A versatile biocatalytic platform for therapeutic oligonucleotide synthesis

Therapeutic oligonucleotides are short DNA analogues which selectively bind to target mRNA through Watson-Crick base pairing, and regulate the production of disease related proteins. Following the award of the 2006 Nobel Prize for RNA interfering technology and recent FDA approvals of several RNA-based therapeutics for the treatment of rare diseases, there has been significant investment into therapeutic oligonucleotides as a new drug modality. There are currently more than 160 oligonucleotide products in clinical trials including those for population based indications. The increase in the number of potential therapeutics, including those for common diseases, creates a significant manufacturing challenge as existing methods of chemical synthesis are restricted to 10Kg batches and are not suitable for large scale applications (>100Kg). Moreover the process uses prohibitively large volumes of acetonitrile (1000L per Kg of oligonucleotide) which presents a threat to the security of oligonucleotide supply, given recent world shortages of acetonitrile.
This project will investigate scalable biocatalytic approaches to therapeutic oligonucleotide synthesis based on the polymerase chain reaction. Therapeutic oligonucleotides require chemical modifications (Fig. 1) to confer improved efficacy, selectivity, metabolic stability, and toxicity profiles. While natural polymerases are tolerant to some nucleotide modifications their activity is compromised, and therefore directed evolution will be used to improve polymerase activity towards nucleotides containing pharmaceutically relevant modifications.
Substitution of the phosphodiester linkage with a phosphorothioate (PS) linkage is a common modification which results in the formation of monomers with two distinct stereochemical configurations. However, due to limitations of existing synthetic methods, current marketed RNA-based therapeutics are typically supplied as complex mixtures of diastereoisomers and there remains very limited understanding of the bioactivity of individual stereoisomers. The biocatalytic process developed within this project will be used to produce individual stereoisomers which will be evaluated for biological activity. NMR spectroscopy techniques including phosphorous NMR, will be used provide detailed structural characterization of single stranded oligonucleotide stereoisomers and of the duplexes formed with mRNA targets.
This project merges the complimentary disciplines of biocatalysis, synthetic chemistry and analytical chemistry and is thus perfectly aligned with the strategic research priorities of the iCAT programme.

iCAT will work with industry partners to create an holistic approach to the training of students in biocatalysis, chemocatalysis, and their process integration. Traditional graduate training typically focuses on one aspect of catalysis and this approach can severely restrict innovation and impact. Advances in technology and fundamental reaction discovery are rendering this silo-approach obsolete, and a new training modality is needed to produce the next generation of chemists and engineers who can operate across a far broader chemical continuum. iCAT will meet this challenge with a state-of-the-art CDT, equipping the next generation of scientists and engineers with the skills needed to develop future catalytic processes and create the functional molecules of tomorrow.

The UK has one of the world's top-performing chemical industries, achieving outstanding levels of growth, exports, productivity and international investment. The UK's chemical industry is a significant provider of jobs and creator of wealth, with a turnover in excess of £50 billion and a contribution of over £15 Billion of value to the UK economy [2015 figures]. iCAT will deliver highly skilled people to lead this industry across its various sectors, achieving impact through the following actions:

1. Equip the next generation of science and engineering leaders with the interdisciplinary skills and knowledge needed to work across the bio and chemo catalytic remit and build the functional molecules we need to structure society.

2. Provide a highly skilled workforce and research base, skilled in the latest methodologies, strategies and techniques of catalysis and engineering that is crucial for the UK's Chemical Industry.

3. Build the critical mass necessary to support effective cohort-based training in a world-class research environment.

4. Develop and disseminate new catalytic technologies and processes that will be taken up by industrial and academic teams around the world.

5. Encourage Industry to promote research challenges within the CDT that are of core relevance to their business.

6. Provide cohesion in the integration of biocatalysis, engineering and chemocatalysis to create a more unified voice for strategic dialogue with industry, funders and policy makers, and more generally outreach and public engagement.

7. Draw-in and bring together Industrial partners to facilitate future Industrial collaborations.

8. Benefit Industrial scientists through interactions with the CDT (e.g. training and supervisory experience, exposure to cutting-edge synthesis and catalysis etc).

9. Link with other activities in the landscape: bringing unique expertise in catalysis to, for example, externally-funded University-led initiatives, EPRSC Grand Challenge Networks, and the National Catalysis Hub.