Oligonucleotide therapeutics are short sequences of nucleic acids, typically around 20 nucleotides long, designed to modulate the production of specific proteins by binding to target messenger RNAs. Their effectiveness hinges on the precise sequence and chemical modifications they carry. While oligonucleotide therapies were initially developed for rare diseases, recent approval of inclisiran for atherosclerotic cardiovascular disease signals a shift toward using nucleic acid therapeutics for chronic diseases, affecting millions of individuals. With significant advancements in nucleic acid chemistry and innovative delivery modalities, these therapeutic agents are ushering in a transformative era in the management of conditions ranging from cancers to genetic disorders and beyond. As the number of approved oligonucleotide therapeutics continues to rise and a robust pipeline of promising candidates awaits clinical evaluation, the landscape of oligonucleotide manufacturing is poised for a fundamental transition towards significantly larger-scale production methodologies to meet the expected escalating demand. Historically, oligonucleotide synthesis has relied on solid-phase phosphoramidite chemistry. This process involves iterative coupling, capping, oxidation, and deprotection steps for each nucleotide addition. Although powerful, this method has limitations for large-scale production, limiting batches to less than 10 kilograms. Additionally, excessive use of protected monomers and chromatographic purification results in a substantial waste of resources. Consequently, existing methods generate products in less than 50% yield with modest purities, and when dealing with phosphorothioate (PS)-modified sequences oligonucleotides are produced as mixtures of stereoisomers.
In response to these challenges, various innovative approaches to oligonucleotide production have been explored, including the use of nucleoside 3′-oxazaphospholidine derivatives, phosphorus(V) reagents, chiral phosphoric acid catalysts, and enzymatic methods. These approaches offer improvements in stereocontrol, solvent economy, and reduced waste but still follow the fundamental stepwise chain extension model. Furthermore, alternative strategies for constructing oligonucleotides through fragment ligation have been developed but still rely on solid-phase phosphoramidite chemistry.
Recognizing the need of better oligonucleotide manufacturing methods and understanding its challenges, a new study published in the prestigious Journal Science by PhD candidate Ewan Moody, Postdoctoral fellow Dr. Richard Obexer, Florian Nickl, Reynard Spiess, and led by Dr. Sarah Lovelock from the Manchester Institute of Biotechnology, School of Chemistry at the University of Manchester in the UK, introduced a biocatalytic platform that revolutionizes the oligonucleotide manufacturing process. This innovative method uses DNA polymerases and nucleoside triphosphate (NTP) building blocks to extend a catalytic self-priming hairpin template. A crucial component of the process is an endonuclease (EndoV) that selectively cleaves a single strand of the duplex DNA downstream of an inosine base within the hairpin sequence, releasing the product and regenerating the template. A main advantage of this new platform is increased efficiency and its ability to operate under aqueous conditions without the need for solid supports or large volumes of acetonitrile, addressing scalability and sustainability challenges that have plagued conventional methods.
The authors meticulously validated the biocatalytic platform. Initial assessments of the extension and cleavage steps in isolation demonstrated the efficiency of the DNA polymerase and endonuclease. Encouragingly, in one-pot cascade reactions, the target sequence was produced with high selectivity and efficiency. Furthermore, by optimizing reaction conditions and scale, the University of Manchester research team achieved excellent results, including the production of various modified sequences and clinically relevant oligonucleotides. Importantly, these achievements were obtained with an impressive reduction in the environmental footprint, as indicated by a lower Process Mass Intensity (PMI) compared to traditional methods.
One of the key advantages of this biocatalytic platform is its ability to accommodate various chemical modifications important in the oligonucleotides chemistry, including phosphorothioate (PS) linkages, 2′-fluoro, 2′-methoxy, and locked nucleic acid (LNA) modifications. The authors believe that with further enzyme engineering, the new platform’s applicability can be extended to a wider range of nucleotide modifications, enhancing the diversity of therapeutic oligonucleotides available for medical use.
According to the authors, the biocatalytic platform presented offers a new paradigm for therapeutic oligonucleotide manufacturing. It can replace multi-step chemical synthesis with a streamlined, more efficient, and environmentally friendly sustainable process. This breakthrough could enable synthesis of oligonucleotides on a much larger-scale, making them more accessible. Additionally, the platform opens the door to the production of highly pure, stereospecific oligonucleotides, which may have improved safety and efficacy profiles. This is particularly relevant for PS-modified oligonucleotides, as the study demonstrated the ability to produce all-Rp oligonucleotides as single stereoisomers.
The development of this biocatalytic platform is only the beginning for Dr. Sarah Lovelock’s research group. As researchers continue to optimize and expand its capabilities, we can anticipate even greater advancements in the field of oligonucleotide therapeutics. Specific directions for future research include the engineering of polymerases with extended substrate scopes and the development of scalable methods for producing NTP building blocks. In conclusion, the study led by Dr. Sarah Lovelock and her team is considered a major advancement the field of oligonucleotide therapeutics. Their biocatalytic platform offers a scalable, efficient, and sustainable alternative to traditional chemical synthesis methods.
Moody ER, Obexer R, Nickl F, Spiess R, Lovelock SL. An enzyme cascade enables production of therapeutic oligonucleotides in a single operation. Science. 2023 Jun 16;380(6650):1150-1154. doi: 10.1126/science.add5892.