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 was easier to develop drugs with inhibitory or antagonistic effects. With innovations in large-scale recombinant protein production and purification technologies, protein replacement therapy, such as recombinant insulin, has been successful clinically in treating diseases like diabetes. However, this approach is primarily applicable to secreted proteins or enzymes and is hindered by the complex pharmacokinetics and cost-related issues associated with these molecules. Moreover, synthetic proteins are unlikely to fully represent the diversity of endogenous protein functionality arising from selective splicing, post-translational modifications, and other regulatory mechanisms.
In recent years, various nucleic acid-based therapies (NBT) have emerged as effective and specific activators of endogenous gene expression. Unlike gene therapy approaches supplementing gene expression, RNA-targeted therapy enhances protein production by selectively modulating cellular mechanisms mediated by endogenous RNA, such as transcription, splicing, translation, mRNA stability, and subcellular localization.
Currently, the US Food and Drug Administration (FDA) and the European Medicines Agency have approved several therapies targeting splicing mechanisms to regulate mutations in exons. These methods can increase the expression of coding and non-coding genes specifically and controllably, reducing development and manufacturing costs and thus expanding the range of treatable diseases. Based on the demonstrated therapeutic potential and significant unmet medical needs, NBT is poised for rapid advancement in the future.
Biological Upregulation of Protein Expression
The upregulation of therapeutic proteins can be achieved by modulating any stage of the biological processes involved in protein production within 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 amenable to modulation by NBT.
Transcriptional activation is the most extensively studied method for increasing protein expression abundance. Distal enhancer elements recruit transcription factors, chromatin modifiers, mediator complexes, and RNA polymerase II (Pol II) to the active gene promoter through physical interactions with chromatin loops, resulting in transcriptional activation. Bidirectional transcription from the promoter region and enhancers produces non-coding promoter RNA (pRNA) and enhancer RNA (eRNA), respectively. Natural antisense transcripts (NATs) originating from protein-coding loci, antisense long non-coding RNAs (lncRNAs), pRNAs, and eRNAs transcribed from the opposite strand contribute to epigenetic modifications and transcriptional regulation of target genes.
A key feature of NATs is their ability to specifically regulate the transcription, RNA processing, and translation of their sense genes in a cis- or trans-acting manner. Additionally, NATs have various regulatory functions, including sequestering miRNAs and pairing with mRNAs to enhance their stability. However, most of them act to inhibit the expression of their target genes through coordinated suppressive factors. Therefore, targeting NATs with antisense oligonucleotides (ASOs) can lead to derepression of sense genes and increased protein expression.
Furthermore, naturally occurring modified nucleotides, such as N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, pseudouridine, and 2’-O-methylation of riboses, occur during the transcription of mRNA and lncRNA. Modulating these modifications through NBTs can influence transcription and protein expression. Translation efficiency may also be influenced by mRNA structural features, and using NBTs to disrupt or enhance the activity of these structures may lead to upregulation of therapeutic protein expression.
Clinical Stage Nucleic Acid-based Therapies (NBT)
In the past 5-7 years, NBT has experienced explosive growth, with some already approved and various others in clinical trials being explored. The table below displays approved NBTs.
NBTs Regulating Splicing
RNA splicing abnormalities caused by mutations often lead to the rapid degradation of non-functional transcripts mediated by nonsense-mediated decay (NMD), resulting in the deficiency of affected proteins, which forms the basis of many diseases. Additionally, normal selective splicing of pre-mRNAs can include so-called “toxic exons,” leading to rapid transcript degradation via NMD, thereby reducing protein levels. Antisense oligonucleotides (ASOs) binding to specific sequences on pre-mRNAs that regulate splicing events can prevent the production of mutant or naturally non-productive transcripts, thereby increasing target protein expression levels.
Vesletplirsen (SRP-5051) is the next antisense oligonucleotide drug targeting exon skipping therapy for patients with Duchenne muscular dystrophy (DMD) amenable to 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 protein levels. However, the trial was temporarily paused due to observed severe hypomagnesemia, later resumed with expanded monitoring of urinary biomarkers and magnesium supplementation.
WVE-N531, a novel systemic antisense oligonucleotide therapy, is currently in clinical trials (NCT04906460) in 15 DMD patients predisposed to exon 53 skipping. Mid-term results indicate high muscle concentrations of the drug, with an average exon 53 skipping rate of 53%. Pharmacokinetic data shows a half-life of 25 days, potentially supporting monthly dosing. Preliminary clinical results of WVE-N531 suggest it may have pharmacological improvements compared to the first-generation DMD splicing switching NBT and knockdown NBT seen in the Huntington’s disease program.
In splicing-regulating NBTs used for treating other diseases, sepofarsen (QR-110) showed positive results in early clinical trials for Leber congenital amaurosis (NCT03913143). Sepofarsen targets the c.2991+1655A>G variant in intron 26 of CEP290. STK-001, an antisense oligonucleotide targeting SCN1A splicing for Dravet syndrome developed by Stoke Therapeutics, is currently in phase I/IIa clinical trials (NCT04442295 and NCT04740476).
mRNA Delivery
Compared to injecting purified proteins, introducing exogenous mRNA into diseased cells has several advantages, including correct post-translational modifications and subcellular localization of the resulting protein products, lower immunogenicity, simpler manufacturing processes, and lower costs.
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 unmodified CFTR mRNA transcribed in vitro, encapsulated in lipid nanoparticles and delivered to the lungs via nebulization. Mid-term results of the Phase I/II clinical trial of MRT5005 in cystic fibrosis patients showed good tolerability. Lung function results varied, with some patients showing no significant improvement in ppFEV1, while in the 16 mg dose group, three patients exhibited an average maximum increase of 15.7% from baseline to day 8.
mRNA-3927, developed by Moderna, provides dual mRNAs for propionic acidemia patients, encoding the alpha and beta subunits of propionyl-CoA carboxylase. In an ongoing Phase I/II clinical trial (NCT04159103), 10 patients showed good tolerability, and preliminary data indicated a reduction in the number of clinical crises occurring during the course of the disease.
Small Molecules Facilitating Ribosomal Translation
While NBT interacts with RNA based on their sequence complementarity, RNA-targeting small molecules (rSMs) target the 3D structure of RNA. Advantages of using rSMs to modulate RNA targets include oral availability and, in some cases, blood-brain barrier permeability.
RIBOTACs, a small molecule mechanism similar to proteolysis-targeting chimeras (PROTACs) for protein degradation, are designed with an rSM linked to a portion recruiting RNases, which can degrade targeted RNA, thereby upregulating disease-associated proteins. A drug approved by the regulatory body was identified by a design team using the online computational platform INFORNA. Dovitinib, a selective binder of pre-cancerous miR-21, was transformed into a RIBOTAC, dovitinib-RIBOTACs, which could inhibit the metastasis of breast cancer cells to the lungs in a mouse model.
Additionally, DT-216, an rSM for treating Friedreich’s ataxia, showed good tolerability in Phase I/II clinical trials (NCT05285540 and NCT05573698). After a single dose administration, DT-216 increased frataxin (FXN) mRNA levels by 1.2 to 2.6 times within 24 hours.
miRNA-targeted NBT
In recent years, miRNAs have become highly attractive therapeutic targets. Synthetic oligonucleotides have been used to interfere with this pathway, such as miRNA mimics (promirs) and miRNA inhibitors (antagomirs). Both intervention types could potentially lead to upregulation of target proteins based on the mechanism of miRNAs.
Remlarsen (MRG-201) is a promirs of miR-29, and studies have found that the accumulation of scar tissue in lung is associated with decreased miR-29 levels. Therefore, creating a molecule similar to miR-29 might reverse this scarring. However, due to high toxicity, studies in humans were quickly terminated. Subsequently, miRagen/Viridian developed a new, improved molecule, MRG-229, where they chemically modified the molecule for increased stability and added a peptide for more targeted delivery. MRG-229 showed good tolerability in animal models without any adverse reactions.
Development of miRNA inhibitors has also been ongoing for some time, but the field has been plagued by multiple failures. Regulus halted clinical trials of RGLS4326, which inhibits miR-17, for treating autosomal dominant polycystic kidney disease. Next-generation candidate molecule RGLS8429 was developed to replace RGLS4326, without observing off-target central nervous system events seen with RGLS4326, and is currently in Phase Ib clinical trials (NCT02855268).
Additionally, lademirsen, an antiviral drug targeting miR-21, was tested in a Phase II clinical trial (NCT05521191) for Alport syndrome, a rare kidney disease. Although the drug showed good tolerability, mid-term efficacy analysis results led to the termination of the study.
Advantages and Challenges of NBT
Some advantages of NBT make it highly suitable for treating protein deficiencies caused by many known diseases. NBT features high target specificity, good stability, significantly prolonged half-life (such as weeks or months), and less frequent dosing. Several approved NBTs in clinical trials have demonstrated good tolerability. The short development cycle and low cost of NBT enable personalized treatments for rapidly developing cancers and rare genetic diseases.
However, compared to traditional treatment modalities, NBTs also face some challenges. A common drawback of current NBTs is their inability to penetrate the intestinal wall or blood-brain barrier. Currently, this issue can be addressed through invasive methods such as intravenous, subcutaneous, intracerebral, intraventricular, or intrathecal administration. However, these methods are invasive and may potentially cause adverse effects, especially in central nervous system delivery. Therefore, extensive research is needed to develop chemically modified and carrier-mediated delivery technologies to reduce invasive delivery routes. Additionally, compared to most small molecules, NBTs have complex and currently poorly understood pharmacokinetics and pharmacodynamics. Despite these limitations, the overall success rate of NBT development reportedly matches or exceeds the industry average. An analysis of 7,455 drug development projects conducted between 2006 and 2015 found that only about 6% of new molecular entities entering clinical trials were approved. In this analysis, the success rate of novel biologics was 11.5%.
Recently, advances in NBT have opened up significant new opportunities for upregulating protein expression to treat diseases. The success of mRNA vaccines for COVID-19 has brought RNA-targeted protein upregulation technology into the spotlight and may accelerate major breakthroughs in the field. Improvements in gene sequencing technology and 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 increased the number of diseases with known genetic causes, all of which increase the applicability of NBT for upregulating proteins.
Reference
Khorkova, Olga, et al. “Amplifying gene expression with RNA-targeted therapeutics.” Nature Reviews Drug Discovery 22.7 (2023): 539-561.