Addressing RNA Research Challenges

RNA-based therapeutics include an ever-expanding repertoire of messenger RNAs, microRNAs, small interfering RNAs, antisense oligonucleotides and aptamers. RNA-based therapeutics can target diverse cellular molecules, including those deemed “undruggable” – pathways or molecules that are not amenable to targeting by conventional drugs.

The messenger RNA (mRNA) vaccines developed for protection against COVID-19 are perhaps the most widely recognized RNA-based intervention, but other RNA therapeutics have been approved for the treatment of muscular dystrophy, macular degeneration and other rare diseases, with even more currently in clinical trials.

Technology Networks had the pleasure of speaking to Dr. Eliza Small, Senior Manager at Azenta Life Sciences to find out about the major challenges facing researchers developing RNA-based therapeutics and how these challenges can be tackled.

Katie Brighton (KB): How is RNA technology currently being used for the development of therapeutics? What are the emerging trends within this field?

Eliza Small (ES): After its use in developing vaccines addressing COVID-19, a renewed interest in RNA technologies has surfaced in advancing therapies for diseases and disorders such as cancer, cystic fibrosis, amyloidosis and diabetes, among others. RNA-based therapeutics target diseases that cannot be treated by other conventional drug groups and several clinical studies are underway for a variety of therapeutics treating a series of incurable diseases.

RNA-based therapies modify gene expression or introduce new genes to a target cell. They can be a few dozen nucleotides in length – such as antisense oligonucleotides, small interfering RNAs and microRNAs – or up to thousands of bases, in the case of messenger RNA (mRNA). The smaller RNAs are primarily used to silence gene expression or modulate splicing. The larger mRNA therapies can replace defective proteins in the cell, generate antigens for vaccination or edit the genome via CRISPR technology.

RNA therapeutics have two major advantages over conventional pharmaceuticals; firstly, most therapeutic targets are undruggable by small molecules or antibodies, which need a favorable binding site on a protein. RNA therapies have no such limitation since they use the cell’s genetic machinery to effect changes. Secondly, RNA therapies can be designed and manufactured quickly. Technologies for RNA synthesis are well established, enabling rapid and cost-effective production of virtually any sequence.

KB: What are the typical challenges researchers face when working with RNA?

ES: For mRNA therapeutics, common issues include stability, translation efficiency and purity of the final product. An mRNA molecule is inherently unstable, inside and outside the cell. Structural elements – such as a 5’-cap, poly(A) tail, and untranslated regions – boost the translation efficiency and stability of the transcript.

Researchers can face serious limitations in the quality, scalability or speed of mRNA production if the process is not sufficiently optimized. Synthetic mRNA is generally produced by in vitro transcription, which requires a DNA template that is often a product of artificial gene synthesis. Once the mRNA is produced, quality control is critical to verify that the sequence is accurate and the full transcript is intact. Otherwise, downstream experiments may yield poor or inconsistent results.

KB: How does Azenta’s experience with RNA technologies address these research challenges?

ES: Over the past two decades, Azenta Life Sciences has supported the scientific community with its robust capabilities for nucleic acid sequencing and synthesis. Understanding the vast opportunities offered by the advancement of RNA therapeutics, we’ve continued to invest in furthering this expertise to offer researchers an end-to-end workflow that seamlessly combines gene synthesis and in vitro transcription. Researchers simply provide a nucleotide sequence as an input and we generate a high-quality mRNA product that is ready for research use with rapid turnaround. Our process efficiently adds a 5’ cap and poly(A) tail to the transcript to increase its stability and functionality.

Stringent quality control protocols measure accuracy throughout the workflow to ensure the fidelity and integrity of the final product at every step of the process. The accuracy of the DNA template used for in vitro transcription is fully verified by Sanger sequencing. The purity and size of the mRNA product is analyzed by UV spectroscopy, gel electrophoresis and bioanalyzer. Additionally, researchers can use our next-generation sequencing (NGS) capabilities to read full-length contiguous sequences of the mRNA molecules. This approach captures the poly(A) tail and enables you to measure the distribution of tail lengths.

KB: Could you elaborate on how Azenta’s full-length RNA-sequencing resolves poly(A) tails? Why might this be important when designing RNA therapeutics?

ES: Existing NGS-based methods to evaluate mRNA molecules have major trade-offs. Standard isoform sequencing (Iso-Seq) on the PacBio® platform can read individual transcripts end-to-end, but the poly(A) tail is largely removed prior to sequencing. Assays on the Illumina® short-read platform measure the distribution of polyadenylation length; however, the data lacks the context of the rest of the transcript.

To address these shortcomings, Azenta Life Sciences developed a library preparation protocol for PacBio sequencing that retains the poly(A) tail, enabling full-length RNA sequencing (RNA-Seq). You can see exactly what RNA molecules are present in the sample and their frequency. Mixed populations that include truncated products can be characterized with single-nucleotide resolution. Since short poly(A) tails can destabilize the mRNA in vivo, thorough characterization of an mRNA product is strongly recommended prior to use in experiments. Full-length RNA-Seq provides an effective and high-throughput solution for quality control of these therapies.

KB: What benefits does Azenta's RNA expertise bring to a researcher looking to advance their therapeutic discoveries?

ES: Interest in RNA therapies is surging, especially with the high-profile success of the mRNA COVID vaccines. Over a dozen RNA-based therapeutics have been approved by the FDA. About 30 are currently in clinical trials and many more are in early development. It’s becoming a very competitive space. Researchers prize speed and reliability and need a collaborative partner that shares the same professional ethic. These shared priorities make Azenta communication and research progress seamless, operating as a single, cohesive research group from anywhere in the world.

By partnering with Azenta, they can access tools to develop RNA therapies faster and better. Our genomics services like mRNA synthesis and full-length RNA-Seq have been purpose-built for scientists working on RNA therapeutics. They help increase reproducibility, reduce troubleshooting and free up time for critical experiments.

KB: What challenges remain when developing RNA therapeutics? How does Azenta envision overcoming such challenges in the future?

ES: The stability, delivery and immunogenicity of RNA-based drugs can still be improved. The coming years will likely bring major innovations to make these therapies more effective and better targeted. At Azenta, we’re excited by the growth and progress of RNA therapeutics thus far and look forward to seeing how the field matures. We’ll continue to monitor the latest developments and build solutions to help customers bring RNA therapies to market faster.

Dr. Eliza Small was speaking to Katie Brighton, Scientific Copywriter for Technology Networks.