The success of mRNA vaccines during the COVID-19 pandemic highlighted the broader potential of nucleic acid therapeutics (NATs). This class of medicines includes siRNA, antisense oligonucleotides and guide RNAs for gene editing, alongside increasingly sophisticated mRNA constructs for oncology, rare disease and infectious disease.
This momentum has exposed a tension in the sector, with discovery capabilities outpacing the ability to manufacture these modalities at scale. Each modality has its own chemistry demands, yet successful manufacturing depends universally on the quality of the raw materials and the reliability of the synthesis methods used to make them.
The industry must acknowledge that manufacturing infrastructure built for low-volume, rare disease programs is not well-positioned to supply the scales that large-volume commercial-stage programs require. In many ways, the conventional synthesis methods that underpinned the first generation of approvals are reaching a critical threshold. Addressing these bottlenecks requires the industry to advance chemistry innovation and scalable infrastructure in tandem, rather than treating manufacturing as an afterthought.
Have the limits of scalability been reached in oligonucleotide manufacturing?
Solid-phase oligonucleotide synthesis (SPOS) has been the standard method for manufacturing oligonucleotides for four decades and works effectively for short, chemically modified sequences. Yet as the field looks toward large-patient indications with more complex molecules, the operational limitations become apparent. Flow-through synthesizers employed in SPOS are typically limited to batches of around 10 kg, and producing larger quantities means running multiple campaigns and pooling batches (scaling out). This scale-out approach is inefficient, driving up cost, complexity and waste. For siRNA drugs targeting cardiometabolic diseases, where global patient populations can number in the tens of millions, demand for a single successful product could eventually reach ton-scale API requirements annually, a scale that SPOS was never designed to meet.
Long oligonucleotides, such as guide RNA molecules used in gene editing drugs, also present problems. Synthesizing these molecules, which can exceed 100 nucleotides in length, by sequential phosphoramidite coupling means that impurities accumulate with every step. These cumulative errors result in truncated sequences and other byproducts that are difficult to remove during purification, making batch-to-batch consistency challenging.
Chemoenzymatic ligation is a scalable solution for the next stage of oligonucleotide manufacturing
Chemoenzymatic ligation addresses these constraints by combining chemical and enzymatic synthesis. Rather than building the full-length oligonucleotide in one synthesis campaign, the molecule is assembled from shorter SPOS-derived fragments that are joined using T4 RNA ligase. The shorter fragments are synthesized more cleanly and the ligation reaction is highly selective, proceeding preferentially with correctly positioned, viable substrates. Many fragment impurities are therefore excluded from the final product, reducing truncation sequences, simplifying downstream processing and enhancing purity.
The ligation step is performed in aqueous buffer, making it compatible with batch reactors and single-use bioreactors, which are inherently more scalable than flow-through synthesizers. The industry is currently exploring two distinct workflows for this emerging method: sticky end ligation for double-stranded molecules such as siRNA, and splinted ligation for single-stranded oligonucleotides such as guide RNAs. Both approaches support the full range of chemical modifications found in NAT development candidates, including phosphorothioate, phosphodiester and phosphoramidate backbones, 2′-O-methyl and 2′-fluoro ribose modifications and GalNAc conjugates.
The chemoenzymatic ligation process also offers flexibility in how manufacturers balance purity, yield and cost. Fragments sufficiently pure in crude form can be processed by ultrafiltration and diafiltration without chromatography, increasing yield without sacrificing purity. This workflow is called C-to-P (crude-to-purified). This lays the foundation for a fully chromatography-free C-to-C (crude-to-crude) process, which is a potentially game-changing advance for high-volume cardiometabolic programs where cost of goods is a key commercial consideration.
Realizing the potential of this approach also requires a more robust enzymatic toolkit. Wild-type T4 RNA ligase has limitations around operating temperature, and RNA substrates can form secondary structures that reduce ligation efficiency. To address this, the development of thermostable T4 RNA ligase variants that function at temperatures up to 57°C can help reduce secondary structure formation, improving substrate alignment and ligation efficiency. This advance is expected to be particularly beneficial for enabling C-to-C processes and for the synthesis of very long constructs such as prime editing guide RNA (pegRNA). The challenge now is to show that these gains can be delivered consistently across diverse sequences, modification patterns and commercial-scale processes.
Cap analog chemistry and optimizing mRNA performance
Innovation in NAT manufacturing extends beyond oligonucleotides. In the mRNA field, considerable effort is focused on optimizing the 5′ cap structure. The chemistry of the cap directly influences IVT performance, translation efficiency, transcript stability and innate immune recognition. The conventional Cap1 structure, which includes a 2′-O-methylation at the first nucleotide (N1), has known limitations, including the ability of the innate immune protein IFIT1 to bind Cap1 RNA under inflammatory conditions, sterically blocking translation initiation. In repeat-dose or oncology settings where the immune environment is active, this can reduce protein expression.
Novel cap analogs address this through rational structure-based design. By substituting a bulkier 2′-O-ethyl group at the N1 ribose position, the cap introduces a steric clash with key IFIT1 residues, destabilizing the IFIT1-RNA interaction while maintaining compatibility with standard IVT workflows. Both trinucleotide Cap1 analogs and tetranucleotide variants have been developed, improving capping efficiency and broadening applicability across mRNA chemistries.
In IFNα-stimulated cell models and LPS-challenged mice, these novel analogs maintain protein expression at levels that conventional cap structures cannot match under the same conditions. In a melanoma tumor inhibition model, mRNA using this modified cap structure achieved approximately 60 percent tumor growth inhibition versus around 30 percent for a conventional Cap1 comparator. The recent advance of mRNA compositions containing one of these novel cap analogs into investigator-initiated clinical trials for solid tumors, acute myeloid leukemia and post-surgical pancreatic cancer is an important translational milestone.
The vulnerability of fragmented raw material supply chains
Phosphoramidites, loaded solid supports, GalNAc, enzymes, nucleotide triphosphates and cap analogs are complex inputs whose quality directly impacts NAT drug substance quality and therapeutic success. Even trace impurities in phosphoramidites can react, propagate and accumulate during SPOS, producing a distribution of related impurities in the oligonucleotide product. Controlling these impurities to ensure batch-to-batch consistency requires institutional knowledge, combined with robust processes and dedicated infrastructure.
Despite this, it is typical for NAT developers to source individual raw materials from multiple vendors, distributing quality risk across organizations and requiring time-consuming material handoffs. A vertically integrated supply model consolidates the supply chain from nucleoside building blocks through to cGMP drug substance and drug product, reducing handoffs and concentrating accountability. When a raw material or process changes, the investigation, corrective action and revalidation all happen within one organization, removing the delays and misalignment that characterize fragmented supply chains. That continuity of control is critical for allowing chemistry innovation to translate reliably into successful and efficient manufacturing outcomes.
Advancing chemistry and infrastructure in tandem
The NAT field is not short of excitement and promise: siRNA programs are advancing toward commercial volumes that will test existing infrastructure; gene editing medicines are moving into the clinic with increasingly complex guide RNA constructs; mRNA therapeutics are demanding improved performance, including sustained protein expression in immunologically challenging environments. However, developers are still largely using a manufacturing toolkit designed for a previous era. Each new modality creates specific manufacturing requirements that current tools were not designed for.
Chemistry innovation and manufacturing capability must develop together. Innovation without infrastructure stays in the lab, and infrastructure without innovation delivers the same products without improvements in quality or efficiency. The next generation of NATs will be defined by organizations that invest seriously in both, with the foresight to overhaul their chemistry and manufacturing platforms together.
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