A versatile biocatalytic platform for therapeutic oligonucleotide synthesis

Lead Research Organisation: University of Manchester
Department Name: Chemistry

Abstract

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.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/S023755/1 01/04/2019 30/09/2027
2279351 Studentship EP/S023755/1 01/10/2019 30/09/2023 Ewan Robert Moody