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Journal articleBarton RD, Tregoning JS, Wang Z, et al., 2025,
A sort and sequence approach to dissect heterogeneity of response to a self-amplifying RNA vector in a novel human muscle cell line
, Molecular Therapy: Nucleic Acids, Vol: 36, ISSN: 2162-2531Self-amplifying RNA (saRNA) is an extremely promising platform because it can potentially produce more protein for less RNA. We used a ‘sort and sequence’ approach to identify host cell factors associated with transgene expression from saRNA; the hypothesis was that cells with different expression levels would have different transcriptomes. We tested this in CDK4/hTERT immortalised human muscle cells transfected with VEEV derived saRNA encoding GFP. Cells with the highest expression levels had very high levels of transgene mRNA (5-10% total reads); the cells sorted with low and negative levels of GFP protein also had detectable levels of both VEEV and GFP RNA. To understand host responses, we performed RNASeq. Differentially expressed gene (DEG) patterns varied with GFP expression; GFP high cells, had many more DEG, these were associated with protein synthesis and cell metabolism. Comparing profiles by an unsupervised approach revealed that negative cells expressed higher levels of cell intrinsic immunity genes such as IFIT1, MX1, TLR3 and MyD88. To explore the role of interferon, cells were treated with the Jak inhibitor Ruxolitinib, this reduced DEG number but differences between cells sorted by expression level remained. These studies demonstrate the complex interplay of factors, some immune related, affecting saRNA transgenes.
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Journal articleBalmes A, Rodriguez JG, Seifert J, et al., 2024,
Role of the NO-GC/cGMP signaling pathway in platelet biomechanics
, PLATELETS, Vol: 35, ISSN: 0953-7104 -
Journal articleSymes OL, Ishikura H, Begg CS, et al., 2024,
Harnessing oxetane and azetidine sulfonyl fluorides for opportunities in drug discovery
, Journal of the American Chemical Society, ISSN: 0002-7863Four-membered heterocycles such as oxetanes and azetidines represent attractive and emergent design options in medicinal chemistry due to their small and polar nature and potential to significantly impact the physiochemical properties of drug molecules. The challenging preparation of these derivatives, especially in a divergent manner, has severely limited their combination with other medicinally and biologically important groups. Consequently, there is a substantial demand for mild and effective synthetic strategies to access new oxetane and azetidine derivatives and molecular scaffolds. Here, we report the development and use of oxetane sulfonyl fluorides (OSFs) and azetidine sulfonyl fluorides (ASFs), which behave as precursors to carbocations in an unusual defluorosulfonylation reaction pathway (deFS). The small-ring sulfonyl fluorides are activated under mild thermal conditions (60 °C), and the generated reactive intermediates couple with a broad range of nucleophiles. Oxetane and azetidine heterocyclic, -sulfoximine, and -phosphonate derivatives are prepared, several of which do not have comparable carbonyl analogs, providing new chemical motifs and design elements for drug discovery. Alternatively, a SuFEx pathway under anionic conditions accesses oxetane-sulfur(VI) derivatives. We demonstrate the synthetic utility of novel OSF and ASF reagents through the synthesis of 11 drug analogs, showcasing their potential for subsequent diversification and facile inclusion into medicinal chemistry programs. Moreover, we propose the application of the OSF and ASF reagents as linker motifs and demonstrate the incorporation of pendant groups suitable for common conjugation reactions. Productive deFS reactions with E3 ligase recruiters such as pomalidomide and related derivatives provide new degrader motifs and potential PROTAC linkers.
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Journal articlePybus H, Dangarh P, Ng MYM, et al., 2024,
Mechanistic modelling of allergen-induced airways disease in early life
, Scientific Reports, ISSN: 2045-2322Asthma affects approximately 300 million individuals worldwide and the onset predominantly arises in childhood. Children are exposed to multiple environmental irritants, such as viruses and allergens, that are common triggers for asthma onset, whilst their immune systems are developing in early life. Understanding the impact of allergen exposures on the developing immune system and resulting alterations in lung function in early life will help prevent the onset and progression of allergic asthma in children. In this study, we developed an in silico model describing the pulmonary immune response to a common allergen, house dust mite, to investigate its downstream impact on the pathophysiology of asthma, including airway eosinophilic inflammation, remodelling, and lung function. We hypothesised that altered epithelial function following allergen exposure determines the onset of airway remodelling and abnormal lung function, which are irreversible with current asthma therapies. We calibrated the in silico model using age appropriate in vivo data from neonatal and adult mice. We validated the in silico model using in vivo data from mice on the effects of current treatment strategies. The in silico model recapitulates experimental observations and provides an interpretable in silico tool to assess airway pathology and the underlying immune responses upon allergen exposure. The in silico model simulations predict the extent of bronchial epithelial barrier damage observed when allergen sensitisation occurs and demonstrate that epithelial barrier damage and impaired immune maturation are critical determinants of reduced lung function and asthma development. The in silico model demonstrates that both epithelial barrier repair and immune maturation are potential targets for therapeutic intervention to achieve successful asthma prevention.
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Journal articleTriantafyllou E, Gudd CLC, Possamai LA, 2024,
Immune-mediated liver injury from checkpoint inhibitors: mechanisms, clinical characteristics and management.
, Nat Rev Gastroenterol HepatolImmunotherapy has changed the treatment landscape for patients with cancer in the past decade. Immune checkpoint inhibitor (ICI)-based therapies have proven effective in a range of malignancies, including liver and gastrointestinal cancers, but they can cause diverse off-target organ toxicities. With the increasingly wider application of these drugs, immune-mediated liver injury from ICIs has become a commonly encountered challenge in clinical hepatology and gastroenterology. In this Review, we discuss the evidence from human and animal studies on the immunological mechanisms of immune-mediated liver injury from ICIs and summarize its clinical features and practical considerations for its management.
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Journal articleMohammed Abdul Wajid L, Saglani S, Nagakumar P, et al., 2024,
Managing preschool wheeze: a qualitative study of parents' views and experiences.
, Arch Dis ChildOBJECTIVE: The aim of this study was to explore parents' views and experiences of managing preschool wheeze, including opinions on the use of investigations to inform treatment pathways. DESIGN: Purposive sampling was used to recruit 16 participants from 14 families across England and Wales. Qualitative data were collected via semi-structured interviews with parents of children aged 1-5 years with preschool wheeze, conducted on Microsoft (MS) Teams. Data were transcribed and analysed using thematic analysis, facilitated by NVivo software package. RESULTS: Analysis generated four themes: (1) pathway to diagnosis, (2) medication management, (3) living with preschool wheeze and (4) improving preschool wheeze healthcare. Findings suggest a negative impact of preschool wheeze on families' lives, including high levels of worry and limiting capacity for work and travel. Barriers to effective management of preschool wheeze included inconsistent terminologies and diagnostic uncertainty alongside limited education and management support. Other barriers included parental concerns about medications, delayed investigations and challenges with accessing specialist care. Parents were in favour of performing investigations to guide treatment pathways. CONCLUSION: Parents' views highlight the problem of diagnosing and treating preschool wheeze at multiple system levels. To improve management and ensure that services for children with preschool wheeze are effective, there is an urgent need for consistent terminology, a unified approach to guide investigations and treatments and for upskilling healthcare professionals in primary and secondary care.
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Journal articleFenn J, Madon K, Conibear E, et al., 2024,
An ultra-early, transient interferon-associated innate immune response associates with protection from SARS-CoV-2 infection despite exposure.
, EBioMedicine, Vol: 111BACKGROUND: A proportion of individuals exposed to respiratory viruses avoid contracting detectable infection. We tested the hypothesis that early innate immune responses associate with resistance to detectable infection in close contacts of COVID-19 cases. METHODS: 48 recently-exposed household contacts of symptomatic COVID-19 cases were recruited in London, UK between May 2020 and March 2021 through a prospective, longitudinal observational study. Blood and nose and throat swabs were collected during the acute period of index case viral shedding and longitudinally thereafter. Magnitude of SARS-CoV-2 exposure was quantified, and serial PCR and serological assays used to determine infection status of contacts. Whole-blood RNA-seq was performed and analysed to identify transcriptomic signatures of early infection and resistance to infection. FINDINGS: 24 highly-exposed household contacts became PCR-positive and seropositive whilst 24 remained persistently PCR-negative and seronegative. A 96-gene transcriptomic signature of early SARS-CoV-2 infection was identified using RNA-seq of longitudinal blood samples from PCR-positive contacts. This signature was dominated by interferon-associated genes and expression correlated positively with viral load. Elevated expression of this 96-gene signature was also observed during exposure in 25% (6/24) of persistently PCR-negative, seronegative contacts. PCR-negative contacts with elevated signature expression had higher-magnitude SARS-CoV-2 exposure compared to those with low signature expression. We validated this signature in SARS-CoV-2-infected individuals in two independent cohorts. In naturally-exposed healthcare workers (HCWs) we found that 7/58 (12%) PCR-negative HCWs exhibited elevated signature expression. Comparing gene-signature expression in SARS-CoV-2 Controlled Human Infection Model (CHIM) volunteers pre- and post-inoculation, we observed that 14 signature genes were transiently upregulated as soon as 6 hr post
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Journal articleBradford T, Summers PA, Majid A, et al., 2024,
Imaging G-Quadruplex Nucleic Acids in Live Cells Using Thioflavin T and Fluorescence Lifetime Imaging Microscopy.
, Anal ChemVisualization of guanine-rich oligonucleotides that fold into G-quadruplex (G4) helical structures is of great interest in cell biology. There is a large body of evidence that suggests that these noncanonical structures form in vivo and play important biological roles. A promising recent development highlighted fluorescence lifetime imaging microscopy (FLIM) as a robust technique for the direct and quantitative imaging of G4s in live cells. However, this method requires specialized, bespoke synthetic dyes that are not widely available. Herein, we demonstrate that the fluorescence lifetime of commercially available environmentally sensitive dyes Thioflavin T (ThT) and Thiazole Orange (TO) is strongly dependent on the type of DNA topology they bind to, with G4s showing long and distinctive decay times that should allow G4 detection in the biological environment. We applied this observation to visualize G4s in live U2OS cells using FLIM of ThT, upon alteration in G4 levels due to competitive binding or nuclease treatment of cells.
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Journal articleDabas R, Alan K, David C, et al., 2024,
Redox-responsive polymeric nanogels as efficient mrNRA delivery vehicles in Caenorhabditis elegans
, microPublication Biology, ISSN: 2578-9430Efficient delivery of sensitive nucleic acid payloads, including mRNA, in Caenorhabditis elegans remains challenging, especially with traditional, labor-intensive transgenesis methods. We addressed these challenges using polymeric nanogels (NGs) as an advanced platform for mRNA delivery in C. elegans. These polymeric delivery vehicles can be engineered to suit desired applications owing to their chemical versatility, resulting from the ability to conjugate multiple functional groups onto the same backbone. Here, we validate the in vivo RNA delivery potential of redox-responsive NGs. The NGs showed up to 72.4 % RNA encapsulation and 6.61 % loading efficiencies and facilitated the controlled release of the mRNA payloads at intracellular concentrations of the reducing agent glutathione, where most of the RNA was released within 24 hours. As a proof of concept, we successfully delivered green fluorescent protein (GFP)-expressing mRNA using NGs in C. elegans for the first time. Physicochemical characterization revealed uniform NG size and charge, and fluorescence microscopy confirmed GFP expression in the gut after 24 hours of treatment. Our findings show NGs' potential as an mRNA delivery system in C. elegans.
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Journal articleClark TW, Tregoning JS, Lister H, et al., 2024,
Recent advances in the influenza virus vaccine landscape: a comprehensive overview of technologies and trials.
, Clin Microbiol Rev, Vol: 37SUMMARYIn the United Kingdom (UK) in 2022/23, influenza virus infections returned to the levels recorded before the COVID-19 pandemic, exerting a substantial burden on an already stretched National Health Service (NHS) through increased primary and emergency care visits and subsequent hospitalizations. Population groups ≤4 years and ≥65 years of age, and those with underlying health conditions, are at the greatest risk of influenza-related hospitalization. Recent advances in influenza virus vaccine technologies may help to mitigate this burden. This review aims to summarize advances in the influenza virus vaccine landscape by describing the different technologies that are currently in use in the UK and more widely. The review also describes vaccine technologies that are under development, including mRNA, and universal influenza virus vaccines which aim to provide broader or increased protection. This is an exciting and important era for influenza virus vaccinations, and advances are critical to protect against a disease that still exerts a substantial burden across all populations and disproportionately impacts the most vulnerable, despite it being over 80 years since the first influenza virus vaccines were deployed.
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