Nucleic acids represent a new class of therapies that specifically target the gene’s associated with disease. They are applicable to treat both rare and common diseases and can be developed in a shorter period of time than conventional medicines, NATA will enable the nucleic acid technology platform to advance by overcoming current scientific challenges.

Cells have specialised machinery - called ribosomes - for making proteins. When cells are dividing, they put a lot of resources into manufacturing proteins to make more cells. Much research has focussed on how ribosomes make cellular proteins to support cell division to make new cells, and this has been important to understand diseases in which cell division is uncontrolled - such as cancer. However, cells don't divide much in healthy adults, so protein production tends to be devoted to tissue maintenance - in which old cells are replaced with new ones - and wound healing/tissue repair processes. Indeed, during wound healing cells start to make lots more proteins; not for cell growth but for secretion from the cells. Many of these secreted proteins make up the connective tissue, or extracellular matrix, that repair wounds, and we term this connective tissue - the 'extracellular matrix'. Moreover, extracellular matrix proteins need to be transported through a series of compartments in the cell to be secreted in the appropriate manner, and this complex transport process is termed intracellular protein 'trafficking'. In healthy individuals, extracellular matrix production is controlled and timed appropriately, so that it can be turned-off when the wound is repaired and/or an old cell is replaced with a new one. However, following certain toxic insults - such as paracetamol poisoning - cells in the liver become damaged in such a way as to make them 'senescent' - a damaged state which is akin to premature ageing. Damaged 'senescent' cells then start to make lots of extracellular matrix proteins which they release in an excessive and uncontrolled way leading to scar formation and, ultimately to liver fibrosis and cirrhosis. This is a key concern, because this cirrhotic scar tissue forms a breeding ground for other diseases, such as liver cancer. We propose that damaged/senescent liver cells must re-structure their ribosomes and their intracellular protein trafficking machinery to enable the production and release of all these extracellular matrix proteins. This research programme will combine state-of-the-art methodologies, such as advanced 3-dimensional microscopy, to investigate the mechanisms linking increased ribosome function with alterations to protein trafficking machinery as liver cells become senescent. Specifically, we will elucidate the cellular mechanisms that link the production of the various extracellular matrix proteins by ribosomes with the trafficking machinery that takes them to the cell surface. Then we will investigate how these processes may be coordinated as cells attempt to maintain homeostasis in the face of increased demand to secrete proteins in senescent cells. Integral to this research programme will be the use of laboratory mouse models in which senescence has been induced in the liver, by dietary, toxic and targeted changes to the DNA. We will then pursue these studies to find out how these alterations to the protein synthesis and secretory machinery that occur during liver senescence contribute to the initiation of liver cancer. The combination of these approaches will enable us to assemble a detailed understanding of the relationship between secretory protein production, protein trafficking and build-up of extracellular matrix proteins outside cells during liver fibrosis/scarring and the initiation of liver cancer. We anticipate that this detailed mechanistic understanding will facilitate identification of individuals particularly at risk of fibrotic liver disease and cancer and assist with preventative interventions.

Genetic information is encoded in DNA as an ordered sequence of nucleotide units. Cells can retrieve this information by transcribing DNA into protein-coding messenger and noncoding RNAs. We have previously identified the pyrimidine-rich noncoding transcript PNCTR and showed that it is widely expressed in cancer cells. This RNA originates from an intergenic spacer between repeated genes encoding 47S precursors of ribosomal RNAs in genomic regions called ribosomal DNA (rDNA) arrays. PNCTR can promote cancer cell survival, at least in part, by sequestering multiple copies of the RNA-binding protein PTBP1 in a membraneless perinucleolar compartment (PNC). Cancers cells tend to accumulate mutations, which often make cancers more challenging to treat. Such genetic instability is commonly observed, for example, in breast cancers, the most common cancer type in the UK. Notably, many breast cancers produce the PNC through yet-to-be-understood mechanisms. Our new data suggest that cancer cells may transcribe PNCTR from genetically rearranged rDNA sequences, emerging as a result of error-prone repair of DNA double-strand breaks (DSBs). We also hypothesize that the assembly of the PNC around nascent PNCTR molecules segregates genetically compromised rDNA loci away from the nucleolus. Finally, we propose that PNCTR plays a key part in the breast cancer biology, and that its knockdown may reduce the ability of cancer cells to thrive and metastasize. We will explore these intriguing possibilities by pursuing three distinct but interrelated objectives. 1. Elucidating the role of rDNA rearrangements in PNCTR expression: We will test if PNCTR is commonly produced from genetically rearranged rDNA by sequencing PNCTR-enriched RNA fractions from breast cancer cell lines and patient samples and mining publicly available sequencing datasets. The proposed analyses will also illuminate the role of recurrent rDNA DSBs and different DSB repair pathways in the emergence of PNCTR-encoding loci. 2. Dissecting the mechanisms of PNC assembly: Our new data suggest that the PNC assembles near the PNCTR transcription site. We will test this prediction using proximity DNA labeling and chromatin immunoprecipitation with PTBP1-specific and control antibodies. To examine the PNC assembly dynamics, we will perform live imaging of cancer cells retrofitted with fluorescent markers. By combining these experiments with appropriate knockdown and knockout approaches, we will be able to distinguish between co- and post-transcriptional mechanisms of PNC assembly and find out if this process facilitates the segregation of rearranged rDNA loci to the nucleolar periphery. 3. Understanding PNCTR functions: We will employ appropriate knockdown or/and knockout approaches in both 2D and organoid cultures to investigate the role of PNCTR in sustaining the viability and metastatic properties of breast cancer cells. To understand the underlying mechanisms, we will evaluate the impact of PNCTR/PNC on PTBP1 activity and the integrity of the nucleolus. These studies will yield fundamental insights into the role of genome instability in noncoding RNA production and subnuclear compartmentalization. Furthermore, they are expected to contribute to the development of innovative research tools for the broader biomedical community. We anticipate that this research trajectory will ultimately pave the way for transformative diagnostic approaches and precision therapies for cancer patients.

CONTEXT: At the forefront of biological research is understanding how cells interact with their environment. Selective interaction with the extracellular milieu and the recognition of molecular signatures presented on a cell surface enables organisms to respond to environmental changes, which is often altered in disease states. Many cell targeting molecules are advancing our understanding of how these interactions influence different cell phenotypes. However, distinct challenges in the development of these targeting molecules (e.g., small molecules, antibodies, aptamers) necessitates the need for innovative bioanalytical infrastructure to dissect and characterise their binding profile in live cells. AIMS & OBJECTIVES: The purpose of this equipment grant is to establish the UK's first Real-Time Interaction Cytometry (RT-IC) facility, which will enable users to measure the binding properties of molecules (e.g., kinetic rates; affinity, avidity) directly on live cells. The Glas-cyto facility will be part of Glasgow's wider biophysical centre of excellence formed between the Universities of Strathclyde (UoS) and Glasgow (UoG), enabling the UK userbase integrated access to a breadth of bioanalytical and biophysical facilities to examine cellular binding interactions. The heliXcyto equipment will be suitable for a wide range of analyses for eukaryotic cell types and will be underpinned by support from dedicated research technical professionals (RTPs) within the Strathclyde Centre for Molecular Bioscience (UoS) and the Integrated Protein Analysis Facility (UoG). THE RESEARCH THAT THE EQUIPMENT WILL ENABLE: Our dedicated facility will enable users the unique opportunity to culture live cells alongside instrumentation that will quantify binding interactions in less than 30 min. Access to such a facility will enable our user base to develop better cell-targeting biologics (e.g., antibodies, aptamers, Theme 1), deliver a new design proxy for the development of cell targeting molecules for the development of diagnostic platforms (Theme 2), and further our understanding of protein trafficking and cell-selective recognition of G-protein coupled receptors by small molecules (Theme 3). POTENTIAL APPLICATIONS & BENEFITS: The current state-of-the-art in the biophysical analysis of extracellular interactions have predominantly focused on low throughput assays (e.g., CETSA, In Cell Pulse DiscoverX, SPR), which require the downstream isolation of analytes, or qualitative analysis of interactions by flow cytometry. The major drawback of these techniques is that binding is inferred rather than quantified. Detailed quantitative knowledge of spatial patterns of receptor expression, and reconciling these data with binding interactions can accelerate the translational potential of novel targeting molecules in early discovery. Establishing this dedicated facility within Glasgow will provide, for the first time, the ability to quantify binding interactions on live cells, under conditions more closely mimicking their native environment. The Glas-cyto facility will enable new training opportunities across the breadth of the UK's biological user base, and infrastructure to enhance interactions with industrial partners.

Nucleic acid therapeutics (NATs) offer great potential to treat rare diseases (RDs) by addressing their genetic causes in a target-specific manner. The exponential increase in NAT clinical trials in the last few years clearly demonstrates the role of these molecules in translational research and unique opportunities for investigator-led preclinical and clinical studies, in which the UK has particular track record strengths. To further promote the development of NATs for RD patients in the UK, we are creating the node entitled, 'Establishing a UK Platform for the Development of Nucleic Acid Therapy for Rare Disease' (UPNAT). UPNAT will bring together relevant stakeholders, comprising of scientists, clinicians, geneticists, trainees, patient advocacy groups and charities, industrial partners, international non-profit organizations and regulatory bodies, to establish and coordinate a national network that will facilitate the exploitation of the rapid development of NATs. UPNAT intends to address a number of challenges that NAT research and development is currently encountering in the RD field within the UK, including 1) a lack of a national infrastructure for cross-disciplinary knowledge exchange and expertise sharing between centres leading NAT preclinical and clinical development; 2) a clear path for systematically linking patients carrying unique mutations to NAT expertise; and 3) the need for continuous dialogue between regulators and researchers to streamline the process of regulatory approvals, monitoring of outcomes, and accelerating the clinical translation of RD-specific NATs. To tackle these challenges, UPNAT will create the following networking opportunities: 1) scientific symposia to promote cross-disciplinary knowledge exchange between researchers, clinical and industry stakeholders; 2) webinars and activities between patient advocacy groups, charities, and researchers to promote public engagement; 3) training schemes to educate and equip the next generation of scientists and clinicians with the knowledge and skills to lead future NAT research programs. The node encompasses three complementary projects, to address the overall objectives and crucial bottlenecks. These projects focus on 1) Target selection, NAT strategy design and pre-clinical development; 2) Enhancing UK's capability in NAT scale-up synthesis and pilot toxicology studies tailored for RD; 3) NAT clinical trial design and regulatory approval. Collectively these work packages will enable a robust framework for the design, development, and clinical translation of NAT to be adopted by RD centres in the UK. UPNAT will focus on areas of unique strength in rare paediatric and adult disorders, including six paediatric highly specialised services provided by the partner organisations in London, Oxford, Cambridge, Birmingham, Liverpool and Sheffield, and the adult expert centres at the University College London (UCL) Institute of Ophthalmology, Institute of Neurology, Moorfields Eye Hospital and University College Hospital. The node will be focused on neurological, neurodegenerative, metabolic and ophthalmological diseases which are uniquely conducive for NAT applications and remain open to other disease areas as NAT technology rapidly advances and Node develops. UPNAT will be led by investigators and collaborators from UCL Great Ormond Street Institute of Child Health, Institute of Ophthalmology, Institute of Neurology, Great Ormond Street Hospital (GOSH), Moorfields Eye Hospital, UKRI NATA (Oxford) and investigators from Oxford, Cambridge and Birmingham. Node members will work in partnership with Genomics England, five NIHR BRCs (GOSH, UCLH, Moorfields, Oxford and Cambridge), industry, patient advocacy groups and charities, the UK regulators, and the international consortia on NAT in RD. Collectively, we are well equipped and determined to maximise the transformative potential NATs offer for the RD patient community within the UK and beyond.