Noncoding RNA Therapeutics — Challenges and Potential Solutions
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PERSPECTIVES siRNAs or ASOs that influence only a single target gene. Noncoding RNA therapeutics — The diverse functional repertoire of lncRNAs reveals various opportunities for challenges and potential solutions their therapeutic targeting, the means of which need to be adjusted to the mode of Melanie Winkle, Sherien M. El-Daly, Muller Fabbri and George A. Calin action of the lncRNA. LncRNA targeting may include transcriptional inhibition, Abstract | Therapeutic targeting of noncoding RNAs (ncRNAs), such as microRNAs post-transcriptional inhibition, steric (miRNAs) and long noncoding RNAs (lncRNAs), represents an attractive approach hindrance of secondary structure formation for the treatment of cancers, as well as many other diseases. Over the past decade, or protein interactions, introduction of substantial effort has been made towards the clinical application of RNA-based synthetic (for example, circular) lncRNAs, and modulation of lncRNA genomic loci or therapeutics, employing mostly antisense oligonucleotides and small interfering expression patterns via CRISPR–Cas9 and RNAs, with several gaining FDA approval. However, trial results have so far been CRISPR–Cas13, respectively (reviewed in ambivalent, with some studies reporting potent effects whereas others REF.17). An interesting development is the demonstrated limited efficacy or toxicity. Alternative entities such as antimiRNAs exploration of natural antisense transcripts are undergoing clinical testing, and lncRNA-based therapeutics are gaining (NATs): lncRNAs that are transcribed in interest. In this Perspective, we discuss key challenges facing ncRNA therapeutics the antisense direction to coding genes, and negatively regulate them in cis. ASOs — including issues associated with specificity, delivery and tolerability — and focus that target NATs, termed ‘antagoNATs’ on promising emerging approaches that aim to boost their success. have shown very promising preclinical results for gene reactivation in the central Noncoding RNAs (ncRNAs) are generated (TABle 1), aiming at gene modifications nervous system. AntagoNATs successfully from the larger part of the genome that in liver, muscle or the central nervous upregulated brain-derived neurotrophic does not encode proteins but produces system. All these therapeutics are either factor (BDNF), a protein highly involved in noncoding transcripts that regulate gene siRNAs or ASOs that cause specific gene memory formation18, as well as the healthy expression and protein function. The two downregulation, or ASOs that target allele of SCN1A, the haploinsufficiency major classes of ncRNA are the well-studied pre-mRNA splicing (that is, inducing of which causes the brain disorder Dravet short microRNAs (miRNAs) and the exon skipping or inclusion). In addition, syndrome19. Notably, BDNF-AS-targeting more recently identified long ncRNAs a plethora of RNA therapeutics are in antagoNATs were successfully delivered (lncRNAs) (BOx 1). Deregulation of both phase II or III clinical development, including across the blood–brain barrier using a types of transcript has been linked to every newer entities such as miRNA mimics and minimally invasive nasaldepot (MIND) in a cancer investigated to date and affects all antimiRs (TABle 2), but no lncRNA-based mouse model. MIND is aimed at directing major cancer hallmarks1–5. In addition, they therapeutics have entered the clinic. drug delivery to the olfactory submucosal have been linked to complex biological The use of miRNA-based therapeutics space and achieved approximately 40% processes such as immune cell development has dual advantages10,13,14. First, miRNAs are efficacy compared with riskier invasive and function, immune disorders6, neural naturally occurring molecules in human delivery techniques20. Such promising development and neurological diseases7–9. cells, unlike man-made chemotherapy developments suggest that the entrance of Therapeutic targeting of such naturally compounds or ASOs, and therefore have lncRNA-based therapeutics into clinical occurring ncRNAs thus represents a very all the mechanisms in place for their testing is imminent. promising approach for the treatment of processing and downstream target selection The translation of all RNA-based various diseases. (Fig. 1). Second, miRNAs act by targeting therapeutics into the clinic has been Various RNA-based therapies, including multiple genes within one pathway, thus hampered by issues associated with specificity, antisense oligonucleotides (ASOs), small causing a broader yet specific response. delivery and tolerability. Specificity issues interfering RNAs (siRNAs), short hairpin The miR-15–miR-16 cluster, downregulating include undesired on-target effects due RNAs (shRNAs), ASO anti-microRNAs multiple anti-apoptotic factors including to uptake in cells other than the cells of (antimiRs), miRNA mimics, miRNA BCL-2 and MCL1, represents an excellent interest, or off-target effects caused by sponges, therapeutic circular RNAs example of a miRNA acting at multiple either sequence similarities or overdosing (circRNAs) and CRISPR–Cas9-based levels to affect the same cancer hallmark15,16. to levels much higher than expected gene editing have been developed and The use or targeting of naturally occurring endogenously. Delivery issues are related to several excellent reviews describe these miRNAs could therefore represent a three major points: the instability of ‘naked’, agents10–12 (BOx 2). Currently, 11 RNA-based promising alternative to existing RNA-based chemically unmodified RNA structures; therapeutics are approved by the FDA and/or therapies and may potentially boost their inefficient intracellular delivery, which the European Medicines Agency (EMA) therapeutic effects compared with synthetic requires the exploitation of endosomal NATURE REVIEWS | DRUG DISCOVERY VOLUME 20 | AUGUST 2021 | 629 0123456789();: PERSPECTIVES Box 1 | Main characteristics of microRNAs and long noncoding RNAs siRNA was first shown not to trigger an interferon response in animals30, intraocular MicroRNAs (miRNAs) are highly conserved, small, 17- to 25-nucleotide (nt), single-stranded injections of anti-vascular endothelial ncRNAs that act as gene regulators. Their biogenesis is a multistep process (see Fig. 1) involving, growth factor receptor 1 (VEGFR1) siRNAs first, the production of long primary miRNA transcripts (pri-miRNA) by RNA polymerases II and III; bevasiranib (NCT00499590) and AGN second, the processing of pri-miRNAs by the nuclear ribonuclease Drosha and DGCR8 into a 70 nt long precursor miRNA (pre-miRNA) with a stem–loop structure; third, the nuclear export of 211745 (NCT00395057) were later shown pre-miRNAs by exportin 5 and Ran-GTPase; and fourth, the cleavage of pre-miRNAs by RNase III to owe their anti-angiogenic effects to direct enzyme Dicer to yield a mature, double-stranded miRNA. Gene regulation is mediated through the TLR3 stimulation instead of to the desired unwinding of the miRNA by RNA helicase and the incorporation of the miRNA guide strand into silencing effect31, resulting in termination the RNA-induced silencing complex (RISC)274–277. Post-transcriptional gene silencing is mediated of clinical development (TABle 3). The via nucleotide complementarity to mainly the 3′ UTR, or less prevalently the 5′ UTR or coding predominant pathway recognizing RNA region, of target mRNAs278,279. The miRNA ‘seed sequence’ is situated from nucleotides 2 to 7 at therapeutics is TLR signalling, which is the 5′-end of the miRNA sequence. Seed sequence binding with perfect complementarity results mediated through myeloid differentiation in the deadenylation and degradation of the targeted mRNA, whereas binding with imperfect factor 88 (MyD88) and activates various complementarity, which is more common, results in translational inhibition, both of which are pathways, resulting in nuclear factor-κB facilitated by RISC. (NF-κB) activation and the production of Long noncoding RNAs (lncRNAs) are larger transcripts (>200 nt in size) that are synthesized similarly to mRNAs, but not translated into protein280. LncRNAs contain two types of functional pro-inflammatory cytokines (IL-6, IL-8, element, the interactor elements involved in direct physical interaction with other nucleic acids, IL-12 and TNF), or a type I interferon with proteins or lipids, and the structural elements, leading to the occurrence of secondary response, which ultimately leads to the and/or tertiary 3D RNA structures, which direct their functional interactions281. It is this capacity activation of diverse downstream immune to interact with DNA and RNA as well as proteins via base pairing in linear form or chemical responses32. TLR7 and TLR8 stimulation by interactions in secondary structures, that allows lncRNAs to function in more variable ways than miRNA was shown to be dependent on the miRNAs. Gene-regulatory roles have been identified for many lncRNAs, for example, by influencing presence of GU-rich sequences (for example, transcription factor binding or epigenetic marks. Interactions with mRNAs may influence their 5′-UGUGU-3′ or 5′-GUCCUUCAA-3′) stability or rate of translation. Similarly, lncRNA–protein interactions may influence the stability, and caused a IFNα-mediated inflammatory activity or localization of the protein282,283. Furthermore, circular RNAs, which have a similar length range to lncRNAs, have become known for their potent roles as miRNA sponges179,284. response, activation of NF-κB and production of inflammatory cytokines (for example, IL-6 and TNF)