Introduction
In the ever-evolving landscape of medicine, nucleic acid therapeutics stand at the forefront of innovation. These groundbreaking treatments hold the promise of targeting diseases at their genetic roots, offering gene inhibition, addition, replacement or editing. They offer advantages in terms of target specificity aiming at proteins that were once ‘undruggable’, broad therapeutic potential through multifactorial targets, and potential for personalised medicine based on the genetic blueprint of a disease. Besides addressing protein-coding RNAs, the Encyclopedia of DNA Elements (ENCODE) project has shown that a significant part of the human genome encodes for non-protein coding RNAs (1). Among those, long noncoding RNAs are of particular interest from a clinical viewpoint (2).
This article explores the multifaceted journey of short-coding, synthetic RNAs, from their discovery to regulatory challenges, manufacturing advancements and challenges, and opportunities for digitalisation and automatisation. These molecules come with ongoing challenges to survive on their way to the inside of cells due to their size and physicochemical properties, immune responses leading to potential adverse effects, and off-target effects resulting in unintended consequences or toxicity.
Discovery
The history of nucleic acid therapeutics is deeply intertwined with scientific milestones, including Rosalind Franklin’s groundbreaking X-ray diffraction images of DNA, followed by Watson and Crick’s discovery of the double helix structure, which laid the foundation for understanding genetic information.
A new era in medicine with the treatment of diseases at their genetic roots started with antisense oligonucleotides (ASOs), which aimed to inhibit gene expression by binding to target RNA molecules, and shortly after with RNAi technology, a breakthrough that enabled specific gene silencing through siRNA molecules. This key milestone was the work of Fire and Mello on the RNA interference (RNAi) pathway for gene regulation performed in Tuschl’s laboratory (3). It demonstrated the ability of synthetic small interfering RNA (siRNA) to modulate protein expression in mammalian cells, including human cells.
Since the first RNA-based therapeutic, fomivirsen (Vitravene), was approved by the FDA in 1998 for treating cytomegalovirus retinitis, nucleic acid therapeutics have expanded to today more than 20 approved therapeutics, with hundreds of ongoing clinical trials worldwide.
Advancements and discoveries in genetics have significantly contributed to the development of these treatments. Morten Lindow, PhD, with experience in both academia and industry, commented in the interview that currently most targets being worked on in the field are genetically defined, which means that they have a high degree of validation and that many players are working on the same targets.
However, optimisation of the pharmacological properties and therapeutic efficacy of nucleic acid therapeutics is an ongoing effort, with an outlook to advancing their development for a wide range of diseases. In this respect, significant effort has been put into developing various delivery technologies (4). After approaches focused on lipid nanoparticles (LNPs) and synthetic nanoparticles, siRNA conjugate with a trimer of N-acetylgalactosamine (GalNAc), which is a ligand for the asialoglycoprotein receptor (ASGPR), proved to be a solution to the delivery problem for liver hepatocytes (5).
The GalNAc-siRNA approach resulted in 5 approvals, including the approval of Leqvio (Inclisiran). It is a first-in-class siRNA lipid-lowering therapy that targets the liver, inhibits the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9), and so reduces low-density lipoprotein cholesterol (LDL-C) and the risk of heart disease. Also, it is the first nucleic acid therapeutic for a disorder as common as high cholesterol.
Morten Lindow commented in the interview that it’s time for discovery to move from the beachhead of genetically defined diseases towards multifactorial and more common diseases.
The GalNAc-siRNA conjugates are a solution to the siRNA delivery problem for liver hepatocytes and have shown the path forward for targeting cells in multiple organs. Interest is expanding to extrahepatic organs, and many different delivery and distribution strategies are currently under exploration (e.g., nanoparticles, conjugation with antibodies and lipids like C16 for central nervous system (CNS) distribution.
Depending on the strategic business focus, companies can focus on disease biology rather than specific targets. Morten Lindow commented in the interview that once you have developed a sound disease model system (e.g., an animal model), the fast and generic nature of oligonucleotide discovery allows companies to test multiple target hypotheses in parallel or in combination.
Manufacturing
Innovation in the manufacturing of synthetic oligonucleotides is catalysed by progress in discovery, research and development (R&D), environmental sustainability and regulatory requirements. Holistic approaches with advancements in chemistry, biocatalysis, and process engineering technologies play a pivotal role in driving these innovations forward.
The synthesis of RNA API (Active Pharmaceutical Ingredient) typically starts with the automated solid-support phosphoramidite-based synthesis, which was developed more than 50 years ago. While starting materials with different chemical modifications and diverse reagents and reactants can be used, the synthesis method is well-established, predictable, and reliable. However, this widely used manufacturing process does not necessarily address all future expectations and demands. First, it requires a facility with specialized equipment, and the current technology has a batch size limitation. Additionally, atom efficiency is low since phosphoramidites are used as starting materials. Furthermore, solvent usage and, accordingly, waste generation is not favoring current greenness and sustainability considerations (6).
For Leqvio (Inclisiran), a collective demand projected on metric tons of quantities underscored the pressing need to enhance the scalability of manufacturing processes for improved efficiency, affordability, sustainability, and accessibility. Manufacturing approaches are being explored for larger quantities of potential therapeutics in a cost-effective and sustainable manner (7). Nowadays, more such therapeutic candidates are in the preclinical and clinical phases. On the other hand, the rapid evolution of gene editing technologies highlights a parallel need for smaller quantities of long RNAs with optimal yield and purity. This dual emphasis on quantity and quality underscores the ongoing pursuit of innovation in manufacturing.
Jale Muslehiddinoglu, PhD, with experience in both the pharmaceutical industry and CDMOs (Contract Development and Manufacturing Organizations), commented in the interview that the current solid-supported synthesis approach may pose limitations for large quantities. While proposals for technology modifications exist, such as continuous operation and increased automation, further exploration is needed. Evaluation of the stirred-bed approach is underway, particularly in leveraging peptide manufacturing capacity. The phosphorous five P(V) chemistry shows promise as a potential game changer, but its implementation maturity is still pending.
Besides advancements in alternative approaches to synthesis, other aspects of the manufacturing process, like purification, isolation strategies and quality control methodologies, continue to drive progress in the field, enabling consistent and reproducible quality of the final products. In purification, advanced chromatography systems could integrate robotics, online monitoring, and Quality Control (QC). Continuous purification systems are gaining popularity for oligonucleotide purification, offering advantages such as higher throughput, reduced processing times, reduced use of resin and solvent, and improved scalability. Advances in maximising the use of analytical tools and techniques and their capabilities are focused on providing valuable insights into sequence, structure, and purity.
While solid-supported synthesis is looking into alternative approaches, liquid-phase approaches are undergoing extensive evaluation (8). Linear or convergent synthesis, soluble support or enzymatic methods, and utilising hubs and membranes are among the alternative strategies being considered. Some emerging insights include the preference for convergent synthesis, the potential benefits of hybrid approaches, and the ongoing pursuit of enhanced efficiency and sustainability.
Still, the prevalent synthesis approach is Solid-Phase Oligonucleotide Synthesis (SPOS) and integrating Liquid-Phase Oligonucleotide Synthesis (LPOS) would require additional investments in equipment and facility infrastructure. It should be noted that the implementation of Process Analytical Technology (PAT) tools facilitates quality control and optimisation of manufacturing processes for both SPOS and LPOS approaches.
In conclusion, advancements in technology and holistic approaches to integrating science, automation, and engineering are instrumental in driving the rapid progress of manufacturing.
Developments in chemical synthesis, biocatalysis, and process engineering, coupled with innovative purification techniques and quality control, hold great promise for the sustainable production of diverse products, further propelling the field toward improved therapeutic outcomes and broader clinical applications.
Regulatory Landscape
With the COVID-19 pandemic, the mRNA vaccine demonstrated potential and brought global attention to nucleic acid therapeutics, streamlining the path to market approval. This pivotal moment has catalysed advancements in the field and propelled nucleic acid therapeutics into the forefront of medical innovation.
Bringing nucleic acid therapeutics from the laboratory to clinical trials and marketing authorisation is not without its challenges. Regulatory agencies such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) play a critical role in ensuring the safety, efficacy, and quality of New Molecular Entities (NME). Historically, regulatory pathways have primarily focused on small-molecule drugs, synthetic-peptide therapeutics, and biologics.
As advances in molecular biology and nucleic acid chemistry accelerated, so did the momentum in developing nucleic acid therapeutics, evidenced by the increasing number of regulatory approvals. Due to their size, structural complexity, and diverse chemical properties, these novel therapeutic modalities posed new regulatory challenges.
Dominik Altevogt, PhD, with experience in both the pharmaceutical industry and CDMOs, shared in the interview specific considerations that apply to this class of therapeutics:
The evolving regulatory landscape for synthetic oligonucleotides reflects their unique position at the interface between small molecules and large molecules (biologics). While general concepts for small molecules apply, specific guidelines for synthetic oligonucleotides are still limited.
The International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines do not specifically address the particular quality aspects of oligonucleotide drugs. Although ICH Q3A, ICH Q6A, and M7 specifically exclude oligonucleotides, the spirit of these guidelines nevertheless applies.
U.S. Food and Drug Administration (FDA): Synthetic oligonucleotides are regulated as drugs by the Centre for Drug Evaluation and Research (CDER).(9) FDA’s quality-related guidance for submission of Investigational New Drug (IND), New Drug Application (NDA) or supplements are applicable. Product-specific guidance is available.
European Medicines Agency (EMA): The guideline on the Chemistry of Active Substances (EMA/454576/2016) applies. In September 2022, EMA published a concept paper on the establishment of a guideline for the development and manufacture of synthetic oligonucleotides. The guideline will address the quality aspects specific to synthetic oligonucleotides.
Pharmaceuticals and Medical Devices Agency (PMDA): Oligonucleotides specific guidance is available: Points to Consider (PtC) on Chemistry, Manufacturing, and Controls (CMC) released in September 2018.
Furthermore, independent of regulatory authorities, industry stakeholders initiated the harmonisation of CMC aspects and developed strategies under the umbrella of the European Pharma Oligonucleotide Consortium (EPOC) (10).
Over the years, the regulatory landscape has evolved to accommodate the unique characteristics of nucleic acid therapies. However, navigating this evolving regulatory landscape remains complex, requiring rigorous preclinical testing, robust clinical trials, and close collaboration between industry stakeholders and regulatory authorities. Despite the challenges, as of 2023, 18 nucleic acid therapies have made it to the market. Among these, ASOs and siRNAs comprise a major segment (11).
Digitalisation
As this technological trend is reshaping all sectors, the life science industry is not excluded, although it was not initially established as digital. However, as it is driven by innovation and processes and is a crucial part of the healthcare ecosystem, technological incorporation and adaptation are inevitable.
The digital transformation of the life science industry involves leveraging digital hardware tools, such as robotics and automatization, alongside software solutions, such as artificial intelligence (AI), data analytics, and advanced modelling software. The potential advantages of digitalisation within life science companies are endless. They encompass innovation, flexibility, cost-effectiveness, efficiency, and productivity in all aspects of research and development, manufacturing, clinical trials, supply chain management, and the broader ecosystem.
In the context of nucleic acid therapeutics, one example where digital technology plays a crucial role is in the fast and efficient screening of molecules with variable sequences and different chemistries. After acquiring reliable and error-free data, AI models can be trained. Trustworthy AI models can guide the selection of optimal candidates for further development and offer valuable insights into structure-activity relationships (12).
Ming Wang, PhD, with experience in both academia and industry, elaborated in the interview that high-throughput screening generates a significant volume of data, exceeding the capacity of conventional tools like Excel for data processing. In this case, advanced data analysis platforms, ones that are designed to automate complex data processing steps and have inbuilt quality control features, can ensure data quality and consistency whilst saving time for researchers by removing many of the manual inputs. The integrity and consistency of processed data are important not only for immediate decision-making but also as reliable data inputs for training AI models.
In addition to newly generated data, historical data can also be repurposed and used as data inputs for model generation. However, it is important that such data is appropriately reformatted and quality controlled. Model generation is an iterative and dynamic process that relies on continuously updating and refining AI models with high-quality data. Thus, a cohesive data strategy is vital, fostering collaboration between wet lab and dry lab to ensure the acquisition, processing, and utilisation of reliable data throughout the drug discovery pipeline.
With digitalisation, hardware can be seamlessly scaled up with robotics, minimising errors and enhancing efficiency. Furthermore, digital technology can facilitate the integration of automated hardware with automated software data processing pipelines, ensuring seamless connectivity and data flow. As more data is generated, data storage, particularly in a structured manner, becomes important, especially if there is an incentive to use the new data for data science purposes.
In the ideal case, data which includes results from all forms of assays used in discovery, from in vitro screening assays to in vivo experiments, is mapped to compound identity and includes metadata that gives more description of the experimental procedures. For this purpose, digital tools can be adopted and, thereby, facilitate the registration of compounds, the processing of data, and the generation of reports in a more automated and, therefore, efficient way.
Conclusion
Nucleic acid therapeutics represent a transformative shift in the treatment of diseases. From their humble beginnings in the laboratory to the forefront of modern medicine, these innovative therapies hold immense potential to save and improve the lives of patients worldwide. To realise this potential, we must navigate various challenges, from discovery and delivery to manufacturing and regulatory domains. The rapid pace of progress in the field underscores growing confidence in the potential of these therapies as a cornerstone of 21st-century medicine. As research progresses and technology advances, the future holds immense promise for the continued evolution of these therapeutics, bringing in a new era of personalised medicine and broader clinical applications.
References and Notes
- The ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 2012; 489: 57–74.
- Mattick JS, Amaral PP, Carninci P, et al. Long non-coding RNAs: definitions, functions, challenges and recommendations. Nat Rev Mol Cell Biol. 2023; 24: 430–447.
- Fire A, Xu S, Montgomery M, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998; 391: 806–811.
- Paunovska K, Loughrey D, Dahlman JE. Drug delivery systems for RNA therapeutics. Nat Rev Genet. 2022; 23: 265–280.
- Nair JK, et al. Multivalent N-Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated Gene Silencing. J Am Chem Soc. 2014; 136: 16958–16961.
- Andrews BI, et al. Sustainability Challenges and Opportunities in Oligonucleotide Manufacturing. J Org Chem. 2021; 86; 49−61.
- Obexer R, Nassir M, Moody ER, Baran PS, Lovelock SL. Modern approaches to therapeutic oligonucleotide manufacturing. Science (New York, NY). 2024; 384; 6692: 4015.
- Creusen G, Akintayo CO, Schumann K, Walther A. Scalable One-Pot-Liquid-Phase Oligonucleotide Synthesis for Model Network Hydrogels. J Am Chem Soc. 2020; 142: 16610−16621.
- Platform Technology Designation Program for Drug Development Guidance for Industry, Draft Guidance, U.S. Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER), May 2024.
- Tivesten A, et al. European Pharma Oligonucleotide Consortium: A Move to Consolidate Oligonucleotide Knowledge and Share Experience Within the Community. Therapeutic Innovation & Regulatory Science. 2018; 52; 6: 687-688.
- Zhu Y, Zhu L, Wang X, et al. RNA-based therapeutics: an overview and prospectus. Cell Death Dis. 2022; 13: 644.
- Sciabola S, et al. PFRED: A computational platform for siRNA and antisense oligonucleotides design. Plos. 2021, January 22.
