Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

Inflammatory diseases are responsible for significant death, disability and poor outcomes and are a major financial burden on the health service. Despite decades of research and billions of pounds of R&D investment, no targeted therapeutics exist that modulate neutrophils (a key cell involved in inflammation and also the major cell that surrounds lung cancers and promotes invasiveness and poor prognosis). Acute and chronic inflammatory diseases such as asthma, COPD and lung injury are common and are now increasing in incidence and severity due to the aging population. Hence, this research addresses, one of the biggest challenges facing modern drug development, the need to develop in-human assay systems that provide confidence in early trials to either continue progressing or terminating drug development programmes. A major cause for failures of drug development include the historical reliance on animal models of disease which do not accurately reflect human disease. It is essential to develop new technologies to understand and evaluate disease and drug effectiveness in vivo in situ in humans. This research proposal will develop to near-clinical readiness, novel state of the art engineering and mathematical approaches to improve the quality of the data received from a sensing system called Kronoscan which is able to image and sense in real-time at microscopic detail in new dimensions using some of the world's fastest detector technology, measuring fluorescence lifetime data of inflammatory biomarkers at video rate (>10fps). Fluorescence lifetime overcomes the significant limitations of intensity fluorescence imaging and improves quantification. In patients, we will enable this through a method called microendoscopy suited to diseases that affect the lungs and gastrointestinal tracts and other areas where we can pass small imaging fibres deep into tissue. This method will be coupled alongside chemical SmartProbes which "light" up when they interact with inflamed cells and tissues. The project will be undertaken in partnership with GlaxoSmithKline who will provide "tool" compounds in development for clinical trials. GSK already use other imaging methods such as CT Scans and PET Imaging but see this approach of adding in high resolution ultra sensitive microscopic imaging to the evaluation of drug action as a major addition to the drug development process and an essential step to improving a currently expensive and poorly productive drug development pathway. Work on the different elements needed to create this technology platform will be undertaken by investigators spanning signal processing, electrical engineering, chemistry and clinical science at the University of Edinburgh in collaboration with GSK divisions. This project will be based in the Proteus interdisciplinary "hub" to ensure rapid product development. The researchers will spend time in each others labs in Edinburgh and GSK as well as arranging an open network meeting to ensure broader engagement. The scientists (researcher co-investigators) in the proposal will benefit significantly from networking and establishing the area of next-generation in vivo pharmacology. A key ambition of the research will be to pave the way for subsequent clinical and commercial impact and as such user (clinical and regulatory) input will be paramount during the development of the technology. The team will leverage existing capability and expertise in manufacturing, regulatory and commercialisation support to expedite development. In summary, this project will generate; 1) A cutting edge point-of-care technology platform which will help drug developers, patients, doctors and health care workers throughout the world. 2) Career development of the researcher Co-Is. 3) Develop an entirely new theme with "Big Pharma". 4) A sustainable network to disseminate the technology through GSK's imaging franchise.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

When RNA polymerase starts to transcribe a gene into mRNA, the sequence and thus the activity of the protein encoded by the RNA depend on the pattern in which large portions of the RNA are spliced out. The processes by which the sites of splicing are selected are very complex, and they are still understood poorly. Understanding them is hugely important both because splicing is an essential process that, more than anything else, enables highly complex organisms such as ourselves to have developed despite having only the same number of genes as much simpler organisms and because, by controlling splicing, we could shift the expression of a gene from one type of protein to another for therapeutic purposes. Indeed, the first drugs targeting splicing in muscle and the CNS have been approved recently by the US FDA and others are in trials. There has been great excitement recently over the discovery that quadruplexes (G4s) might regulate splicing. G4s are small, four-stranded structures that can form in the RNA from four sequences of GG or GGG in close proximity. They could open up new ways of understanding and manipulating splicing. However, it has been very difficult to prove that they form in long RNA molecules in functional splicing conditions, and nothing is known of how they might affect splicing. We have recently published a new method, called FOLDeR, that enables us to map the regions of a pre-mRNA that form G4s in splicing conditions. We have applied this to Bcl-X, a gene expressing two isoforms of protein: one promotes cell survival and the other promotes apoptosis. The difference results from the choice between two 5' splice sites. We have shown that there are two G4-forming sequences in Bcl-X, one close to each splice site. Many small molecules are known to bind to and stabilize G4s. We have tested a range of 33 of these on Bcl-X. Both in nuclear extracts and in cells, one reagent shifts splicing so much that the usually minor pro-apoptotic isoform becomes predominant, and we have shown that it affects the structures of the two G4-forming regions in the RNA, probably by enhancing G4 formation. Moreover, it switches the splicing of another gene crucial for some cancers, Mcl-1, to express only the pro-apoptotic isoform. Accordingly, it promotes apoptosis. The same reagent has no effects on some other alternative splicing events, and others that affect different alternative splicing events have no effects on Bcl-X. Most of the other 32 compounds show mild or no effects. Importantly, the effects of each one on Bcl-X splicing are similar in nuclear extracts with purified pre-mRNA and in cells, showing that the molecules affect splicing directly. This suggests that G4 stabilizers might each target a defined set of genes. Are these genes in sets with common biological functions? If not, could we investigate how the small molecules and their cognate G4s work so that we can prevent unwanted effects and develop useful and selective drugs? Could we predict the sites of action of such molecules? Could we use their target sequences to develop ligand dependent splicing switches, enabling a gene to switch from one function to another? We propose to address these exciting possibilities using four approaches. (i) The first is use high-throughput sequencing to identify all the changes in expression and splicing brought about by several G4-binding molecules in cells, which will inform us about the range of effects and the common features associated with the targets of each molecule. (ii) We will identify and test the exact nucleotides and contacts associated with G4 formation and small molecule binding by methods including NMR and X-ray crystallography. (iii) We will synthesize and test a range of new analogues to help in defining interactions and, using structural information, to improve selectivity. (iv) We will determine how G4s affect splicing and use this to test whether G4s can be inserted as switches into new positions.