RNA Therapeutics at a Cossroads: Innovation Meets Infrastructure
The therapeutic potential of RNA is no longer unproven or speculative — in the US, there are at least 21 U.S. Food and Drug Administration (FDA) approved, on-market, RNA-based therapies, as well as a strong pipeline of products in clinical development.10 Oligonucleotide-based modalities such as small interfering RNAs (siRNA) and antisense oligonucleotides (ASO) have achieved clinical success, and RNA interference (RNAi) based treatment approvals extend beyond rare genetic disorders to chronic diseases like cardiovascular conditions. The global RNA therapeutics market is projected to reach $18 billion by 2028, reflecting growing pipelines, expanded indications and the demand for targeted gene-silencing approaches like RNAi.1, 2
This shift from rare to chronic conditions has significant implications for manufacturing. Earlier RNA therapies served small patient populations with limited dosing needs. However, the approval of LEQVIO® (inclisiran), an siRNA for atherosclerotic cardiovascular disease, marked a turning point. Designed for millions of patients requiring ongoing treatment, inclisiran exemplifies the scale and reliability now expected of therapeutic RNA production systems.3
Yet the manufacturing systems behind these therapies remain under strain. Most RNA drugs are still produced via phosphoramidite chemistry (PAC) — a method originally developed for research, not large-scale production. As therapeutic complexity and volume requirements rise, PAC faces growing scrutiny over its scalability, cost, and environmental impact.
PAC workflows require specialized infrastructure (e.g. solvent tank farms) and are inherently subject to cumulative yield loss intensifying with sequence length. For instance, producing multi-kilogram quantities of RNA involves managing large volumes of flammable solvents such as acetonitrile and toluene, along with costly waste treatment. These pressures have prompted developers to explore alternative manufacturing approaches that can reduce costs, simplify workflows, and support greener chemistry without compromising quality or yield.4
A Closer Look at Conventional RNA Synthesis
PAC has been the standard method for oligonucleotide synthesis for decades. It enables high-fidelity construction of RNA sequences and has become deeply embedded in development workflows. However, as RNA therapeutics move into higher-volume indications, the limitations of PAC become harder to ignore, particularly around scalability, cost efficiency, and environmental impact.
The stepwise nature of PAC requires repeated cycles of nucleotide coupling and deprotection, each dependent on protected nucleosides and solvent-heavy conditions. While coupling efficiency is typically high, the cumulative demands of large-scale synthesis introduce practical challenges, including increased reagent use, prolonged cycle times, and stricter infrastructure requirements.4
The synthesis, cleavage and deprotection processes generate undesirable by-products such as truncated strands, bases impacted by depurination and residual protecting groups, requiring extensive chromatographic cleanup to meet therapeutic-grade purity. These processes also depend heavily on hazardous organic solvents like acetonitrile, toluene, and dichloromethane, which further complicate residual solvent limits and ESG compliance due to storage, disposal, and worker safety concerns.5,6
While innovations such as liquid-phase and semi-continuous synthesis offer incremental improvements, they do not alleviate ESG demands nor resolve PAC’s limitations in scaling synthesis batches beyond ~10 kg for chronic disease programs.7
Why Enzyme Engineering is Gaining Ground
While PAC will likely retain value for specific applications, particularly for low demand approved therapies, developers pursuing scalable, cost-efficient, and sustainable RNA manufacturing are turning to more flexible alternatives. Enzyme-catalyzed synthesis is emerging as a viable alternative to PAC, operating in mild, aqueous buffers and eliminating the need for protecting groups or hazardous solvents. Enzyme-based RNA synthesis simplifies workflows and reduces environmental burden.8
Enzymatic RNA synthesis typically employs template-independent polymerases, and RNA ligases to assemble oligonucleotides from shorter fragments. These enzymes are inherently compatible with a variety of RNA formats, including antisense oligonucleotides (ASOs) and siRNAs.
However, wild-type enzymes often fall short when applied to large scale pharmaceutical manufacturing. Their performance tends to be narrow in scope, with limited tolerance for modified backbones (e.g., 2'-OMe, 2'-F, phosphorothioates), reduced activity at high substrate concentrations, and slower kinetics under process-relevant conditions. For complex therapeutic siRNA constructs that feature chemical modifications, and terminal modifications (e.g. tissue targeting moieties), these limitations become acute, resulting in poor conversion, process variability, and scale-up challenges.
To meet the demands of large-scale RNA production for therapeutic formats such as siRNA, enzymes must be optimized not just for activity, but for robustness, specificity, and manufacturability.
Crucially, advances in enzyme engineering have enabled these systems to meet the rigors of industrial-scale manufacturing. Using directed evolution and machine learning informed design, enzymes can be optimized for:
- Substrate flexibility: Enabling modifications such as 2’-OMe, 2’-F, and phosphorothioate bonds, which are commonly used to improve the stability, safety, and efficacy of siRNA therapeutics.
- Enhanced kinetics: Faster ligation without compromising fidelity.
- Process robustness: Stability across wider temperature and pH ranges.
Engineered enzymes also support modular manufacturing strategies. Scientists can rapidly screen libraries of enzyme variants to identify optimal enzyme-substrate combinations for a specific RNA sequence or modification pattern. This approach enables tailored workflows that reduce the need for process re-engineering with each new molecule, ultimately accelerating development and scale-up.
Unlike PAC which often forces developers to adapt their design to the limits of the chemistry, enzyme engineering enables workflows to be built around the molecule. This flexibility reduces development time and supports broader therapeutic exploration.
With mounting pressure to accelerate timelines, cut costs and reduce environmental impact, enzyme-enabled synthesis offers a platform that is not just technically sound but also strategically aligned with the direction of the field.
Engineering for Complexity: Ligation-Based Assembly
Ligation-enabled RNA manufacturing typically involves a two-step process; the synthesis of fragment RNA strands (either chemically or enzymatically), followed by using a ligase enzyme to assemble longer or duplexed sequences. This modular strategy differs from traditional PAC synthesis, which builds the entire RNA strand step by step on a solid support.
In the enzymatic route, these short fragments are synthesized using template-independent polymerases under mild, aqueous conditions. This sequential extension process avoids the need for protecting groups or harsh solvents and is particularly well suited for producing highly modified RNA formats.
While enzymatic synthesis can be used alone, a hybrid approach is also possible, where short PAC generated fragments are ligated enzymatically to overcome length and complexity constraints.
Today’s RNAi therapeutics increasingly involve duplexed or chemically modified structures that strain the limits of conventional synthesis due to their length, double-stranded architecture, and reliance on site-specific chemical modifications. As sequences grow longer and incorporate backbone or sugar modifications, PAC struggles with yield loss, impurity accumulation, and purification complexity e.g. additional polishing steps.
In these cases, enzymatically generated fragments can offer advantages over PAC derived strands, including improved sustainability, greater batch scalability, and easier adaptation to complex modifications. Ligation-based assembly, where short RNA fragments are joined enzymatically offers a scalable and modular strategy, particularly well-suited to constructs like siRNAs.9
This approach avoids the cumulative yield loss associated with stepwise synthesis. By starting with high-purity oligonucleotide fragments produced via chemical or enzymatic means, ligation-based assembly simplifies or even eliminates purification steps.
However, the success of ligation hinges on the ligase. Wild-type ligases typically struggle with modified bases, tolerate only narrow reaction conditions, and perform poorly at the high substrate concentrations needed for commercial manufacturing.
Through advances in protein engineering, double stranded RNA (dsRNA) ligases have been developed to overcome the limitations of wild-type enzymes. Engineered dsRNA ligases now exhibit significantly improved conversion rates, compatibility with high substrate loads, and tolerance to broader pH and temperature ranges while retaining activity with chemically modified RNA inputs such as 2'-OMe, 2'-F, and phosphorothioate linkages.
Some engineered ligases can even ligate fragments directly from crude diafiltered input pools, reducing intermediate purifications and accelerating production timelines.
These improvements have the potential to reduce process complexity, increase batch yields and expand adaptability across different RNA formats. As therapeutic designs grow more structurally diverse, ligation provides a high-fidelity, scalable path forward that aligns with modern expectations for process efficiency, flexibility, and throughput.
Comparative Benefits: From Technical Gains to Strategic Advantage
The move to enzyme-enabled RNA synthesis offers more than incremental improvements, it opens the door to operational advantages that directly support commercial scalability, regulatory alignment, and long-term sustainability goals.
Cost and productivity gains
Phosphoramidite synthesis suffers from declining yields as sequence length increases, driving up the cost per gram and complicating batch planning. By contrast, ligating oligonucleotide fragments enables fast, high-conversion assembly of duplex RNA, even at high substrate concentrations. The elimination of purification steps result in higher yields reducing the number of batches required for a given output.
For commercial developers, this means fewer consumables, lower enzyme loading and the potential to run at higher volumes or integrate semi-continuous production factors that directly affect the cost of goods.
Improved robustness and fewer failure points
PAC based workflows are sensitive to protecting group chemistries, which can affect both reaction efficiency and byproduct formation, especially when scaling or modifying sequences. These dependencies increase risk during tech transfer, scale-up and GMP validation, where process reproducibility is critical.
Wild-type ligases also present challenges at scale, particularly when working with modified substrates or under variable conditions. Engineered enzymes are developed to overcome these constraints. They are designed to tolerate structural modifications and maintain activity across a range of process environments, helping simplify development and improve process robustness. This level of robustness becomes especially valuable when scaling across multiple sites or transferring processes to contract manufacturing organization (CMO) partners.
Sustainability
The environmental footprint of PAC is substantial. Solvents used such as acetonitrile, toluene, dichloromethane, and pyridine, generate chemical waste that must be stored, treated, and disposed of in compliance with safety and environmental regulations.
Enzyme-catalyzed platforms eliminate the need for many of these reagents by operating in aqueous buffers under mild conditions. The result is a cleaner, safer process that aligns with ESG commitments and supports sustainable pharmaceutical manufacturing practices.
Building a More Flexible Manufacturing Future
As RNA therapies diversify the ability to incorporate a broad range of constructs reliably and efficiently, economical manufacturing has become a core differentiator.
Traditional synthesis platforms often constrain RNA design, limiting sequence length, chemical modifications, and structural complexity. In contrast, enzyme-enabled manufacturing offers greater adaptability. Optimized enzymes can support a wide range of formats, including 2'-OMe and 2'-F substitutions, phosphorothioate backbones, and site-specific conjugates.
Enzymatic platforms also support faster process development. Enzyme libraries can be screened early to identify catalysts that match each molecule’s specific structural and chemical characteristics, reducing optimization timelines and scale-up risk. Their compatibility with aqueous systems allows online mass spectrometry analysis and reduced reliance on specialized infrastructure, facilitating smoother tech transfer between internal teams and external partners.
Importantly, enzymatic synthesis often produces cleaner output, reducing side products and simplifying impurity profiling.
For RNA innovators navigating complex pipelines, enzymatic synthesis provides the modularity, scalability and process control needed to adapt quickly without compromising on safety, quality, or efficacy.
Reframing RNA Manufacturing for What Comes Next
RNA therapeutics have matured from niche innovations to essential components of modern medicine. However, realizing their full potential requires more than innovative targets or delivery platforms; it demands a manufacturing foundation that can keep pace with therapeutic ambition.
PAC has brought the field far, but its constraints are increasingly out of step with the scalability, speed and sustainability RNA developers now require. Enzymatic synthesis offers a new model: modular, efficient, and adaptable by design. Advances in enzyme engineering are now making it possible to tailor manufacturing processes to the molecule.
For developers navigating compressed timelines, diverse modalities and global supply expectations, the shift from PAC to enzyme-catalyzed synthesis isn’t optional; it’s a strategic necessity and a defining moment for RNA manufacturing.
References
- Markets and Markets. RNA Therapeutics Market by Type, Application & Region – Global Forecast to 2028. https://www.marketsandmarkets.com/Market-Reports/rna-therapeutics-market-235963408.html
- Martin Egli, Muthiah Manoharan, Chemistry, structure and function of approved oligonucleotide therapeutics, Nucleic Acids Research, Volume 51, Issue 6, 11 April 2023, Pages 2529–2573, https://academic.oup.com/nar/article/51/6/2529/7070965
- Novartis. FDA approves inclisiran (Leqvio) for LDL-C reduction in patients with ASCVD or HeFH. 2021. https://www.novartis.com/news/media-releases/fda-approves-novartis-leqvio-inclisiran-first-class-sirna-lower-cholesterol-and-keep-it-low-two-doses-year
- Beaucage SL. Sustainability Challenges and Opportunities in Oligonucleotide Manufacturing. J Org Chem. 2021;86(1):49–61.
- Impurities in Oligonucleotide Drug Substances and Drug Products Daniel Capaldi, Andy Teasdale, Scott Henry, Nadim Akhtar, Cathaline den Besten, Samantha Gao-Sheridan, Matthias Kretschmer, Neal Sharpe, Ben Andrews, Brigitte Burm, and Jeffrey Foy Nucleic Acid Therapeutics 2017 27:6, 309-322
- International Council for Harmonisation (ICH). (2024). Q3C(R9) Guideline for Residual Solvents – Minor Revision. Step 4 version dated 24 January 2024. https://database.ich.org/ sites/default/files/ICH_Q3C(R9)_Guideline_MinorRevision_2024_2024_Approved.pdf
- Katayama, S., & Hirai, K. (2018). Liquid-phase synthesis of oligonucleotides. In S. Obika & M. Sekine (Eds.), Synthesis of therapeutic oligonucleotides (pp. 83–95). Springer Singapore. https://link.springer.com/chapter/10.1007/978-981-13-1912-9_5
- Alnylam Pharmaceuticals. (2025, February 25). Alnylam R&D Day 2025 [PowerPoint slides, slide 171]. Capella. https://capella.alnylam.com/wp-content/uploads/2025/02/Alnylam-RD-Day-2025-Slides.pdf
- Recent Advances in Biocatalytic and Chemoenzymatic Synthesis of Oligonucleotides Pierre Nicolas Bizat, Nazarii Sabat, Marcel Hollenstein First published: 24 January 2025 https://doi. org/10.1002/cbic.202400987
- Avalere Health. (2024, June 3). Lilly RNA-Based Therapies White Paper. Avalere Health. https:// advisory.avalerehealth.com/wp-content/uploads/2024/06/20240522-Lilly-RNA-Based-Therapies-White-Paper-vFINAL.pd
Author Details
Kevin Norrett, Chief Operating Officer, Codexis Inc.
Publication Details
This article appeared in Pharmaceutical Outsourcing:Vol. 26, No.2 Apr/May/June 2025Pages: 37-39