Reversible Phosphorothioate Backbone Modification for Oligonucleotide Delivery and Control

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.

This programme is focused on a new cohort-driven approach to the training of next-generation doctoral scientists in the practice of novel and efficient chemical synthesis coupled with an in-depth appreciation of its application to biology and medicine.

This collaborative academic-industrial SBM CDT will have long-term benefit to the chemical industry, including the pharmaceutical, agrochemical and fine chemical sectors. These industries will benefit through: (i) the potential to employ individuals trained with broad and relevant scientific and transferable skills; (ii) new approaches to the investigation of complex biological and medical problems through novel chemistry; and (iii) better and more efficient synthetic methods.

We will link the work of DSTL, and our pharmaceutical and agrochemical partners (GSK, UCB, Vertex, Evotec, Eisai, AstraZeneca, Syngenta, Novartis, Takeda, Sumitomo and Pfizer) through a common theme of synthesis training. The design and synthesis of new compounds is essential for disease treatment and prevention, and for maintaining food security. Synthesis contributes significantly to UK tax revenue and results in sustained employment across a number of sectors. Employers in the finance, law, health, academic, analytical, government, and teaching professions, for example, also recognise the value of the translational skill-sets possessed by synthesis postgraduates, which this programme will provide.

The SBM CDT training programme will adopt an IP-free model to enable completely free exchange of information, know-how and specific expertise between students and supervisors on different projects and across different industrial companies. This will lead to better knowledge creation through unfettered access to information from all academics, partners and students involved in the project. By focussing on basic science, we will engender genuine collaboration leading to enabling technology that will be of use across a wide range of industries.

We will train the next generation of multidisciplinary synthetic chemists with an appreciation of the impact of synthesis in biology and medicine. Their unconstrained view of synthesis will aid in new scientific discoveries leading to new products, which (with appropriate inward investment), can lead to the formation of new companies and new UK employment.

We will, in part through an alliance with the Defence, Science and Technology Laboratory, engage with policy-makers to influence future policy issues involving chemistry such as food security and the rise of antibiotic resistance (both of which are relevant to our programme and are important for society as a whole).

Outreach and public engagement will be a key aspect of our programme; and all students in the proposed SBM CDT will take part in at least one outreach activity. Typical activities include: open days in the Chemistry Department through the 'Outreach Alchemists', engaging with the Oxfordshire Science Festival and participation in the various other activities already in place through the public engagement programme of the Department of Chemistry.

The research output of the students will be disseminated via high impact international publications and lectures; these will be of value to other academics in relevant fields and will be of value in the development of further research funding applications. Outreach activities and research output will also be advertised on a website dedicated to the proposed SBM programme.