What’s next for nucleic acid therapeutics?

Dozens of oligonucleotide-based therapeutics are already on the market, helping to treat a range of life-threatening conditions. As we explored in our previous blog post, these drugs take several different approaches to disrupt disease-causing processes. In this post, we will look at some of the cutting-edge technologies in development that could form the treatments of the future. 

Circular RNA

Stability is one of the challenges for mRNA therapeutics, requiring expensive capping agents, careful storage and higher doses to overcome. Circular RNAs, as the name suggests, avoid having 5ʹ or 3ʹ ends that are vulnerable to exonuclease degradation.^1,2 These single-stranded RNA rings can be modified to enable protein translation similarly to modified linear mRNAs but for a longer duration.   

Single-strand circular DNA

Like circRNA, circular single-stranded DNA is extremely stable and protected from exonucleases, presenting opportunities for a range of therapeutics. This has included creating aptamers with greater stability, affinity and intracellular delivery and using circDNA as a scaffold for DNA origami to form 3D nanostructures for use as drug delivery vehicles.^3,4,5  

This DNA origami approach has also been used to develop a conformationally switchable genetic vector that offers reversible control of gene expression in mammalian cells.⁶ In addition, other researchers have investigated using circDNA as the donor DNA in CRISPR-Cas gene editing to improve the efficiency of HDR-mediated insertion.⁷  

Research has also shown that circDNA can bind and inhibit microRNAs. By synthesising single-stranded circDNA containing miR-9 complementary sites, Meng et al. reduced silencing of multiple, co-silenced tumour suppressor genes.⁸ This prevented tumour progression and lung metastasis in patient-derived xenograft models.  

While this work is still in the early stages, the range of uses for single-strand circular DNA is an exciting prospect for advancing therapeutics. 

Transfer RNA

tRNA fulfils one of the final steps in the translation from DNA to protein by supplying the corresponding amino acid based on the codon presented by the mRNA. While this has received little attention in the therapeutics space, research is beginning to show the potential of engineered tRNA to overcome diseases like cystic fibrosis.  

An estimated 11% of genetic diseases are caused by mutations that introduce stop codons in the mRNA sequence, leading to truncated proteins.⁹ For instance, a CGA codon that codes for arginine can be mutated to UGA, a stop codon. By engineering tRNA molecules that can bind to the UGA stop codon and transfer an arginine amino acid to the ribosome, scientists could suppress the mutation and produce normal, full-length protein. 

Several biotech companies, including Alltrna, ReCode Therapeutics, Shape Therapeutics, Tevard Biosciences and hC Bioscience, are studying the potential of these molecules. It is still early days for this technology, but the potential for one engineered tRNA to overcome the same codon change in different genes offers an opportunity to treat multiple orphan diseases. 

tRNAs are usually 70 to 80 bases long and fold into an L-shaped structure. However, there is still a lot to learn about how best to deliver these molecules into the target cells at the right dose.¹⁰ 

miRNA mimics and inhibitors 

MicroRNAs are small, single-stranded noncoding RNAs that play an important role in regulating gene expression.¹¹ They can downregulate their target genes by either inducing mRNA degradation or inhibiting translation. miRNAs typically regulate gene networks or pathways to control biological functions, offering the opportunity to treat diseases that cannot be controlled by targeting single genes.

Oligonucleotides can be designed to mimic the activity of miRNAs, however this approach has yet to yield a successfully approved drug. One compound in the pipeline, MRG-229, is an oligonucleotide designed to mimic the activity of miR-29 and is currently being studied as a treatment for pulmonary fibrosis.¹² 

Alternatively, oligos can be created to inhibit miRNAs. The compound miravirsen is a modified oligonucleotide that selectively hybridises with miR-122. This was tested in phase II clinical trials as a treatment for Hepatitis C, but the developers discontinued it following the success of new antiviral treatments.¹³

Small activating RNA 

Although they have the same structure as siRNAs, small activating RNAs have the opposite effect and up-regulate target gene expression through a process called RNA activation. This involves recruiting the transcriptional machinery to the gene promoters. 

This innovative approach has not yet delivered any approved drugs, although MiNA Therapeutics has a candidate in phase II clinical trials for treating liver cancer. MTL-CEBPA restores expression of the C/EBP-α protein, with the aim of countering the cancer’s immune suppression activity and making existing drug treatments more effective. 

While small activating RNA could offer a powerful way to specifically activate genes, developers face challenges in getting the therapies into the cell without being degraded and eliminated.¹⁴ 

Base and prime editing

Building on the success of using single guide RNA to create CRISPR-based therapeutics, scientists are developing more refined approaches. Base editing and prime editing do not create double-strand breaks in the DNA, so avoid the potential safety risks due to repair errors.^15,16  

Rather than inserting or deleting stretches of DNA, these techniques allow individual bases to be precisely edited. Both use guide RNA to direct Cas9 to the target sequence and, in the case of prime editing, the RNA acts as the template for reverse transcription. The first clinical trial results were only reported in 2023, so it is still too early to understand how these approaches will develop.¹⁷ 

RNA editing

Another future alternative to CRISPR-based therapeutics is to edit disease-causing mutations at the RNA level rather than the cell’s DNA. Modifying the short-lived RNA before it is translated into protein has the same ultimate effect as gene editing, but without changing the DNA. 

This transience could be an advantage. If there are any unforeseen adverse effects, the treatment can be stopped or reduced, whereas edits to DNA can be permanent. This could help RNA editing therapeutics to advance more quickly through testing and regulatory stages. 

A promising approach uses enzymes known as adenosine deaminases acting on RNA (ADARs). These convert adenosines in double-stranded RNA to inosine, which is recognised as guanosine by the translational machinery. This can help to treat G-to-A mutations, which make up 28% of pathogenic single-nucleotide variants.¹⁸ 

The first RNA editing therapeutic entered testing in clinical trial participants in the UK in December 2023. The drug, WVE-006, is an RNA oligonucleotide designed to target a mutation that causes alpha-1 antitrypsin deficiency. WVE-006 binds to the mutation site in the mRNA, recruiting ADAR to modify an adenosine base, enabling the functional protein to be produced. The oligo, developed by Wave Therapeutics, has a GalNAc modification to help it reach the liver. 

In January 2024, the FDA cleared the first RNA exon editor for clinical testing. ACDN-01 interferes with the splicing process, removing exons with disease-causing mutations and replacing them with corrected copies. Ascidian Therapeutics is testing this to treat Stargardt disease, a rare genetic eye condition caused by over 1,000 different mutations across the ABCA4 gene. This variation makes it extremely difficult to treat with standard gene therapy or editing, so RNA editing offers a potential way to create a single treatment for multiple patients. 

There are many more innovative approaches in development at the moment, but this snapshot shows the scale of ambition for the sector. As knowledge of how to deliver the best therapies evolves, LGC Biosearch Technologies can offer custom modifications that scales with your needs. 

References

1. Liu X et al. (2022) Circular RNA: An emerging frontier in RNA therapeutic targets, RNA therapeutics, and mRNA vaccines. J Control Release. 348:84-94. doi: 10.1016/j.jconrel.2022.05.043  
2. Abe H et al. (2009) Synthetic nanocircular RNA for controlling of gene expression. Nucleic Acids Symp. Ser. Oxf. 65–66. doi: 10.1093/nass/nrp033  
3. Kuai H et al. (2017) Circular Bivalent Aptamers Enable in vivo Stability and Recognition. J Am Chem Soc. 139:9128–31. doi: 10.1021/jacs.7b04547 
4. Pan X et al. (2020) A bispecific circular aptamer tethering a built-in universal molecular tag for functional protein delivery. Chem Sci. 11:9648–54. doi: https://doi.org/10.1039/D0SC02279A 
5. Jiang Q et al. (2012) DNA origami as a carrier for circumvention of drug resistance. J Am Chem Soc. 134:13396–403. doi: 10.1021/ja304263n 
6. Tang L et al. (2023) Circular single-stranded DNA as switchable vector for gene expression in mammalian cells. Nat Commun 14, 6665. doi:10.1038/s41467-023-42437-6 
7. Iyer S et al. (2022) Efficient Homology-directed Repair with Circular ssDNA Donors. CRISPR J. 5(5):685-701. doi: 10.1089/crispr.2022.0058 
8. Meng J et al. (2018) Derepression of co-silenced tumor suppressor genes by nanoparticle-loaded circular ssDNA reduces tumor malignancy. Sci Transl Med. 10(442):eaao6321. doi: 10.1126/scitranslmed.aao6321 
9. Temaj et al. (2023) Recoding of Nonsense Mutation as a Pharmacological Strategy. Biomedicines 11(3):659. doi: 10.3390/biomedicines11030659 
10. Coller J and Ignatova Z (2023) tRNA therapeutics for genetic diseases. Nature Reviews Drug Discovery 23:108–125. doi: 10.1038/s41573-023-00829-9 
11. Bartel DP (2018) Metazoan MicroRNAs. Cell. 173:20–51. doi: 10.1016/j.cell.2018.03.006 
12. Chioccioli et al. (2022) A lung targeted miR-29 mimic as a therapy for pulmonary fibrosis. eBioMedicine 85:104304. doi: 10.1016/j.ebiom.2022.104304 
13. Sparmann A and Vogel J (2023) RNA‐based medicine: from molecular mechanisms to therapy. EMBO J 42:e114760. doi: 10.15252/embj.2023114760 
14. Ghanbarian H et al. (2021) Small Activating RNAs: Towards the Development of New Therapeutic Agents and Clinical Treatments. Cells 10(3):591. doi: 10.3390/cells10030591  
15. Rees HA and Liu DR (2018) Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet 19: 770–788. doi: 10.1038/s41576-018-0059-1 
16. Anzalone AV et al. (2019) Search‐and‐replace genome editing without double‐strand breaks or donor DNA. Nature 576: 149–157. doi: 10.1038/s41586-019-1711-4 
17. Booth BJ et al. (2023) RNA editing: Expanding the potential of RNA therapeutics. Molecular Therapy 31(6):1533-1549. doi: 10.1016/j.ymthe.2023.01.005 
    

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