Reversible Phosphorothioate Backbone Modification for Oligonucleotide Delivery and Control

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Background:Oligonucleotide therapeutics show great promise to treat a variety of diseases on a genetic level. One major problem current oligonucleotide therapeutics face is poor delivery. Oligonucleotides are large, highly negatively charged biomolecules with poor bioavailability and ineffective cell delivery. Due to their highly anionic nature, these drugs poorly bind to plasma proteins, which reduces tissue distribution. Additionally, internalisation into cells is hindered, as they do not spontaneously cross cellular membranes. It is well known that modification of oligonucleotides is essential to improve their pharmacological properties, as first generation oligonucleotide therapeutics suffered from fast turnover and the inability to achieve sufficient intracellular concentrations. To improve stability and delivery of oligonucleotide therapeutics, a large number of modifications are employed, the most common of which is the phosphorothioate backbone. These modifications allow for better distribution, while still retaining the desired activity; however, tissue levels are still low and off-target effects are often observed.Project Summary:This project aims at exploiting the inherent reactivity of the phosphorothioate for chemical modification with stimuli-responsive chemical groups. These groups can then be selectively removed by application of the stimulus, allowing for spatial and temporal control of oligonucleotide release. It is believed that through the masking of the charge and the increase in lipophilicity with chemical modification, the pharmacological properties of distribution and cell penetration should be improved. Additionally, through the stimuli-responsive regeneration of the unmodified oligonucleotide, off-target effects will be reduced.It has been shown that the phosphorothioate is reactive towards chemical groups and that modification increases its lipophilicity. However, there are no accounts yet on the reversible masking of phosphorothioates and their applications in living systems. To study this, a variety of reactive stimuli-responsive chemical groups will be synthesised and their reactivity towards phosphorothioates studied. Following evaluation of the chemistry of these modifications, known therapeutic sequences will be modified as above and their delivery into living cells and their biological activity will be investigated. These biological studies will aid in the design of further modifications. These modified oligonucleotides will then be used on cellular disease models to measure their efficacy against current oligonucleotide therapies.This project is undertaken in the group of Dr. Michael Booth within the Synthesis for Biology and Medicine Centre for Doctoral Training (SBM CDT) at the University of Oxford. The project falls within the EPSRC chemical biology and biological chemistry research area.