July 23, 2024

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What is RNA-targeted gene activation therapy?

What is RNA-targeted gene activation therapy?


What is RNA-targeted gene activation therapy?

In the early 21st century, after the completion of the Human Genome Project, it was discovered that many diseases were caused by gene mutations leading to insufficient expression of functional proteins.

However, in drug development, it is easier to develop drugs with inhibitory or antagonistic effects.

With the innovation of large-scale recombinant protein production and purification technology, protein replacement therapy, that is, recombinant protein has achieved clinical success, such as insulin for the treatment of diabetes

. However, this approach is mainly applicable to secreted proteins or enzymes and is hampered by complex pharmacokinetic and cost-related issues of these molecules.

Furthermore, synthetic proteins are unlikely to fully represent the diversity of endogenous functions of proteins caused by alternative splicing, post-translational modifications, and other regulatory mechanisms.


What is RNA-targeted gene activation therapy?


In recent years, a variety of nucleic acid-based therapeutic ( NBT ) modalities have emerged as potent and specific activators of endogenous gene expression.

Unlike gene therapy approaches that supplement gene expression, RNA-targeted therapies enhance protein production by selectively modulating endogenous RNA-mediated cellular mechanisms such as transcription, splicing, translation, mRNA stability, and subcellular localization .


What is RNA-targeted gene activation therapy?


Currently, the U.S. Food and Drug Administration ( FDA ) and the European Medicines Agency have approved several therapies that target the splicing machinery to regulate mutant exons.

These approaches can specifically and controllably increase the expression of coding and noncoding genes, reduce development and manufacturing costs, and thereby increase the range of treatable diseases.

Based on the proven therapeutic potential and huge unmet medical needs, NBT will usher in more rapid development in the future.



The biology of protein expression upregulation

Upregulation of therapeutic proteins can be achieved by modulating biological processes at any stage of protein production in cells, including transcription, splicing, translation, or post-translational modifications.

Since many of these processes involve DNA or mRNA and are regulated by ncRNA networks, they are particularly susceptible to regulation by NBTs .


What is RNA-targeted gene activation therapy?


Transcriptional activation is the most studied method for increasing protein expression abundance. Distal enhancer elements recruit transcription factors, chromatin modifiers, the Mediator complex, and RNA polymerase II (Pol II) through physical interaction of chromosomal loops with active gene promoters, resulting in transcriptional activation .

Bidirectional transcription of the promoter region and enhancer generates non-coding promoter RNA ( pRNA ) and enhancer RNA ( eRNA ), respectively.

Natural antisense transcripts ( NATs ) transcribed from the antisense strands of protein-coding loci, trans-acting long noncoding RNAs ( lncRNAs ), pRNAs, and eRNAs can carry out epigenetic modification and transcriptional regulation of target genes.


A key feature of NATs is that they can specifically regulate transcription, RNA processing, and translation of their sense genes in cis or trans.

In addition, NAT also has multiple regulatory functions, including adsorbing miRNAs and pairing with mRNAs to improve their stability.

But most of them inhibit the expression of their target genes by coordinating repressors. Thus, targeting NAT with antisense oligonucleotides ( ASOs ) can lead to derepression of sense genes and increased protein expression.


In addition, naturally occurring modified nucleotides, such as N6- methyladenosine, 5- methylcytosine, N1 -methyladenosine, pseudouridine, and 2′-O- methylated ribose, occur in mRNA and lncRNA during the transcription process.

These modifications can be regulated by NBTs to affect transcription and protein expression.

Translation efficiency may also be affected by mRNA structural features, and blocking or enhancing the activity of these structures using NBTs may lead to upregulation of therapeutic protein expression.


NBT in the clinical stage

Over the past 5-7 years, NBTs have experienced explosive growth, some of which have been approved, while a variety of other NBTs are being explored in clinical trials, the table below shows the approved NBTs .


What is RNA-targeted gene activation therapy?



NBT that regulates splicing

Aberrant RNA splicing caused by mutations often results in the rapid destruction of non-functional transcripts by nonsense-mediated degradation ( NMD ), resulting in a shortage of affected proteins that underlies many diseases.

In addition, normal alternative splicing of pre-mRNAs can contain so-called “toxic exons”, leading to rapid degradation of transcripts by NMD, thereby reducing protein levels.

Binding to specific sequences on pre-mRNAs that regulate splicing events, ASOs can prevent mutations or the production of naturally unproductive transcripts and increase target protein expression levels.



Vesletplirsen (SRP-5051) , a next-in-line oligonucleotide drug for exon skipping therapy, targets Duchenne progressive muscular dystrophy (DMD) patients who can be treated with exon 51 skipping.

In a clinical trial ( NCT04004065 ), vesletplirsen treatment resulted in an 18-fold increase in exon skipping and an 8-fold increase in dystrophin levels.

However, the trial was temporarily put on hold due to observations of severe hypomagnesemia, and later resumed with expanded monitoring of urinary biomarkers and magnesium supplementation.


WVE-N531 , a novel systemically administered antisense oligonucleotide therapy, is currently in a clinical trial in 15 DMD patients prone to exon 53 skipping (NCT04906460 ) .

Interim results showed high muscle concentrations of the drug with an average exon skipping rate of 53%. Pharmacokinetic data showed a half-life of 25 days, which may support monthly dosing.

Preliminary clinical results of WVE-N531 suggest possible pharmacological improvements compared to first-generation DMD splice-switch NBTs and knockdown NBTs in the Huntington’s disease program.


Among splicing-modulating NBTs for the treatment of other diseases, sepofarsen (QR-110) showed positive results in an early clinical trial for Leber congenital amaurosis ( NCT03913143 ).

Sepofarsen targets the c.2991+1655A>G variant in intron 26 of CEP290.

STK-001 is an oligonucleotide developed by Stoke Therapeutics for SCN1A-targeted splice switching in Dravet syndrome and is currently in phase I/IIa clinical trials ( NCT04442295 and NCT04740476 ).


Delivery of mRNA

Introduction of exogenous mRNA into diseased cells has several advantages over injection of purified protein, including correct post-translational modification and subcellular localization of the resulting protein product, lower immunogenicity, simpler manufacturing process, and lower the cost of.



One of the representative clinical applications of therapeutic mRNA delivery technology is MRT5005 , a CFTR mRNA therapy 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, encapsulated in lipid nanoparticles, nebulized to allow inhalation delivery to the lungs. Interim results from a Phase I/II clinical trial of MRT5005 in cystic fibrosis patients showed good tolerability.

Lung function results are mixed,ppFEV1 did not improve significantly in some patients,while 3 patients in the 16 mg dose group from baseline to day 8,Average maximum increase of 15.7%.


mRNA-3927, developed by Moderna, provides dual mRNAs for propionyl-CoA carboxylase subunits α and β in patients with propionic acidemia.

In the ongoing Phase I/II clinical trial ( NCT04159103 ), 10 patients were well tolerated, with preliminary data showing a reduction in the number of clinical crises that occur during the natural course of the disease.


Small molecules that promote ribosomal translation

The interaction of NBTs with RNAs is based on their sequence complementarity, while RNA-targeting small molecules ( rSMs ) target the 3D structure of RNAs. Advantages of using rSMs to modulate RNA targets include oral availability and, in some cases, blood-brain barrier permeability.



RIBOTACs is a small molecule mechanism similar to proteolytic targeting chimeras ( PROTACs ) for proteasomal degradation.

It designs rSM linked to RNase recruitment moiety, which can target RNA for degradation and can upregulate disease-associated proteins .

A design team identified an approved drug through the online computing platform INFORNA, the receptor tyrosine kinase inhibitor dovitinib is a selective binder of pre-oncogenic miR-21.

By engineering dovitinib into RIBOTACs, the dovitinib-RIBOTACs were able to inhibit the metastasis of breast cancer cells to the lungs in a mouse model.


In addition, DT-216 is an rSM for the treatment of Friedrich’s ataxia. In phase I/II clinical trials ( NCT05285540 and NCT05573698 ), DT-216 showed good tolerance, and after a single dose, Can increase frataxin ( FXN ) mRNA by 1.2 to 2.6 times within 24 hours.


miRNA targeting NBT

In recent years, miRNAs have become very attractive therapeutic targets. Synthetic oligonucleotides have been used to interfere with this pathway, such as miRNA mimics ( promirs ) and miRNA blockers ( antagomirs ).

Depending on the miRNA’s mechanism of action, both types of intervention may lead to upregulation of the target protein.



Remlarsen (MRG-201) , a promirs of miR-29, was found to be associated with the reduction of miR-29 in the accumulation of lung scar tissue.

Therefore, creating a miR-29-like molecule might reverse this scarring. However, studies in humans were quickly terminated due to high toxicity.

miRagen/Viridian then developed the new, improved MRG-229 molecule, which they chemically modified to make it more stable and added a peptide that allowed for more targeted delivery.

MRG-229 was well tolerated in animal models without any adverse effects.


The development of miRNA blockers has also been underway for a long time, but the field has been plagued by multiple failures.

Regulus discontinued clinical trials of RGLS4326 , which inhibits miR-17, for the treatment of autosomal dominant polycystic kidney disease.

The next-generation candidate molecule RGLS8429 was then developed to replace it. RGLS8429 did not observe the off-target CNS events of RGLS4326. Currently, a Phase Ib clinical trial is underway ( NCT02855268 ).


Separately, lademirsen , an antiviral drug against miR-21, was tested in a phase II clinical trial of the rare kidney disease Alport syndrome ( NCT05521191 ). Although the drug was well tolerated, an interim futility analysis led to study termination.



Advantages and challenges of NBT

Several advantages of NBT make it well suited for the treatment of protein underexpression caused by many known diseases.

NBT is characterized by high target specificity, good stability, greatly extended half-life ( such as weeks or months ), and no frequent administration.

Multiple clinical trials of approved NBT demonstrated its good tolerability. The short development timeline and low cost of NBTs allow personalized treatments for rapidly evolving cancers and rare genetic diseases.


However, compared with traditional treatment models, NBT also faces some challenges. A common disadvantage of current NBTs is their inability to cross the gut wall or the blood-brain barrier.

Currently, this problem can be addressed by intravenous, subcutaneous, intracerebral, intraventricular or intrathecal administration.

However, these methods are invasive, especially for CNS delivery, with some potential for adverse effects.

Therefore, more extensive research is needed to develop chemical modifications as well as drug delivery technologies with carriers that allow for less invasive delivery routes.

Furthermore, NBTs have complex and currently poorly understood pharmacokinetics and pharmacodynamics compared to most small molecules. 


Within these limitations, however, overall success rates for NBT development have been reported to be on par with or above average for the pharmaceutical industry.

An analysis of 7,455 drug development programs conducted between 2006 and 2015 showed that only about 6% of new molecular entity drugs entering clinical trials were approved.

The novel biologics had an 11.5 percent success rate in this analysis .




Recently, advances in NBT have opened up enormous new opportunities for the treatment of disease through upregulation of protein expression.

The success of the COVID-19 mRNA vaccine has thrust RNA- targeted protein upregulation technology into the spotlight and may hasten major breakthroughs in the field.

Improvements in gene sequencing technology, as well as new hopes sparked by the feasibility of NBT in genetic diseases, will lead to the expansion of genetic research.

Significant advances in human genomics have resulted in an ever-increasing number of diseases for which genetic causes are known. And these all increase the applicability of NBT up-regulated proteins.


With the continuous development of understanding of disease genomics and pathophysiology , as well as the improvement of NBT technology, manufacturing and regulatory infrastructure, the clinical application of NBT will continue to expand, opening up opportunities for better treatment of ” rare ” diseases and personalized precision medicine the way.





1. Amplifying gene expression with RNA-targeted therapeutics. Nat Rev Drug Discov. 2023 May 30.

What is RNA-targeted gene activation therapy?

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