Where is that RNA we delivered? Searching for delivered RNA in the target cell.

The project aligns to CDT themes: Complex Product Characterisation & Advanced Product Design

This project aims to use molecular biology tools to map the subcellular localisation of RNA drugs following delivery to target cells. RNA therapeutics, including siRNA, antisense oligonucleotide (ASO) and mRNA drugs, have considerable potential for targeted therapy of genetic and other diseases, and the recent success of mRNA vaccines for COVID-19 demonstrates the promise of this technology for new vaccine development. However, delivery of RNA therapeutics to their subcellular site of action remains a major barrier, as most chemical delivery strategies and gymnosis of naked oligonucleotides rely on endocytosis, and endocytic escape of RNA molecules is an inefficient and poorly understood process1. Most existing methods measure cellular uptake of RNA therapeutics but are unable to distinguish the subcellular distribution of the molecules and therefore cannot detect whether the RNA is present in the endosomal or at a functional site within the cell (cytoplasm or nucleus). Improved approaches to determine the subcellular localisation of RNA therapeutics are therefore urgently needed in order to compare and improve delivery strategies. Ideally these approaches should be label-free, quantitative, and scalable to high throughput.

In this project, we will adapt recently developed approaches from the field of molecular biology to address this problem. In particular, the project will focus on the APEX-seq method that was recently developed by the Ting lab2. We will use lentiviral vectors (available from Addgene) to generate cell lines in which the APEX2 engineered peroxidase is localised in the cytoplasm, nucleus or endoplasmic reticulum. Following delivery of an RNA therapeutic, cells will be briefly treated with hydrogen peroxidase and biotin-phenol, leading to proximity biotinylation of RNA in each of the subcellular locations. Biotinylated RNA can then be isolated by streptavidin chromatography and analysed by quantitative real time PCR (qRT-PCR). We will test this approach with different mRNA and siRNA molecules delivered to cells by different delivery platforms, with the aim of developing robust methods that can be applied to quantify nuclear/cytoplasmic distribution of different RNA therapeutics and to compare delivery platforms. Finally, we will use this data to generate predictive models for subcellular localisation and incorporation into functional complexes of therapeutic RNA molecules, and for RNA delivery kinetics.

This project will bring together the complementary expertise of different research teams in the School of Pharmacy in molecular and RNA biology (CJ), drug delivery and siRNA therapeutics (SS) and molecular modelling (NH). It will take the novel approach of applying molecular biology tools that are not widely used in drug development to achieve improved screening of RNA therapeutic delivery, with strong potential for industrial application as transformative pharmaceutical technologies.

Pharmaceutical technologies underpin healthcare product development. Medicinal products are becoming increasingly complex, and while the next generation of research scientists in the life- and pharmaceutical sciences will require high competency in at least one scientific discipline, they will also need to be trained differently than the current generation. Future research leaders need to be equipped with the skills required to lead innovation and change, and to work in, and connect concepts across diverse scientific disciplines and environments. This CDT will train PhD scientists in cross-disciplinary areas central to the pharmaceutical, healthcare and life sciences sectors, whilst generating impactful research in these fields. The CDT outputs will benefit the pharmaceutical and healthcare sectors and will underpin EPSRC call priorities in the development of low molecular weight molecules and biologics into high value products.

Benefits of cohort research training: The CDT's most direct beneficiaries will be the graduates themselves. They will develop cross-disciplinary scientific knowledge and expertise, and receive comprehensive soft skills training. This will render them highly employable in R&D in the pharmaceutical, healthcare and wider life-sciences sectors, as is evidenced by the employment record in R&D intensive jobs of graduates from our predecessor CDTs. Our students will graduate into a supportive network of alumni, academic, and industrial scientists, aiding them to advance their professional careers.

Benefits to industry: The pharmaceutical sector is a key part of the UK economy, and for its future success and international competitiveness a skilled workforce is needed. In particular, it urgently needs scientists trained to develop medicines from emerging classes of advanced active molecules, which have formulation requirements that are very different from current drugs. The CDT will make a considerable impact by delivering a highly educated and skilled cohort of PhD graduates. Our industrial partners include big pharma, SMEs, CROs, CMOs, CMDOs and start-up incubators, ensuring that CDT training is informed by, and our students exposed to research drivers in, a wide cross-section of industry. Research projects in the CDT will be designed through a collaborative industry-academia innovation process, bringing direct benefits to the companies involved, and will help to accelerate adoption of new science and approaches in the medicines development. Benefit to industry will also be though potential generation of IP-protected inventions in e.g. formulation materials and/or excipients with specific functionalities, new classes of drug carriers/formulations or new in vitro disease models. Both universities have proven track records in IP generation and exploitation. Given the value added by the pharma industry to the UK economy ('development and manufacture of pharmaceuticals', contributes £15.7bn in GVA to the UK economy, and supports ~312,000 jobs), the economic impacts of high-level PhD training in this area are manifest.

Benefits to society: The CDT's research into the development of new medical products will, in the longer term, deliver potent new therapies for patients globally. In particular, the ability to translate new active molecules into medicines will realise their potential to transform patient treatments for a wide spectrum of diseases including those that are increasing in prevalence in our ageing population, such as cardiovascular (e.g. hypertension), oncology (e.g. blood cancers), and central nervous system (e.g. Alzheimer's) disorders. These new medicines will also have major economic benefits to the UK. The CDT will furthermore proactively undertake public engagement activities, and will also work with patient groups both to expose the public to our work and to foster excitement in those studying science at school and inspire the next generation of research scientists.