strength of miRNAs is also a weakness because their cellular effects are so numerous that off-target effects can hardly be avoided. In this review, we point out the main challenges and the strategies to specifically address the problems that need to be surmounted in the push toward a therapeutic application of miRNAs. Particular emphasis is given to approaches that have already found their way into clinical studies. What are the promises of miRNA therapeutics?miRNAs (see Glossary) are small, noncoding RNAs that serve as post-transcriptional regulators of protein encoding genes. There are more than 2300 different miRNAs in human cells with time- and tissue-dependent expression patterns [1.Alles J. et al.An estimate of the total number of true human miRNAs.Nucleic Acids Res. 2019; 47: 3353-3364Crossref PubMed Scopus (207) Google Scholar, 2.Kozomara A. et al.miRBase: From microRNA sequences to function.Nucleic Acids Res. 2019; 47: D155-D162Crossref PubMed Scopus (1326) Google Scholar, 3.Ludwig N. et al.Distribution of miRNA expression across human tissues.Nucleic Acids Res. 2016; 44: 3865-3877Crossref PubMed Scopus (529) Google Scholar]. Essential aspects of miRNA biogenesis and its functionality are provided in Box 1. Criteria of miRNA fidelity are addressed below.Box 1Cellular miRNA biogenesismiRNA encoding sequences are located in exons or introns of protein-encoding genes or in intergenic regions. They can be coregulated together with their host genes or can be under the control of their own promoters [103.Olena A.F. Patton J.G. Genomic organization of microRNAs.J. Cell. Physiol. 2010; 222: 540-545PubMed Google Scholar]. During their biogenesis (thoroughly reviewed elsewhere, e.g., [103.Olena A.F. Patton J.G. Genomic organization of microRNAs.J. Cell. Physiol. 2010; 222: 540-545PubMed Google Scholar, 104.Ha M. Kim V.N. Regulation of microRNA biogenesis.Nat. Rev. Mol. Cell Biol. 2014; 15: 509-524Crossref PubMed Scopus (3208) Google Scholar, 105.Winter J. et al.Many roads to maturity: MicroRNA biogenesis pathways and their regulation.Nat. Cell Biol. 2009; 11: 228-234Crossref PubMed Scopus (1970) Google Scholar]), the miRNA encoding sequences are transcribed by RNA polymerase II or III to form primary miRNAs (pri-miRNAs), which are up to several thousand nucleotides in length and shaped as a hairpin structure. pri-miRNAs are further processed by the Drosha-DGCR8 microprocessor complex in the nucleus to generate an miRNA precursor (pre-miRNA) of approximately 70 nucleotides in length. In a noncanonical biogenesis, intron-encoded pre-miRNAs (mirtrons) can be directly processed along with their coencoded transcripts through spliceosomes. The pre-miRNA hairpin is exported by exportin-5 to the cytoplasm, where it is cleaved into an miRNA duplex of approximately 22 nucleotides in length by the RNase Dicer and the double-stranded RNA binding enzyme TRBP. Single miRNA strands are subsequently incorporated into the RISC, allowing the ribonucleoprotein complex to bind to target sequences that are usually located within the 3′ untranslated regions of the mRNAs. Reverse complementary binding takes place in the seed region, which is usually situated at nucleotides 2 to 7 of the miRNA's 5′ end. The binding results in an inhibition or abrogation of the translation process. It is estimated that up to 60% of all protein-encoding genes are subject to miRNA-based post-transcriptional regulation [106.Friedman R.C. et al.Most mammalian mRNAs are conserved targets of microRNAs.Genome Res. 2009; 19: 92-105Crossref PubMed Scopus (6022) Google Scholar], making miRNAs central regulators of cellular signaling with a widespread impact on almost every biological process [6.Gebert L.F.R. MacRae I.J. Regulation of microRNA function in animals.Nat. Rev. Mol. Cell Biol. 2019; 20: 21-37Crossref PubMed Scopus (893) Google Scholar]. Besides their effects on the post-transcriptional level, there is recent evidence that miRNAs can translocate to the nucleus to regulate the transcription efficiency of specific genes, further enhancing their impact on cellular signaling networks [107.Liu H. et al.Nuclear functions of mammalian microRNAs in gene regulation, immunity and cancer.Mol. Cancer. 2018; 17: 64Crossref PubMed Scopus (167) Google Scholar].Physiological changes of miRNA expression are pivotal to regulate complex genetic networks and in consequence cellular signaling cascades. In many disease scenarios, altered miRNA expression plays likewise a central role in modifying the protein expression as part of pathological cellular changes [4.Subramanian S. Steer C.J. Special issue: MicroRNA regulation in health and disease.Genes (Basel). 2019; 10Crossref Scopus (10) Google Scholar]. Besides the diagnostic potential of altered miRNA expression levels, these small RNAs offer themselves for therapeutic purposes toward a targeted manipulation of cell functions that are crucial to a disease phenotype [5.Huang W. MicroRNAs: Biomarkers, diagnostics, and therapeutics.Methods Mol. Biol. 2017; 1617: 57-67Crossref PubMed Scopus (100) Google Scholar]. What makes an miRNA-based intervention most efficient, and consequently especially attractive, is the broad spectrum of targets that can be regulated by a single miRNA [6.Gebert L.F.R. MacRae I.J. Regulation of microRNA function in animals.Nat. Rev. Mol. Cell Biol. 2019; 20: 21-37Crossref PubMed Scopus (893) Google Scholar]. Thus, a single miRNA can direct entire cellular pathways in spite of a relatively moderate effect on each of the targeted genes, as shown for miR-34a-5p that has been identified as a hub of T cell regulation networks [6.Gebert L.F.R. MacRae I.J. Regulation of microRNA function in animals.Nat. Rev. Mol. Cell Biol. 2019; 20: 21-37Crossref PubMed Scopus (893) Google Scholar,7.Hart M. et al.miR-34a as hub of T cell regulation networks.J. Immunother. Cancer. 2019; 7: 187Crossref PubMed Scopus (19) Google Scholar]. Vice versa, one gene or one pathway is typically regulated by several miRNAs, resulting in a complex and powerful regulatory network, potentially addressing the majority of molecular pathomechanisms in humans.Against this background, it is not surprising that, according to PubMed records, since 2015, more than 600 articles have been published under the heading of 'miRNA-based therapeutics'. Although a future therapeutic use of miRNAs is undoubtedly appealing, there are still great practical difficulties to overcome, including the identification of proper administration routes, the control of in-body stability, the targeting of specific tissues and cell types, and the attaining of the intended intracellular effects. Hence, only few miRNA-based drugs have, as of now, entered a clinical test phase (Table 1). In the following sections, we address the different challenges on the way to an effective and nonhazardous use of miRNA therapeutics. We particularly emphasize preclinical studies that developed strategies to address specific challenges associated with using miRNA therapeutics.Table 1Clinical trials with miRNA therapeuticsaNCT numbered trials are registered at ClinicalTrials.gov; EudraCT numbered trials are registered at EU Clinical Trials Register (clinicaltrialsregister.eu).miRNA drug nameTargeted miRNAMode of actionBackground diseaseBody application/permission of cellular uptakeClinical trial number(s)RefsAMT-130bPhase I ongoing.Artificial miRNAamiRNA expressionHuntington diseaseStereotaxic infusion/viral transfer (adeno-associated vector)NCT04120493[23.Keskin S. et al.AAV5-miHTT lowers huntingtin mRNA and protein without off-target effects in patient-derived neuronal cultures and astrocytes.Mol. Ther. Methods Clin. Dev. 2019; 15: 275-284Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 24.Miniarikova J. et al.Design, characterization, and lead selection of therapeutic miRNAs targeting huntingtin for development of gene therapy for Huntington's disease.Mol. Ther. Nucleic Acids. 2016; 5e297Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 25.Samaranch L. et al.MR-guided parenchymal delivery of adeno-associated viral vector serotype 5 in non-human primate brain.Gene Ther. 2017; 24: 253-261Crossref PubMed Scopus (41) Google Scholar]RG-012/lademirsen/SAR339375cPhase II ongoing.miR-21Anti-miRAlport syndromeSubcutaneous injection/chemical modification (phosphorothioate)NCT03373786, NCT02855268[70.Gomez I.G. et al.Anti-microRNA-21 oligonucleotides prevent Alport nephropathy progression by stimulating metabolic pathways.J. Clin. Invest. 2015; 125: 141-156Crossref PubMed Scopus (246) Google Scholar,76.Kelnar K. et al.Quantification of therapeutic miRNA mimics in whole blood from nonhuman primates.Anal. Chem. 2014; 86: 1534-1542Crossref PubMed Scopus (49) Google Scholar,122.Kashtan C.E. Gross O. Clinical practice recommendations for the diagnosis and management of Alport syndrome in children, adolescents, and young adults – an update for 2020.Pediatr. Nephrol. 2021; 36: 711-719Crossref PubMed Scopus (14) Google Scholar,123.Kashtan C.E. Gross O. Correction to: Clinical practice recommendations for the diagnosis and management of Alport syndrome in children, adolescents, and young adults-an update for 2020.Pediatr. Nephrol. 2021; 36: 731Crossref PubMed Scopus (2) Google Scholar]RG-125/AZD4076dPhase I completed.miR-103/107Anti-miRNonalcoholic steatohepatitis (NASH) in patients with type 2 diabetes/prediabetesSubcutaneous injection/biomolecule conjugation (GalNAc)NCT02612662, NCT02826525[76.Kelnar K. et al.Quantification of therapeutic miRNA mimics in whole blood from nonhuman primates.Anal. Chem. 2014; 86: 1534-1542Crossref PubMed Scopus (49) Google Scholar, 77.Drenth J.P.H. Schattenberg J.M. The nonalcoholic steatohepatitis (NASH) drug development graveyard: Established hurdles and planning for future success.Expert Opin. Investig. Drugs. 2020; 29: 1365-1375Crossref PubMed Scopus (16) Google Scholar, 78.Huang Y. Preclinical and clinical advances of GalNAc-decorated nucleic acid therapeutics.Mol. Ther. Nucleic Acids. 2017; 6: 116-132Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar]MRG-110dPhase I completed.miR-92aAnti-miRWoundsSkin injection/chemical modification (LNA)NCT03603431[124.Gallant-Behm C.L. et al.A synthetic microRNA-92a inhibitor (MRG-110) accelerates angiogenesis and wound healing in diabetic and nondiabetic wounds.Wound Repair Regen. 2018; 26: 311-323Crossref PubMed Scopus (46) Google Scholar,125.Abplanalp W.T. et al.Efficiency and target derepression of anti-miR-92a: Results of a first in human study.Nucleic Acid Ther. 2020; 30: 335-345Crossref PubMed Scopus (32) Google Scholar]MesomiR 1dPhase I completed.miR-16miRNA mimicMalignant pleural mesothelioma, non–small cell lung cancerIntravenously/vehicle transfer (nonliving minicells)NCT02369198[56.Reid G. et al.Clinical development of TargomiRs, a miRNA mimic-based treatment for patients with recurrent thoracic cancer.Epigenomics. 2016; 8: 1079-1085Crossref PubMed Scopus (120) Google Scholar,57.van Zandwijk N. et al.Safety and activity of microRNA-loaded minicells in patients with recurrent malignant pleural mesothelioma: A first-in-man, phase 1, open-label, dose-escalation study.Lancet Oncol. 2017; 18: 1386-1396Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar,126.Reid G. et al.Restoring expression of miR-16: a novel approach to therapy for malignant pleural mesothelioma.Ann. Oncol. 2013; 24: 3128-3135Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar]CDR132LdPhase I completed.miR-132Anti-miRHeart failureIntravenously/chemical modification (LNA)NCT04045405[127.Taubel J. et al.Novel antisense therapy targeting microRNA-132 in patients with heart failure: Results of a first-in-human phase 1b randomized, double-blind, placebo-controlled study.Eur. Heart J. 2021; 42: 178-188Crossref PubMed Scopus (47) Google Scholar,128.Batkai S. et al.CDR132L improves systolic and diastolic function in a large animal model of chronic heart failure.Eur. Heart J. 2021; 42: 192-201Crossref PubMed Scopus (22) Google Scholar]Remlarsen/MRG-201ePhase II completed.miR-29miRNA mimicKeloid disorderSkin injection/biomolecule conjugation (cholesterol)NCT02603224, NCT03601052[73.Gallant-Behm C.L. et al.A microRNA-29 mimic (remlarsen) represses extracellular matrix expression and fibroplasia in the skin.J. Invest. Dermatol. 2019; 139: 1073-1081Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar,103.Olena A.F. Patton J.G. Genomic organization of microRNAs.J. Cell. Physiol. 2010; 222: 540-545PubMed Google Scholar,104.Ha M. Kim V.N. Regulation of microRNA biogenesis.Nat. Rev. Mol. Cell Biol. 2014; 15: 509-524Crossref PubMed Scopus (3208) Google Scholar]Miravirsen/SPC3649ePhase II completed., fUnknown status.miR-122Anti-miRChronic hepatitis C virusSubcutaneous injection/chemical modification (LNA)NCT02508090, NCT02452814, NCT01200420, NCT01872936, NCT01727934, NCT01646489[16.Ottosen S. et al.In vitro antiviral activity and preclinical and clinical resistance profile of miravirsen, a novel anti-hepatitis C virus therapeutic targeting the human factor miR-122.Antimicrob. Agents Chemother. 2015; 59: 599-608Crossref PubMed Scopus (130) Google Scholar,129.Gebert L.F. et al.Miravirsen (SPC3649) can inhibit the biogenesis of miR-122.Nucleic Acids Res. 2014; 42: 609-621Crossref PubMed Scopus (228) Google Scholar, 130.Elmen J. et al.LNA-mediated microRNA silencing in non-human primates.Nature. 2008; 452: 896-899Crossref PubMed Scopus (1381) Google Scholar, 131.Lanford R.E. et al.Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection.Science. 2010; 327: 198-201Crossref PubMed Scopus (1427) Google Scholar, 132.Janssen H.L. et al.Treatment of HCV infection by targeting microRNA.N. Engl. J. Med. 2013; 368: 1685-1694Crossref PubMed Scopus (1644) Google Scholar]MRX34gStopped/terminated.miR-34amiRNA mimicSolid tumors (e.g., hepatocellular carcinoma), melanomaIntravenously/vehicle transfer (liposomal)NCT01829971, NCT02862145[29.Beg M.S. et al.Phase I study of MRX34, a liposomal miR-34a mimic, administered twice weekly in patients with advanced solid tumors.Investig. New Drugs. 2017; 35: 180-188Crossref PubMed Scopus (468) Google Scholar,32.Daige C.L. et al.Systemic delivery of a miR34a mimic as a potential therapeutic for liver cancer.Mol. Cancer Ther. 2014; 13: 2352-2360Crossref PubMed Scopus (112) Google Scholar,133.Huang H.Y. et al.miRTarBase 2020: Updates to the experimentally validated microRNA-target interaction database.Nucleic Acids Res. 2020; 48: D148-D154PubMed Google Scholar]RG-101gStopped/terminated.miR-122Anti-miRChronic hepatitis C virusSubcutaneous injection/biomolecule conjugation (GalNAc)EudraCT numbers 2015-001535-21, 2015-004702-42, 2016-002069-77[76.Kelnar K. et al.Quantification of therapeutic miRNA mimics in whole blood from nonhuman primates.Anal. Chem. 2014; 86: 1534-1542Crossref PubMed Scopus (49) Google Scholar,79.van der Ree M.H. et al.Safety, tolerability, and antiviral effect of RG-101 in patients with chronic hepatitis C: A phase 1B, double-blind, randomised controlled trial.Lancet. 2017; 389: 709-717Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar,80.Stelma F. et al.Immune phenotype and function of natural killer and T cells in chronic hepatitis C patients who received a single dose of anti-microRNA-122, RG-101.Hepatology. 2017; 66: 57-68Crossref PubMed Scopus (23) Google Scholar]Cobomarsen/MRG-106gStopped/terminated.miR-155Anti-miRMycosis fungoidesIntravenously/chemical modification (LNA)NCT02580552, NCT03713320, NCT03837457[134.Seto A.G. et al.Cobomarsen, an oligonucleotide inhibitor of miR-155, co-ordinately regulates multiple survival pathways to reduce cellular proliferation and survival in cutaneous T-cell lymphoma.Br. J. Haematol. 2018; 183: 428-444Crossref PubMed Scopus (126) Google Scholar, 135.James A.M. et al.SOLAR: A phase 2, global, randomized, active comparator study to investigate the efficacy and safety of cobomarsen in subjects with mycosis fungoides (MF) [abstract].Hematol. Oncol. 2019; 37: 562-563Crossref Google Scholar, 136.Querfeld C. et al.Preliminary results of a phase 1 trial evaluating MRG-106, a synthetic microRNA antagonist (LNA antimiR) of microRNA-155, in patients with CTCL.Blood. 2016; 128: 1829Crossref PubMed Google Scholar]a NCT numbered trials are registered at ClinicalTrials.gov; EudraCT numbered trials are registered at EU Clinical Trials Register (clinicaltrialsregister.eu).b Phase I ongoing.c Phase II ongoing.d Phase I completed.e Phase II completed.f Unknown status.g Stopped/terminated. Open table in a new tab How to modify cellular miRNA expression?The general aim of miRNA therapeutics is to modify and ideally reverse pathological miRNA expression changes. This includes the enhancement or reconstitution of endogenous miRNAs that act as pathological suppressors and the expressional reduction or functional blocking of miRNAs that act as pathological drivers. To modify miRNA levels, nucleic acids are commonly used (Figure 1), including synthetic miRNAs (miRNA mimics), recombinant expression vectors carrying miRNA encoding sequences, and oligonucleotide-based miRNA inhibitors (anti-miRs) [8.van Rooij E. Kauppinen S. Development of microRNA therapeutics is coming of age.EMBO Mol. Med. 2014; 6: 851-864Crossref PubMed Scopus (433) Google Scholar].One of the currently pursued advanced approaches makes use of small cell permeable molecules. These small molecules exert their function by, for example, the interaction with proteins involved in the process of miRNA biogenesis or via binding to miRNA-specific secondary structures [9.Fan R. et al.Small molecules with big roles in microRNA chemical biology and microRNA-targeted therapeutics.RNA Biol. 2019; 16: 707-718Crossref PubMed Scopus (22) Google Scholar]. Small molecules are designed with the aid of bioinformatics tools or are identified through experimental screening of pharmacologically active chemical compounds [10.Disney M.D. et al.Inforna 2.0: A platform for the sequence-based design of small molecules targeting structured RNAs.ACS Chem. Biol. 2016; 11: 1720-1728Crossref PubMed Scopus (95) Google Scholar,11.Suresh B.M. et al.A general fragment-based approach to identify and optimize bioactive ligands targeting RNA.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 33197-33203Crossref PubMed Scopus (0) Google Scholar]. A recent example is the identification of an inhibitor for the oncogenic miR-21. This inhibitor was identified by a target-oriented screening of various low-molecular-weight chemical compounds [11.Suresh B.M. et al.A general fragment-based approach to identify and optimize bioactive ligands targeting RNA.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 33197-33203Crossref PubMed Scopus (0) Google Scholar]. Natural compounds are also a rich source for miRNA interfering molecules [12.Alnuqaydan A.M. Targeting micro-RNAs by natural products: A novel future therapeutic strategy to combat cancer.Am. J. Transl. Res. 2020; 12: 3531-3556PubMed Google Scholar]. Curcumin has, for example, been shown to act on multiple miRNAs to inhibit breast cancer cell growth [13.Norouzi S. et al.Curcumin as an adjunct therapy and microRNA modulator in breast cancer.Curr. Pharm. Des. 2018; 24: 171-177Crossref PubMed Scopus (35) Google Scholar].A further strategy toward the development of miRNA therapeutics is to combine miRNA-based approaches together with treatments by conventional drugs. The efficiency of drug-based therapies can particularly be improved by miRNA-based interventions that target cellular pathways, which affect therapeutic outcomes [14.Seo H.A. et al.MicroRNA-based combinatorial cancer therapy: Effects of microRNAs on the efficacy of anti-cancer therapies.Cells. 2019; 9: 29Crossref Google Scholar]. Liver-specific miR-122 is considered as a driver of hepatitis C virus (HCV) infection and maintenance in hepatocytes [15.Panigrahi M. et al.miR-122 affects both the initiation and maintenance of hepatitis C virus infections.J. Virol. 2021; 96e0190321PubMed Google Scholar]. In a Phase II clinical trial (ClinicalTrials.gov identifiers NCT01200420, NCT01872936), resistance against HCV treatment has been counteracted by combining conventional viral protein inhibitor drugs with the miR-122 inhibitor miravirsen/SPC3649 [16.Ottosen S. et al.In vitro antiviral activity and preclinical and clinical resistance profile of miravirsen, a novel anti-hepatitis C virus therapeutic targeting the human factor miR-122.Antimicrob. Agents Chemother. 2015; 59: 599-608Crossref PubMed Scopus (130) Google Scholar]. Combined application schemas of chemotherapeutics and miRNA manipulators are especially being developed for the improvement of antitumor therapies, including therapies of common cancers such as breast cancer [17.Gong C. et al.Functional exosome-mediated co-delivery of doxorubicin and hydrophobically modified microRNA 159 for triple-negative breast cancer therapy.J. Nanobiotechnol. 2019; 17: 93Crossref PubMed Scopus (93) Google Scholar,18.Saatci O. et al.Targeting lysyl oxidase (LOX) overcomes chemotherapy resistance in triple negative breast cancer.Nat. Commun. 2020; 11: 2416Crossref PubMed Scopus (62) Google Scholar].The combined use of miRNAs with siRNAs offers another route to improve the efficiency of therapeutic miRNAs. siRNAs constitute a group of small RNAs conceived for the specific regulation of a single or few target genes [19.Lam J.K. et al.siRNA versus miRNA as therapeutics for gene silencing.Mol. Ther. Nucleic Acids. 2015; 4e252Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar]. The establishment of siRNA drugs is in general more advanced than miRNA-based drugs [20.Zhang S. et al.The risks of miRNA therapeutics: In a drug target perspective.Drug Des. Devel. Ther. 2021; 15: 721-733Crossref PubMed Scopus (40) Google Scholar]. A combination of miRNAs and siRNAs can be achieved by coexpression in a recombinant plasmid, as recently shown for human lung cancer cells [21.Petrek H. et al.Bioengineering of a single long noncoding RNA molecule that carries multiple small RNAs.Appl. Microbiol. Biotechnol. 2019; 103: 6107-6117Crossref PubMed Scopus (13) Google Scholar].Artificially designed miRNA constructs, referred to as 'amiRNAs,' promise further advancement toward therapeutic miRNAs. amiRNAs are combinations of siRNA sequences and scaffolds of primary miRNA transcripts. While amiRNAs show high target specificity, because of their siRNA-based design, their cellular processing is ensured by their endogenous miRNA-based structure [22.Kotowska-Zimmer A. et al.Artificial miRNAs as therapeutic tools: Challenges and opportunities.Wiley Interdiscip. Rev. RNA. 2021; 12e1640Crossref PubMed Scopus (3) Google Scholar]. An amiRNA-based drug (AMT-130) that includes a siRNA sequence against the Huntingtin gene together with a pri-miR-451 scaffold is currently being employed in a clinical trial on Huntington's disease (ClinicalTrials.gov identifier NCT04120493) [23.Keskin S. et al.AAV5-miHTT lowers huntingtin mRNA and protein without off-target effects in patient-derived neuronal cultures and astrocytes.Mol. Ther. Methods Clin. Dev. 2019; 15: 275-284Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 24.Miniarikova J. et al.Design, characterization, and lead selection of therapeutic miRNAs targeting huntingtin for development of gene therapy for Huntington's disease.Mol. Ther. Nucleic Acids. 2016; 5e297Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 25.Samaranch L. et al.MR-guided parenchymal delivery of adeno-associated viral vector serotype 5 in non-human primate brain.Gene Ther. 2017; 24: 253-261Crossref PubMed Scopus (41) Google Scholar].miRNA sponges offer an option to manipulate cellular levels of miRNAs. These are RNA constructs harboring multiple miRNA binding sites. miRNA sponges exert their function through sequestration of endogenous miRNAs. The effectiveness of miRNA sponges, including circular RNAs, has been analyzed in several studies. The expression of an artificially designed circular RNA sponge, including six alternating binding sites for the inhibition of miR-132 and miR-212, has been tested, for example, on mouse models for the treatment of cardiovascular diseases [26.Lavenniah A. et al.Engineered circular RNA sponges act as miRNA inhibitors to attenuate pressure overload-induced cardiac hypertrophy.Mol. Ther. 2020; 28: 1506-1517Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar]. Additional studies highlight the potential of naturally occurring transcripts as a source for therapeutically usable miRNA sponges. Recently, the circular RNA hsa_circ_0120472, which includes two predicted miRNA binding sites, has been shown to act as an efficient sponge to inhibit miR-550a in human breast cancer cells [27.Meng L. et al.Circular RNA circCCDC85A inhibits breast cancer progression via acting as a miR-550a-5p sponge to enhance MOB1A expression.Breast Cancer Res. 2022; 24: 1Crossref PubMed Scopus (2) Google Scholar]. The yet increasing number of new strategies that are currently being pursued to modify and reverse pathological miRNA expression changes will certainly promote the development of therapeutic approaches.What are severe side effects of miRNA therapeutics?Depending on the chosen route of administration and the way to warrant an intracellular delivery, the effects of miRNA therapeutics are not necessarily restricted to the intended tissue or cells but can also cause systemic side effects. A prominent example of the occurrence of disastrous side effects is MRX34, a synthetic miR-34a mimic. A clinical study with MRX34 for tumor treatment (ClinicalTrials.gov identifier NCT01829971), including various solid tumors and hematologic malignancies, had to be terminated prematurely because of severe immune-related side effects causing the death of four patients [28.Hong D.S. et al.Phase 1 study of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumours.Br. J. Cancer. 2020; 122: 1630-1637Crossref PubMed Scopus (183) Google Scholar,29.Beg M.S. et al.Phase I study of MRX34, a liposomal miR-34a mimic, administered twice weekly in patients with advanced solid tumors.Investig. New Drugs. 2017; 35: 180-188Crossref PubMed Scopus (468) Google Scholar]. The trial was designed to make use of the properties of miR-34a as a powerful tumor suppressor [30.Saito Y. et al.microRNA-34a as a therapeutic agent against human cancer.J. Clin. Med. 2015; 4: 1951-1959Crossref PubMed Google Scholar]. The miR-34a mimic was systemically administered by a liposomal amphoteric (i.e., pH-dependent) delivery strategy, which was supposed to take effect specifically in the low-pH environment of tumorous tissues [31.Bouchie A. First microRNA mimic enters clinic.Nat. Biotechnol. 2013; 31: 577Crossref PubMed Google Scholar]. Animal models, however, showed an miR-34a mimic uptake not only in tumorous tissues but also in bone marrow and spleen [32.Daige C.L. et al.Systemic delivery of a miR34a mimic as a potential therapeutic for liver cancer.Mol. Cancer Ther. 2014; 13: 2352-2360Crossref PubMed Scopus (112) Google Scholar,33.Kelnar K. Bader A.G. A qRT-PCR method for determining the biodistribution profile of a miR-34a mimic.Methods Mol. Biol. 2015; 1317: 125-133Crossref PubMed Scopus (11) Google Scholar], both of which are known to be involved in the generation and preservation of immune cells. Accordingly, in context with the clinical testing, a dose-dependent modulation of several target genes was observed in white blood cells [28.Hong D.S. et al.Phase 1 study of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumours.Br. J. Cancer. 2020; 122: 1630-1637Crossref PubMed Scopus (183) Google Scholar]. It is now evident that miR-34a not only functions as a tumor suppressor but also impacts the signaling of immune cells, for example, by regulating calcium or chemokine signaling [3