RNA therapeutics represent one of the most promising frontiers in modern medicine, offering highly specific mechanisms of action and the potential to address previously undruggable diseases. Dave Butler, Hongene discusses.
Hongene
The rapid expansion of therapeutic applications is driving unprecedented demand for synthetic oligonucleotides including small interfering RNA (siRNA) and single-guide RNA (sgRNA).
While solid-phase oligonucleotide synthesis (SPOS) has served as the foundation of oligonucleotide drug development for decades, its inherent limitations in scalability, sustainability, and cost-efficiency are increasingly misaligned with the needs of today’s drug developers. These constraints are particularly acute for the production of high-purity, chemically modified RNAs required in both large-scale siRNA applications and advanced gene editing modalities.
Chemoenzymatic ligation has emerged as a transformative alternative, offering a scalable, modular, and high-fidelity approach to manufacturing RNA therapeutics. This article explores the rationale behind its growing adoption, the workflows for siRNA and sgRNA synthesis, and the innovations that are extending its reach.
Limitations of conventional SPOS
SPOS, commonly referred to as Generation 1 technology, has been the foundation of oligonucleotide synthesis for over four decades and remains the standard for producing short, chemically modified oligonucleotides. However, this method is constrained by fundamental limitations in scalability, efficiency, and sustainability. Production of a typical 20-mer oligonucleotide is restricted to ~10 kg per batch using flow-through synthesisers, necessitating costly and operationally complex scale-out strategies to meet increasing global demand, as is anticipated for future siRNA targeting cardiometabolic diseases such as PCSK9, AGT, ApoC3, Lp(a), and INHBE.
SPOS is also resource-intensive, consuming substantial volumes of solvents and reagents and producing significant chemical waste. The iterative coupling steps increase the probability of side-product formation, which is particularly problematic for long RNA constructs such as sgRNA (often ≥ 100 nt). These impurities can accumulate during synthesis and are difficult to remove during purification, resulting in suboptimal purity, poor batch-to-batch consistency, and elevated production costs.
Chemoenzymatic ligation: A hybrid approach
Chemoenzymatic ligation, classified as Generation 2 technology, integrates chemical synthesis with enzymatic precision. In this modular workflow, oligonucleotide fragments, which are synthesised via SPOS, are joined enzymatically using ligases to form full-length siRNA or sgRNA constructs. This approach enables scalable, high-yield, and high-purity oligonucleotide synthesis, while also reducing cost and environmental burden compared to conventional SPOS.
Two main strategies are employed, each optimised for specific molecule types:
1. Sticky end ligation method
Sticky-end ligation is a robust method tailored for the synthesis of double-stranded oligonucleotides, including siRNA. This approach leverages the selectivity of enzymes to improve both product quality and manufacturing efficiency.
Shorter oligonucleotide fragments, synthesised via SPOS, are enzymatically ligated to form full-length siRNA duplexes. Because only fragments with correctly positioned 5′-phosphate and 3′-hydroxyl termini are viable substrates, many impurities generated during chemical synthesis, such as n–X truncations, are inherently excluded from the final product. This selective mechanism enhances purity, batch-to-batch consistency and simplifies downstream purification.
Ligation is performed under mild aqueous conditions, with near-quantitative yields and compatibility with stainless steel batch reactors or single-use bioreactors. Moreover, the method accommodates a wide range of siRNA chemistries, including 2′-OMe, 2′-F, phosphorothioate and phosphodiester backbones, and GalNAc conjugates, making it suitable for most clinical applications.
Sticky-end-ligation has already been deployed under at Hongene to manufacture clinical-grade siRNA, demonstrating its scalability and regulatory readiness.
2. Splinted (template) ligation method
Splinted ligation is particularly well-suited for synthesising long, chemically modified single-stranded oligonucleotides, such as sgRNA used in gene editing applications. The method employs complementary DNA “splints” to guide the precise enzymatic joining of overlapping oligonucleotide fragments.
This modular assembly strategy enables the synthesis of constructs ≥100 nucleotides in length with high fidelity, yield, and chemical compatibility. As with sticky-end ligation, many synthesis-related impurities are excluded during ligation, improving the purity of the final product. Additionally, the ability to ligate multiple fragments provides a scalable path to longer constructs required for next-generation CRISPR modalities such as prime editing.
At Hongene, splinted ligation is an integral part of CDMO manufacturing for sgRNA and other long RNA species, with processes designed for compatibility with both GMP facilities and high-throughput production platforms.
Real-world impact and future potential
Chemoenzymatic ligation is no longer a conceptual alternative, it is now a clinically validated, commercially viable manufacturing platform. Hongene has successfully implemented its sticky-end ligation workflow in its GMP-validated CDMO facilities, producing PCSK9-targeting siRNA drug substance to support human clinical development. This achievement demonstrates the regulatory readiness of the ligation approach and its potential to meet future large-scale production demands associated with cardiometabolic siRNA programs.
In parallel, Hongene is broadening the application of its ligation platform to support the growing needs of gene editing programs. Through a strategic partnership with ReciBioPharm, U.S.-based CDMO infrastructure enables domestic GMP manufacturing of sgRNA and related constructs essential for next-generation CRISPR-based therapeutics.
Looking ahead, the modularity and process flexibility of chemoenzymatic ligation positions it to support a wide variety of oligonucleotide structures and chemistries. As demand increases and therapeutic complexity evolves, this platform offers a scalable and efficient solution for future oligonucleotide manufacturing challenges.
Innovation outlook
To further enhance the reach and efficiency of chemoenzymatic ligation, Hongene is actively investing in three key areas of technological innovation:
1. Scalable Fragment Manufacturing: Efforts are underway throughout industry to overcome the batch-size constraints of flow-through SPOS by developing scalable alternatives. These include transitioning SPOS to batch reactors, evaluating liquid-phase oligonucleotide synthesis (LPOS), and exploring emerging technologies for enzymatic oligonucleotide synthesis (Generation 3 technology).
2. Chromatography-Free Workflows: Hongene has demonstrated that unpurified SPOS fragments can be processed via ultrafiltration/diafiltration (UF/DF), eliminating the need for intermediate chromatography. This ‘C-to-P’ (Crude-to-Purified) workflow improves yields and reduces cost. Future work aims to extend this to a full ‘C-to-C’ (Crude-to-Crude) process, eliminating chromatography entirely for both fragments and final siRNA products, which is an especially attractive solution for high-volume manufacturing.
3. Ligase Engineering: A proprietary thermostable T4 RNA ligase variant has been developed at Hongene to support ligation reactions at elevated temperatures (~45 - 55°C). Higher process temperatures reduce secondary structure formation in RNA substrates, improving alignment and ligation efficiency, which is important for both process robustness and enabling the transition to chromatography-free workflows.
Conclusion: A platform for the future of RNA manufacturing
Chemoenzymatic ligation has evolved from an emerging alternative to a clinically validated, industrially scalable platform that has the potential to reshape how oligonucleotide therapeutics are manufactured. Its ability to deliver high-purity products through modular assembly, while improving yield, reducing waste, and enabling scalability, addresses critical limitations of SPOS for both siRNA and sgRNA production.
As demand accelerates, particularly for cardiometabolic siRNA assets, ligation-based workflows offer a practical, future-ready path to multi-ton manufacturing. At the same time, innovations in fragment synthesis, enzyme engineering, and purification strategies continue to expand the platform’s capabilities.
Through its integrated CDMO model and ongoing technology development, Hongene is advancing chemoenzymatic ligation as a versatile solution which is well-positioned to support a wide range of oligonucleotide structures, modifications, and clinical applications. We expect that this technology will play a foundational role in enabling the next generation of RNA-based medicines.
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