Amplifying gene expression with RNA-targeted therapeutics

RNA-targeted NBTs that are currently being used to upregulate gene expression address both transcriptional and translational mechanisms and can be roughly divided into two groups: (1) NBTs that increase mRNA abundance by enhancing transcription or increasing mRNA stability (Fig. 3) and (2) NBTs that optimize translation (Fig. 4). Strategies in the first group include therapeutic mRNA delivery, NBTs to regulate transcription via modulation of NAT, pRNA, seRNA and eRNA activity, NBTs to regulate RNA stability via modulation of miRNAs and NATs, as well as splice-switching applications that prevent nonsense-mediated decay (NMD) of mRNAs. Strategies in the second group include NBTs modulating uORF translation and activity of other mRNA structural elements and of ncRNAs that regulate translation initiation and elongation.

Fig. 3: NBTs that increase mRNA abundance.
figure 3

a, Therapeutic mRNA transcribed in vitro and encapsulated in lipid nanoparticles (LNPs) is introduced into cells and initiates translation. Targeting molecules (TMs) that bind specific cell surface receptors can be added for cell type-specific delivery. b, Small activating RNAs (saRNAs) are double-stranded synthetic RNAs, formed by guide (red) and passenger (yellow) strands encapsulated in LNPs (known as SMARTICLEs), which are delivered to cells. saRNAs are initially recognized by double-stranded RNA loading factors, followed by argonaute 2 (AGO2) protein binding. The passenger strand of the saRNA is discarded, and a complex consisting of the guide saRNA strand, AGO2 and heterogeneous nuclear ribonucleoproteins (hnRNPs) is imported into the nucleus, where it binds directly to DNA and participates in the RNA-induced transcriptional activation (RITA) complex. RITA interacts with RNA polymerase II (RNA Pol II) to initiate transcription. c, Enhancer RNA (eRNAs), bidirectionally transcribed from an enhancer region, interact with proteins such as BRD4, CREB-binding protein (CBP) or NELF to maintain chromatin in an active state and initiate RNA Pol II pause release. Transcriptional activation of some proteins involves interaction of eRNAs and enhancer-associated natural antisense transcripts (NATs) with the Mediator complex and cohesin. Nucleic acid-based therapeutics (NBTs) mimicking eRNAs or inducing their expression can facilitate transcriptional regulation by blocking eRNA interaction with inhibitory factors. d, NAT scaffold transcriptional repressors at target gene loci. NBT treatment blocks NAT interaction with repressors and/or chromosomes and accelerates transcription. e, NBTs targeting NATs involved in silencing imprinted alleles can derepress transcription of target mRNA. f, NBTs that interact with splice factors that promote or inhibit splicing, or with their binding sites on pre-mRNA, can modulate splice factor binding and/or activity. g, NBTs designed to bind to natural miRNAs (antagomirs) serve as decoys to prevent microRNA (miRNA) binding to mRNAs. Alternatively, NBTs can be designed to function as miRNA mimics (promirs). ASO, antisense oligonucleotide; lncRNA, long non-coding RNA; pRNA, promoter RNA.

Fig. 4: NBTs and small molecules targeting mRNA translation.
figure 4

a, Upstream open reading frame (uORF)-targeting nucleic acid-based therapeutics (NBTs) increase protein expression through blocking uORF translation and thus redirecting the ribosome to the main start site. b, Other regulatory secondary structures in untranslated regions (UTRs) can be targeted. NBTs targeting stem-loop and other secondary structures within 5′ UTRs that inhibit translation can increase protein expression by hybridizing them and releasing inhibitory factors (IFs) that recognize them or by removing obstacles for binding or movement of ribosomes and translation-enhancing factors. c, NBTs targeting polyadenylation (polyA) signals can preferentially prevent expression of mutated or toxic isoforms and redirect expression towards beneficial isoform ratios. d, Small molecules can enable premature termination codon (PTC) readthrough. Upon encountering a PTC, ribosomes recruit proteins such as eukaryotic translation termination factor 1 (eRF1) and eRF3, facilitating premature termination of translation. Small molecules interact with ribosomes to enable recruitment of near-cognate tRNAs and facilitate readthrough, allowing translation of full-length protein. RNA Pol II, RNA polymerase II; TSS, translation start site.

Although the field of NBT-mediated protein upregulation is young, in the last 5–7 years, it has undergone explosive growth and some of these strategies have been approved (Supplementary Table 2), while multiple other NBTs are in clinical trials (Table 1). Examples of these strategies are described below, starting with more advanced clinical cases and moving towards prospective future NBT targets.

Table 1 Selected RNA-targeted drugs in development for gene expression amplification

Clinical-stage NBTs

Several protein-upregulating NBTs have already reached the clinical stage, including both approved drugs and drug candidates in clinical trials. mRNA delivery is perhaps the most clinically advanced modality if COVID-19 vaccines, which induce viral protein synthesis in human cells, are considered. However, development of therapeutic mRNA for the treatment of diseases associated with protein insufficiency is progressing slowly. Several splice-modulating NBTs have been approved for use in genetic diseases and constitute a more clinically mature (in terms of the number of patients treated) and more reversible alternative to gene therapy. Splice-switching NBTs that omit a ‘toxic’ exon, as well as NBTs and small molecules that enhance ribosomal readthrough and other aspects of translation, may be widely used to augment protein production in the absence of insufficiency-causing mutations. Although such NBTs have not yet been approved, some small-molecule readthrough drugs are already on the market.

Splice-modulating NBTs

Aberrant RNA splicing caused by mutations frequently leads to non-functional transcripts that are quickly destroyed by NMD, leading to the shortage of the affected protein, and is known to underlie many diseases. Furthermore, normal alternative splicing of pre-mRNA can lead to inclusion of so-called ‘toxic exons’, resulting in transcripts that are quickly degraded via NMD, thus reducing protein levels. ASOs binding to specific sequences on pre-mRNA that regulate splicing events can prevent the generation of mutated or naturally non-productive transcripts and subsequently increase target protein levels42 (Fig. 3). Given that a significant number of protein-coding genes contain toxic exons, and that many of the known disease-causing mutations can be rescued through skipping mutated exons, this strategy can have wide applicability. Indeed, therapeutic splice-modifying oligonucleotides have received a lot of attention in recent years, with some significant clinical successes (Supplementary Table 2).

Nusinersen, perhaps the most well-known splice-switching ASO, was approved by the FDA in 2016 to treat spinal muscular atrophy (SMA) and has resulted in monumental improvements in duration and quality of life for patients43. SMA is caused by insufficient levels of SMN protein due to loss-of-function mutations in the SMN1 gene. A duplicate gene (SMN2) produces no protein due to aberrant splicing out of exon 7. Nusinersen inhibits skipping of exon 7 in SMN2 by binding to the intronic splicing silencer N1 (ISS-N1) and thus increases production of corrected SMN2 mRNA and SMN protein.

As the pharmacokinetic and pharmacodynamic characteristics of ASO drugs are similar, results obtained with nusinersen can help evaluate future prospects of the protein-upregulating NBT field in general. Overall, the long-term efficacy and safety data for nusinersen are positive44,45,46, although the intrathecal route of administration may be associated with adverse events47,48 and has potentially reduced treatment adherence, highlighting the need to develop non-invasive methods for NBT delivery49.

Notably, both gene therapy and oligonucleotide therapy options are available in SMA. Clinical reports on combined and consecutive treatments with the two modalities may help in the evaluation of the potential of NBTs to compete with gene therapy treatments that have become more widely accepted in recent years. A related question is whether combining NBTs that address independent biological mechanisms leading to upregulation of a target protein would improve patient outcomes. This question might be addressed through clinical experience with nusinersen and gene therapy as these approaches have different gene targets (SMN2 vs SMN1) and could potentially exhibit additive effects. Furthermore, combined treatment with a splice-switching ASO and a small-molecule splicing modifier, RG7800, had an additive effect on SMN protein production, resulting in improved motor unit function in ∆7SMA mice even when given late in development50.

The FDA-approved splice-switching ASOs eteplirsen, golodirsen, viltolarsen and casimersen are intended to treat Duchenne muscular dystrophy (DMD) associated with exon 51, 53 or 45 mutations (Supplementary Table 2). Skipping these exons does not alter dystrophin function, although it possibly leads to partial sequestration and extended half-life of the truncated protein51. Following a rocky start, NBTs targeted at the upregulation of dystrophin in DMD are now showing great promise.

Although, initially, the efficacy of eteplirsen was questioned, evaluation at 6 years post-treatment demonstrated longer median time to loss of ambulation (+2.09 years; P < 0.01) and attenuated rates of pulmonary decline (P < 0.0001) compared to standard-of-care controls52. The biology of the disease, mechanism of action of the particular NBT modality used and the pharmacokinetic properties of NBTs might have all contributed to the extended time needed to observe the clinical effects. These developments also highlight the poor applicability of clinical trial paradigms developed for small-molecule drugs to NBT modalities due to their unusual pharmacokinetic properties and novel mechanisms of action. Importantly, treatment with a next-generation peptide-conjugated phosphorodiamidate morpholino oligomer vesleteplirsen (SRP-5051) led to 18-fold more exon skipping and eightfold higher dystrophin levels compared to eteplirsen in a clinical trial (NCT04004065). The trial was temporarily placed on hold due to observed hypomagnesaemia but later resumed with expanded monitoring of urine biomarkers and magnesium supplementation.

Golodirsen is an NBT designed to induce exon 53 skipping in DMD. Notably, positive clinical results have also been reported for golodirsen after 3-year follow-up., wherein loss of ambulation occurred in 9% of patients treated with golodirsen versus 26% in controls53.

Another treatment option in exon 53-related DMD is a splice-switching phosphorodiamidate morpholino oligomer, viltolarsen. Preliminary trial results after 2-year treatment with viltolarsen demonstrated preservation of motor function, while the DMD natural history controls showed significant functional decline54,55.

The exon 45-skipping ASO casimersen was approved in 2021 with a warning of kidney side effects. However, data from an extension study showed good tolerability in 12 patients followed for a mean period of 139.6 weeks56.

Among the promising novel chemistry investigational treatments for DMD is WVE-N531, a stereopure phosphoramidate splice-modulating oligonucleotide administered systemically, which is in a clinical trial in 15 patients with DMD amenable to exon 53 skipping (NCT04906460). Potential benefits of the stereopure oligonucleotides may involve higher potency. As chemical modifications of oligonucleotides used to increase their nuclease resistance introduce a chiral centre, the standard process leads to the synthesis of a large number of stereoisomers that have variable affinity to the target RNA sequence9,10. An approach developed by Wave Therapeutics leads to selective synthesis of one stereoisomer, thus potentially increasing the concentration of the active ingredient in the oligonucleotide product. Interim results from the initial cohort of three boys evaluated after dose escalation followed for 6 weeks of biweekly injections of WVE-N531 demonstrated high muscle concentration of the drug and 53% mean exon skipping. Pharmacokinetic data demonstrated a half-life of 25 days, which may support monthly dosing. Although mean dystrophin expression was below the limit of quantification, the company expects protein production to lag behind the increase in RNA synthesis. The initial clinical results for WVE-N531 indicate possible pharmacological improvement due to phosphoryl guanidine compared to first-generation DMD splice-switching NBTs and knockdown NBTs in Huntington disease programmes.

Several companies are conducting clinical trials for gene therapy treatments in DMD, but no data comparing their efficacy with exon-skipping oligonucleotides is yet available.

A combination strategy that might mitigate the disadvantages of exon-skipping ASOs and gene therapy approaches in the treatment of DMD with exon 2 duplications is under investigation. ASOs require repeated administration and are not readily taken up by muscle — the target tissue in DMD — while the limited viral vector packaging capacity requires the use of a significantly shortened dystrophin sequence. scAAV9.U7.ACCA, now in a clinical trial (NCT04240314), employs two innovations. It contains four copies of U7 small nuclear RNA, normally involved in 3′-end processing of histone pre-mRNAs. These U7 small nuclear RNAs were modified to replace histone-binding segments with sequences targeting the splice donor (two copies) and splice acceptor (two copies) sites of dystrophin exon 2, which leads to enhanced splicing out of exon 2 and upregulation of dystrophin. Furthermore, these sequences are delivered by a muscle-targeting adeno-associated virus, amplified in situ and incorporated into endogenous small nuclear ribonucleoprotein particles, which protect it from nucleases and facilitate its accumulation in the nucleus, where splicing occurs57. Preliminary data showed that, in the youngest of the three treated patients (7 months old), 99% of muscle fibres produced full-length dystrophin at about 70% of normal amounts. The two other patients (9 and 14 years old) demonstrated stabilization of disease and production of full-length protein (scAAV9.U7.ACCA (See Related links)).

Among splice-modulating NBTs for the treatment of other diseases, sepofarsen (also known as QR-110) is showing positive results in early clinical trials for Leber congenital amaurosis 10 (NCT03913143). Sepofarsen targets the c.2991+1655A>G variant in intron 26 of CEP290 that introduces a cryptic splice donor site and causes insertion of a pseudoexon, resulting in a premature stop codon (p.Cys998*). Sepofarsen is a 17-mer 2′-O-methyl/phosphorothioate-modified single-stranded RNA molecule targeting splicing of CEP290 mRNA, which is delivered by intravitreal injection58. Intravitreal injection offers some advantages, including protection from nucleases and reduced possibility of off-target effects and kidney and liver toxicity, due to sequestration of the NBT in the eye. However, this route of administration is invasive and burdensome for patients, as was illustrated by the clinical trial results. Of the 11 patients, 90.9% developed mild ocular adverse events in the treated eye versus 9.1% in the untreated eye. Eight patients developed cataracts, 75.0% of whom required lens replacement. The effects were dose dependent; therefore; higher doses were discontinued or not initiated. However, given statistically significant improvements in visual acuity and retinal sensitivity, it was concluded that the risk–benefit profile of sepofarsen supports the continuation of clinical development59.

The SCN1A-targeting splice-switching oligonucleotide STK-001 for Dravet syndrome developed by Stoke Therapeutics is currently in phase I/IIa clinical trials (NCT04442295 and NCT04740476)60. This treatment uses a different approach to SMA and DMD therapies, termed targeted augmentation of nuclear gene output (TANGO) platform. TANGO is based on the discovery of ‘toxic’ or ‘poison’ exons naturally occurring in many mRNAs. The majority of mRNA molecules containing such exons are destroyed through the NMD pathway and are not available for translation35,61. ASOs from Stoke Therapeutics are designed to prevent the inclusion of toxic exons, thus increasing the corresponding protein levels60. Preliminary results from the clinical trial show good safety and tolerability of the treatment as well as a 55% median reduction from baseline in convulsive seizure frequency after three doses of 45 mg in six patients refractory to standard medications. Reductions in seizure frequency began after the first dose and were maintained during treatment in the open-label extension study. Trends towards improvement in non-seizure readouts as measured by the BRIEF-P, an assessment of executive function, were also observed (see Related links).

Importantly, Stoke Therapeutics developed a population pharmacokinetic model for intrathecal STK-001 administration using non-human primate data. STK-001 levels in plasma and cerebrospinal fluid of patients treated with STK-001 correlated well with model predictions62. This represents encouraging results for the toxic exon approach, which can be applicable to thousands of disease-relevant genes35.

Therapeutic mRNA delivery

Introducing exogenous mRNA into diseased cells has several advantages compared to injections of purified protein, including correct post-translational modifications and subcellular localization of the generated protein product, lower immunogenicity, a simpler manufacturing process, and lower cost27.

The use of mRNA reduces the chances of insertion mutagenesis, which is a potential risk associated with viral gene therapy. A short half-life makes therapeutic mRNAs well suited for applications that require transient effects such as RNA vaccines. At the same time, for the treatment of genetic diseases, duration of the effect could be extended if the mRNAs are chemically modified or expressed by a plasmid or viral vector (Box 2).

Despite significant advances in chemistry, the use of mRNA as a therapeutic approach has some technical challenges. It is more difficult to synthesize and deliver full-length mRNA than shorter NBTs due to poor cell permeability and instability of exogenous mRNAs63. In vivo delivery of mRNA requires a carrier (Box 3). Furthermore, immunostimulatory properties of mRNAs could be a major safety consideration in gene upregulation applications. Multiple strategies to overcome the immunostimulatory activity are now being developed, including modifications to the RNA backbone, optimization of the manufacturing and purification processes, and supplementation with immune inhibitors64.

The therapeutic mRNA manufacturing and purification processes are also more complex than those of short oligonucleotides that can be manufactured using fully automated chemical synthesis. The currently used manufacturing processes start with in vitro transcription of a linearized plasmid or PCR template containing the gene of interest and a strong promoter region. Transcription is usually conducted by bacteriophage T7, SP6 or T3 RNA polymerases. Recently, significant improvements have been made to this step, including optimization of the cDNA template for transcription, improved mRNA product stability and advanced purification techniques to eliminate dsRNA fragments and other immunogenic by-products. These improvements are crucial for the development of the field as specifics of the manufacturing process significantly contribute to mRNA therapy efficiency (Moderna SEC filing (see Related links)).

The recent innovations in therapeutic mRNA technology (Box 2) resulted in highly positive results for mRNA vaccines in the clinic (for example, COVID-19 vaccines) and in advancement of several protein-replacement therapeutic mRNAs to clinical trials63,65 (Table 1). Progress in the understanding of mRNA biology obtained in the therapeutic mRNA field can be applied to the development of other NBTs.

One of the representative examples of clinical application of therapeutic mRNA technology for protein replacement is the CFTR mRNA therapy MRT5005 for cystic fibrosis developed by Translate Bio/Sanofi, which was tested in a phase II clinical trial (NCT03375047). MRT5005 consists of in vitro-transcribed, unmodified CFTR mRNA encased in lipid nanoparticles and aerosolized to permit inhalation delivery to the lung. The nanoparticles are composed of hyperbranched poly(β-amino esters) (hPBAEs) and enable wide expression of cargo mRNA in lung tissue after inhalation66,67. Importantly, the exogenous mRNA expression is largely localized to the lung, limiting possible off-target effects. The inhalation delivery is non-invasive, and is compatible with frequent dosing and addition of other cargo molecules. The interim results from a phase I/II clinical trial of MRT5005 in patients with cystic fibrosis have shown good tolerability (see Related links). While exploratory efficacy in ppFEV1 (percent predicted forced expiratory volume in 1 s) from the interim analysis of the single-ascending dose portion of the study looked promising, the next set of results, from the multiple-dose portion, did not demonstrate a pattern of increases in ppFEV1. These results underline the necessity of further studies on NBT pharmacokinetics and improvements in drug product composition and clinical study design.

Other examples of therapeutic protein-replacement mRNA in clinical trials further demonstrate rapid progress that is being made in this particular modality. Among them are several early-stage clinical trials of mRNA therapies for rare metabolic diseases conducted by Moderna.

mRNA-3927 developed by Moderna delivers dual mRNAs for propionyl-CoA carboxylase subunits α and β in patients with propionic acidaemia (NCT04159103)68. Repeated intravenous doses were well tolerated in 10 patients, and preliminary data showed a decrease in the number of clinical crises that occur in the natural course of the disease due to build-up of toxic metabolites.

mRNA-3705, delivering mRNA for a mitochondrial enzyme methylmalonyl-coenzyme A mutase, is being tested in patients with methylmalonic acidaemia. The enzyme sequence has been engineered to improve protein translation. In vitro, the therapeutic mRNA showed physiologically correct mitochondrial localization69. mRNA-3705 has replaced mRNA-3704 in clinical trials (NCT04899310) due to its greater potency and extended lowering of methylmalonic acid in mutase-deficient mice compared to mRNA-3704 (Moderna SEC filing (see Related links)). Initial results from the mRNA-3705 study are expected in 2023.

mRNA-3745, also in early clinical trials conducted by Moderna, delivers mRNA for glucose 6-phosphatase in patients with glycogen storage disease type 1a (NCT05095727).

In an alternative approach, mRNA therapeutics are designed to locally or systemically enhance the expression of regulatory proteins that are not mutated in a given disease but needed to induce a desired therapeutic outcome. One example is the investigational cancer therapy mRNA-2752 developed by Moderna, which consists of lipid nanoparticles containing three mRNAs (OX40 ligand, IL-23, IL-36γ). Together, these mRNAs can boost expansion of CD4 and CD8 T cells as well as enhance priming and maturation of dendritic cells. mRNA-2752 is injected directly into tumours to induce a tumour-targeted immune response. A phase I clinical study (NCT03739931) in patients with accessible solid tumours and lymphomas has reported positive preliminary results (see related links). As of July 2022, 88 patients were treated with mRNA-2752, 69 in combination with durvalumab, an immune-checkpoint inhibitor. Analyses of plasma and tumour tissues show that mRNA-2752 treatment was associated with elevated pro-inflammatory cytokines, including IL-23, IL-36γ, IFNγ and TNF, and an increase in proliferating CD8+ T cells, dendritic cell recruitment and T cell activation compared to baseline. Increases in immune response positively correlated with clinical benefit70.

A similar multiplex approach is being advanced by BioNTech/Sanofi in their BNT131 (SAR441000) therapy, which contains a combination of IL-12sc, IL-15sushi, IFNα and GM–CSF nucleoside-modified mRNAs engineered for minimal immunogenicity. BNT131, administered by weekly intratumoural injections in combination with the immune-checkpoint inhibitor cemiplimab, is currently in phase I clinical trials for advanced melanoma (NCT03871348)65,71.

At the same time, as a testament to challenges in NBT clinical development, work on AZD8601, a naked VEGF-A mRNA developed by Moderna/AstraZeneca that was in a phase II trial for enhancing post-surgical angiogenesis after bypass surgery, was discontinued despite showing good safety and positive trends in efficacy end points72.

Overall, direct delivery of protein-encoding mRNA has gained a great deal of attention owing to its utility in generating the COVID-19 vaccines65. Additionally, as demonstrated by the current examples, it also has therapeutic potential for supplementing the insufficient expression of endogenous genes, which will likely significantly expand its applications in the future.

Small molecules facilitating ribosomal readthrough

While NBTs interact with RNA based on their sequence complementarity, RNA-targeting small molecules (rSMs) target the 3D structure of RNAs. RNA molecules form distinct 3D folds similar to pockets of 3D protein structures that can be specifically bound by small molecules to modify biological function. Advantages of using rSMs instead of NBTs to modulate RNA targets include oral availability and, in some cases, blood–brain barrier permeability. However, potential disadvantages include low target specificity and high development costs73.

The first clinical success in the rSM field, ataluren (developed by PTC Therapeutics), was designed to facilitate ribosome readthrough of missense mutations by increasing recruitment of near-cognate tRNAs. The drug received marketing authorization in Europe for the treatment of DMD in 2014. However, it later failed to gain FDA approval and did not show sufficient efficacy in clinical trials in Dravet and CDKL5-deficiency syndromes74.

In 2020, the FDA approved a splicing modifier rSM, risdiplam, for the treatment of SMA. Developed by Roche, risdiplam exerts its unique mechanism of action by interacting with exon 7 of SMN2 pre-mRNA and increasing the binding affinity of spliceosome components. This results in proper inclusion of exon 7 in the final mRNA transcript and subsequent increases in the yield of functional SMN protein. However, risdiplam has significant off-target effects on splicing of non-relevant RNAs, which may increase its toxicity75.

Success of risdiplam has further fuelled interest in the rational design of rSMs. PTC518, a brain-penetrant splice-modifying rSM, is in a phase II clinical trial for Huntington disease (NCT05358717)76. PTC518 degrades huntingtin mRNA by promoting the inclusion of a poison exon (see Related links). Notably, another rSM called TEC-1 has also displayed promising preclinical results for SMA by increasing functional SMN protein. TEC-1 has the same splice-modifying mechanism of action as risdiplam. Yet, unlike risdiplam, TEC-1 did not affect splicing of another well-known target of risdiplam, FOXM1, and did not induce micronucleus formation77.

DT-216 is an rSM in phase I/II clinical trials for Friedreich ataxia (NCT05285540 and NCT05573698). It was developed using a platform known as GeneTAC™ (gene-targeted chimera small molecules). GeneTAC™ molecules comprise a DNA-binding moiety connected via a linker to ligand moieties that engage transcription elongation complexes. A rationally designed DNA-binding moiety binds specifically at the site of the disease-causing nucleotide repeat expansion that normally stalls transcription machinery. The ligand moiety recruits elongation complexes that facilitate transcription through repeat expansion, thus increasing production of the deficient protein. Preliminary results in 39 patients with Friedreich ataxia indicated that treatment with DT-216 was well tolerated. Single doses of DT-216, ranging from 100 to 600 mg, resulted in a 1.2-fold to 2.6-fold increase in frataxin (FXN) mRNA at 24 h post-dose (see Related links).

Other developments in the rSM space are building on the work on riboswitches conducted in the early 2000s78,79. Riboswitches are naturally occurring structures in RNA molecules that change conformation upon binding of small-molecule metabolites such as vitamin derivatives, metal ions and others. They function as endogenous environmental sensors regulating protein expression. Synthetic riboswitches have been applied in the development of biodetectors, molecular diagnostics and computational hardware with a biological component (wetware)80. Endogenous structures similar to riboswitches that can potentially bind rSMs — changing the conformation of the RNA transcript that harbours them and thus affecting its intracellular functions — are present in many RNA transcripts79. Given the unique nature of such interactions, rSMs are typically found via extensive screening. Recent advances in computational modelling of RNA have facilitated in silico identification of rSMs that can directly bind RNA molecules to modify the function of a given RNA structural moiety, inactivate the RNA transcript by generating covalent bonds or even trigger degradation of the RNA transcript81.

Disney’s group used a mechanism similar to proteolysis-targeting chimaeras (PROTACs) or small molecules that tag proteins for proteasomal degradation to design rSMs linked to an RNase-recruiting moiety that can target the RNA for degradation. The RNA-degrading rSMs, termed RIBOTACs, that target inhibitory or repressive RNA molecules, such as miRNAs, are capable of upregulating disease-associated proteins. Expansion Therapeutics, which was founded in 2016 following the work by Costales et al., has been focusing on RIBOTACs that degrade a causative RNA transcript for myotonic dystrophy type 1 (ref. 82). By binding to the hairpin structure of the toxic CUG repeat expansion, which causes myotonic dystrophy type 1, ERX-963 allows translocation of mutant CUG repeat-containing mRNA from the nucleus to the cytoplasm, ultimately stimulating translation of the mutant mRNA83. However, their lead compound, ERX-963, failed to provide any significant therapeutic effect in a clinical trial84.

Disney et al. have further advanced RNA computational modelling by designing an online computational platform, INFORNA, for in silico identification of lead small molecules for RNA targets85. They have utilized INFORNA and more traditional binding assays to identify an approved drug, the receptor tyrosine kinase inhibitor dovitinib, as a selective binder of oncogenic pre-miR-21. By modifying dovitinib into a RIBOTAC, Zhang et al. were able to inhibit metastasis of breast cancer cells to the lungs in a mouse model after 30 days of treatment with the RIBOTAC, with no reports of toxicity86.

As miR-21 represses translation of apoptosis-related genes, including PTEN, TPM1 and PDCD4 (encoding programmed cell death 4), degrading pre-miR-21 with dovitinib-PROTAC derepresses translation of these apoptosis-related genes.

Another rSM designed by Haniff et al. selectively targets miR-200c and represses pancreatic β-cell apoptosis by derepressing translation of miR-200c targets Rps6kb1 and Dnajc3. Surprisingly, the rSM did not inhibit other miR-200 family members while an oligonucleotide targeting the RNA sequence did87.

Arrakis Therapeutics is at the early discovery stage investigating rSMs that modulate intrinsic function of RNA targets, rSMs that form deactivating covalent bonds with RNA targets, and RIBOTACs88 (see Related links). Gene upregulation can be achieved with this approach by deactivating or degrading repressive RNA transcripts.

These developments demonstrate that, although even the most promising rSMs have limitations, there is potential for further development of rSM therapeutics. Notably, identifying molecules that do not have off-target effects resulting in toxicity remains the most pressing challenge for rSMs. Aside from risdiplam, most rSMs are utilized as chemical probes, more useful for experimental, rather than therapeutic, purposes. However, proponents of the approach think that there are specific RNA structures that can be targeted without off-target interactions89.

NBTs in preclinical and early-stage clinical development

miRNA-targeted NBTs

In recent years, miRNAs have emerged as attractive therapeutic targets90. As their activity results in a reduction in abundance of specific mRNAs, blocking this activity leads to an increase in protein production.

miRNAs are initially transcribed as long primary miRNA transcripts containing double-stranded hairpin structures and are then processed to generate RNA duplexes. The duplexes associate with AGO proteins of the RNA-induced silencing complex that remove the ‘passenger’ RNA strand and retain the ‘guide’ strand. The ‘seed’ region of the guide strand (positions 2–8 at the 5′ end) can then anneal to specific response elements in target mRNAs, leading to their degradation directly by AGOs or by additional proteins recruited by AGOs90.

Synthetic oligonucleotides have been used to interfere with this pathway as both miRNA mimics (promirs) and miRNA blockers (antagomirs or antimirs or miRNA sponges; Fig. 3). Depending on the mechanism of action of a given miRNA, both of these intervention types may lead to target protein upregulation.

Promirs are synthetic oligonucleotides that have the same sequence as a naturally occurring miRNA and are used to amplify their effects. In cases when the target mRNA codes for an inhibitory protein, its destruction should lead to de-repression of a disease-relevant protein. Although a similar effect can be achieved by oligonucleotides complementary to any part of the target mRNA sequence because miRNAs usually have multiple targets and can be modulated by endogenous regulatory mechanisms, miRNA mimics could achieve context-dependent pleiotropic effects without the need to apply multiple oligonucleotides91. However, clinical development of such molecules has encountered multiple challenges involving low efficacy and adverse events in clinical trials92.

Antagomirs are single-stranded oligonucleotides that can abolish miRNA activity by competing with target mRNA for binding to the guide strand–AGO complex. miRNA blockers are usually designed with mispairing or modified bases at the AGO2 cleavage site, which inhibits mRNA degradation.

Although development of miRNA blockers has been under way for a long time, the field has been plagued by multiple failures. Regulus discontinued clinical trials of RGLS4326, designed to inhibit miR-17, which suppresses expression of polycystins 1 and 2, for autosomal dominant polycystic kidney disease and replaced it with the next-generation candidate RGLS8429, shown to have a more favourable risk–benefit profile. Off-target CNS events were observed at the top doses of RGLS4326 in chronic preclinical toxicology studies, while RGLS8429 did not show such effects. Notably, miR-17 is known to repress multiple targets besides polycystins and thus has a high possibility of off-target toxicity (see Related links). Topline results from the RGLS8429 study showed good tolerability and an expected pharmacokinetic profile, providing support for initiation of a phase Ib trial (NCT02855268).

At the same time, lademirsen (RG-012/SAR339375), an anti-miR-21 antagomir, was tested in a phase II clinical trial in Alport syndrome, a rare kidney disease (NCT05521191) (Table 1). miR-21 is upregulated in Alport syndrome and blocks the expression of multiple apoptosis-related genes, including PTEN, TPM1, PDCD4 and genes involved in renal tubulointerstitial injury pathways. Although the drug was well tolerated, the results of the interim futility analysis led to study termination.

miRagen/Viridian also halted clinical development of MRG-110, a locked nucleic acid (LNA) ASO against miR-92a-3p for the treatment of heart failure (NCT03601052). Inhibition of miR-92a was shown to improve endothelial cell function and recovery after acute myocardial infarction via endothelial cell-specific regulation of autophagy-related genes93.

miR-155 is a master regulator of inflammation that is overexpressed in multiple cancers and in the spinal cord of patients with amyotrophic lateral sclerosis. In vitro blockade of miR-155 using LNA antagomirs (MRG-107 and cobomarsen/MRG-106) de-repressed numerous direct and downstream miR-155 targets in JAK–STAT, MAPK–ERK and PI3K–AKT pathways, leading to reduced survival signalling and apoptosis94. However, development of cobomarsen in cancer was discontinued due to inconclusive efficacy.

Despite the recent setbacks in the miRNA-targeting NBT space, their preclinical development continues, mostly aimed at gene silencing95,96,97.

Small activating RNAs

Small dsRNAs are widely recognized for their ability to induce degradation of homologous mRNA transcripts. Surprisingly, it was discovered that synthetic dsRNAs containing a guide strand complementary to promoter or enhancer regions of a target gene — coined small activating RNAs (saRNAs) — can induce gene expression98 (Fig. 3).

The mechanisms of action of saRNAs are not completely understood but could be related to the mechanism of promoter activation by endogenous miRNA, referred to as RNA activation99,100. RNA activation could be mediated by miRNA binding to pRNAs (discussed below) and association with the AGO2–TNRC6 complex but does not require pRNA degradation. Using the induction of ZMYND10 expression by miR-34a as a test case, it was shown that this complex interacts with positive transcription elongation factors CDK9 and DDX21, thus bringing them in the vicinity of the target promoter, which leads to the release of paused promoter–proximal RNA Pol II and upregulation of ZMYND10 expression101.

Furthermore, saRNA binding to pRNA or lncRNA in sense and/or antisense orientation was shown to be associated with epigenetic changes within the promoter region, including histone demethylation and acetylation102,103,104. These changes can explain the prolonged upregulation of gene expression induced by saRNAs, which can last several weeks101,105.

Alternatively, saRNAs were proposed to bind AGO2 and hnRNPs in the cytoplasm. This complex can then be imported into the nucleus, where it binds directly to promoter DNA and participates in the RNA-induced transcriptional activation (RITA) complex. RITA interacts with RNA Pol II to initiate transcription. Other components of RITA are RNA helicase A (RHA), RNA polymerase-associated protein CTR9 homologue (CTR9) and DEAD-box helicase 5 (DDX5)99,100.

The saRNA MTL-CEBPA developed by MiNA Therapeutics is being tested as a supplemental therapy to enhance the efficacy of standard-of-care cancer drugs106. The mechanism of action of MTL-CEBPA likely involves inactivation of immune-suppressive myeloid cells by upregulation of its target transcription factor CEBPA107. MTL-CEBPA is composed of amphoteric iminolipid nanoparticles, called SMARTICLES, and a 21-mer 2′-O-methylated RNA oligonucleotide duplex. Interim results showed regression of tumours in 26.7% of patients with hepatocellular carcinoma103. A phase I trial of MTL-CEBPA combined with pembrolizumab, an anti-PD1 immune-checkpoint inhibitor, is ongoing in 50 patients resistant to or ineligible for standard therapies (NCT04105335). The treatment ameliorated immune suppression and improved infiltration of cytotoxic T cells in tumours compared to baseline. Objective tumour responses were observed in four patients who were treated108.

If successful, saRNA treatments could be applicable to a wide range of disorders.

NAT-targeted NBTs

NATs are known to inhibit expression in 50–80% of all gene loci, which gives this NAT-targeted approach high potential in therapeutic protein upregulation2. Several strategies targeting NAT-mediated regulation have been proposed.

Single-stranded NAT-specific oligonucleotides, termed AntagoNATs, targeted to a NAT in the BDNF locus (BDNF-AS) induced gene-specific upregulation of the neurotrophic factor BDNF both in vitro and in vivo14. Furthermore, intranasal delivery via the minimally invasive nasal depot (MIND) technique induced upregulation of BDNF protein in multiple regions, including deep structures, in rat brain109. The MIND procedure involves the use of routine outpatient techniques, representing a significantly safer and more patient-friendly method for oligonucleotide delivery to the brain compared with intracerebroventricular and intrathecal delivery strategies.

The AntagoNAT CMP-SCN targets a NAT in the SCN1A gene locus110. SCN1A codes for the α-subunit of the voltage-gated sodium channel NaV1.1. Heterozygous mutations in SCN1A lead to Dravet syndrome, a rare form of childhood epilepsy. Treatment with CMP-SCN and its mouse analogue induced specific upregulation of SCN1A both in vitro and in vivo and significantly reduced the occurrence of seizures in a mouse model of Dravet syndrome110. Preclinical toxicity studies in non-human primates demonstrated a good safety profile and significant increases in SCN1A mRNA and protein in various brain regions after treatment with CMP-SCN111.

Putative NBTs of the future

pRNAs, eRNAs and seRNAs

Although currently mostly untapped, pRNA, eRNAs and seRNAs present multiple opportunities for therapeutic intervention with saRNA-type molecules17. Interestingly, promoters and enhancers produce a similar repertoire of bidirectionally transcribed short and long non-coding transcripts that can regulate transcription, which could indicate their common evolutionary origins, with enhancers initiating the transcription of the majority of intergenic lncRNAs16. Recent studies have demonstrated the existence of pervasive bidirectional transcription of short ncRNAs from DNase-hypersensitive regions that do not have canonical enhancer or promoter histone modification profiles that could represent other, as yet unidentified, regulatory elements18,112,113. However, the current knowledge of the mechanisms of action and biological effects of transcripts generated by promoters, enhancers, super-enhancers and other regulatory elements is still evolving.

Notably, expression patterns of these RNAs are known to be tissue specific, which makes them attractive as potential therapeutic targets114. Expression of eRNAs is also stimulus specific; for instance, particular subsets of eRNAs are preferentially expressed at inflammation-related genes in response to inflammatory stimuli, in neurons during the stimulus-induced expression of immediate early genes and after exposure to early-life undernutrition115,116,117. An experience-induced enhancer lncRNA, ADRAM (activity-dependent lncRNA associated with memory), is generated by an enhancer of Nr4a2 (nuclear receptor subfamily 4, group A, member 2). ADRAM localized to the Nr4a2 promoter, increased deposition of H3K4me3 marks and coordinated binding of 14-3-3 protein. This facilitated the dissociation of HDAC3 and HDAC4, followed by CBP-mediated activation of Nr4a2 expression. ADRAM was required for the formation of fear extinction memory118. NBTs that act as ADRAM mimics to boost Nr4a2 expression may have therapeutic significance in anxiety disorders.

A possible way to modulate eRNA activity is based on the fact that eRNA expression can be regulated by lncRNAs. For example, the lncRNA KHPS1 (sphingosine kinase 1a antisense transcript) forms a triple-helical RNA–DNA–DNA structure at the sphingosine kinase 1a (SPHK1) enhancer119. The triplex facilitates recruitment of activation factors E2F1 and p300 and expression of eRNA-Sphk1. eRNA-Sphk1 displaces CTCF (CCCTC-binding factor), a zinc finger protein that insulates the SPHK1 enhancer from the SPHK1 promoter and thus upregulates SPHK1 transcription. Interestingly, triplex-forming regions of NATs are sequence specific and can be repurposed to target genes of interest. For example, a KHPS1 transcript engineered to contain the triplex-forming region of lncRNA MEG3 binds to a region in the MEG3 target gene TGFBR1 (ref. 119).

Super-enhancers are known to generate seRNA. seRNA functions include promoting enhancer–promoter looping, recruiting transcription factors and facilitating epigenetic histone modifications. For example, muscle injury triggers expression of Pax7-associated muscle lncRNA (PAM) from a myoblast-specific super-enhancer in muscle satellite cells. PAM interacts with DEAD‐box helicase 5 (Ddx5) to align the super-enhancer to its target genes Timp2 (encoding tissue inhibitor of metalloproteinase 2) and VIM (encoding vimentin). This activates the expression of the extracellular matrix proteins required for satellite cell differentiation into myotubes120. Upregulation of PAM expression may be beneficial in muscle diseases and in promoting muscle regeneration after injury.

Furthermore, hnRNPL has been shown to bind diverse eRNAs and seRNAs in several cell types. In myogenic super-enhancer-generated seRNA1, this binding occurs via a CAAA tract. Disruption of seRNA1–hnRNPL interaction decreases RNA Pol II binding and H3K36me3 deposition and transcription at the myoglobin locus, which impairs muscle formation21. seRNA1 mimics could therefore promote muscle regeneration.

Importantly, activation of seRNAs is cell and tissue specific and can serve as a target for fine modulation of cell fate, essential, for example, in the treatment of cancers, ischaemia and neurodegenerative diseases22,121,122.

Several methods for modulating eRNA and seRNA expression, including ASOs, dsRNAs and repurposed RNA-guided RNA-targeting CRISPR–Cas13 machinery, have been proposed.

Targeting imprinting

In some cases, targeting lncRNAs that are known to participate in silencing of imprinted transcripts can be utilized in disease treatment. In Angelman syndrome, mutations affecting the maternally inherited ubiquitin-protein ligase E3A (UBE3A) can be rescued by de-repression of the paternal copy silenced by Ube3a NAT (Ube3a-ATS). NBTs targeting Ube3a-ATS activated the imprinted copy, increased UBE3A levels in the brain to up to 74% of wild type and normalized phenotypes in a mouse disease model123. Although still in discovery stage, this approach can be applicable in many cases of mutations in imprinted genes and epimutations.

Targeting uORFs

NBTs targeting several translation regulatory elements in 5′ and 3′ UTRs have been proposed as a protein upregulation strategy (Fig. 4). Although none of these approaches is in clinical testing, fine modulation of translation may be beneficial in the treatment of many diseases. Early preclinical studies have shown that ASOs targeting uORFs increase protein expression, likely by base-pairing with the uORF upstream AUG regions and redirecting translation to the main start site36 (Fig. 4).

Targeting regulatory elements in UTRs

Stem-loop and other secondary structures in 5′ UTRs can inhibit translation of the gene. Treatment with ASOs designed to hybridize to such structures was shown to result in up to 2.7-fold increases in the levels of RNASEH1, LDLR and ACP1 proteins in different cell types and species, in vitro and in vivo, with minimal off-target effects as determined by microarray analyses37.

Targeting polyadenylation

Selection of alternative polyadenylation sites during splicing can determine the biological activity and subcellular distribution of proteins produced by mRNA transcripts. Shifting the balance between isoforms with different physiological functions without increasing the overall mRNA levels may have the additional benefit of reducing the possibility of target isoform overexpression.

Such a situation can occur in IgE-mediated allergies that are caused by an increased proportion of secreted versus membrane-bound isoforms of IgE, which differ in their polyadenylation sites40. Morpholino ASOs targeting the polyadenylation signal of IgE that are coupled to Pip6a, an arginine-rich cell-penetrating peptide, alter IgE transcript polyadenylation to produce more membrane-bound isoform and less secreted isoform. This leads to decreased IgE secretion and increased apoptosis of IgE+ antibody-secreting cells in transgenic mice expressing humanized IgE (Fig. 4). The change in IgE isoform composition may be beneficial for the reduction of IgE-related allergic symptoms and could promote allergen tolerance through apoptosis of IgE+ antibody-secreting cells40.

Another example is NEAT1_2, one of the isoforms of lncRNA NEAT1. NEAT1_2 is known to scaffold paraspeckles, essential membrane-less nuclear structures thought to sequester RNAs and proteins. Another isoform, NEAT1_1, is not involved in this process but promotes the proliferation of cancer cells. Treatment with ASOs that block a NEAT1 polyadenylation site results in a shift in the ratio of NEAT1_2 to NEAT1_1 towards the NEAT1_2 isoform, increased paraspeckle formation, induction of differentiation and suppression of tumorigenesis in vitro41.

Combinatorial treatments

Using combinations of NBTs targeting different biological pathways with synergistic effects is an approach that is receiving considerable attention. In a clinically relevant example, it has been shown that treatment with a combination of ASOs that bind to FXN 5′ or 3′ UTR can enhance the upregulation of FXN protein in vitro compared to individual ASO treatment. The mechanism is possibly related to the increased stability of FXN mRNA without changes in transcription of the FXN gene38.

An interesting case of NBT–small molecule synergy occurs after combined treatment with a 5′ UTR-targeting ASO and a small-molecule drug Symdeko (ivacaftor plus tezacaftor). Tezacaftor facilitates the folding and membrane localization of CFTR, and ivacaftor shifts the chloride channel encoded by CFTR towards the open state, while treatment with the ASO increases CFTR protein production. The combination treatment increases CFTR function in primary human bronchial epithelial cells with multiple cystic fibrosis genotypes compared to treatment with Symdeko alone39.

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