tbuckley/cabot-search huggingface dataset:
O17 Highly efficient base editing of homozygous and heterozygous <i>COL7A1</i> variants in fibroblasts from patients with recessive dystrophic epidermolysis bullosa using adenine base editor 8e mRNA system delivered via novel lipid nanoparticle formulations
Authors: Ina Guri, Imogen Brooks, Yara Alrokh, Aidni Kazamizadeh, Carina Graham, Steven Hart, John McGrath, Joanna Jackow
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims Adenine base editors (ABEs) present an attractive approach to gene editing in the inherited blistering disease recessive dystrophic epidermolysis bullosa (RDEB), which Results from pathogenic variants in COL7A1. We previously showed that ABE8e, a new ABE variant, confers remarkable efficiency (94.6%) and a favourable safety profile in correcting variants and restoring collagen 7 (C7) expression when electroporated into RDEB fibroblasts. With a goal to develop topical base editing creams, we now explore use of lipid nanoparticle (LNP)-encapsulated ABE8e mRNA system to edit two distinct variants in RDEB fibroblasts. Methods Primary patient fibroblasts harbouring a homozygous G&amp;gt;A mutation (exon 42) or a heterozygous C&amp;gt;T mutation (exon 54) were gene edited using ABE8e mRNA, delivered via lipofectamine or six different LNPs. To assess the gene editing efficiency, Sanger sequencing and next-generation sequencing were employed. Cell viability was evaluated via a lactate dehydrogenase cytotoxicity assay, while functional restoration of C7 expression was assessed via Western blotting of cell lysate and supernatant. Finally, topical delivery was tested on normal human skin equivalents (HSEs). Results We identified an optimal ratio of 1 : 7 for sgRNA : ABE8e, at which editing efficiency increases with ABE8e dosage, with 400 ng being sufficient for 90% biallelic editing. Formulations containing DTDTMA (C14) and DHDTMA (C16) LNPs demonstrated approximately 90% and approximately 80% editing (with and without the peptide, respectively), in addition to reduced cytotoxicity compared with lipofectamine. Depending on the nature of the mutation, the edit corresponded to an increase in C7 expression in supernatant only or cell lysate as well, whereby C14 LNPs enabled twofold C7 restoration. Finally, our HSEs demonstrated viability of topical delivery of lipofectamine-encapsulated green fluorescent protein mRNA. Conclusions We demonstrated the potential value of base editing as a gene therapy for RDEB in addition to the potential of these LNPs for clinical translation of the therapy. Nonetheless, further safety and functionality tests still need to be conducted.
O08 Exploring the effect of skin ageing-related basement membrane collagen loss on nuclear mechanics
Authors: Simranpreet Summan, Andrew Mayes, David Gunn, Pradeepa Madapura, Julien Gautrot, Matthew Caley, Edel O’Toole
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims Skin ageing is a universal process that can increase susceptibility to inflammation and disease. Basement membrane collagens (BMCs) are reduced during ageing and the consequences are poorly understood. BMC sensing of mechanical stress, vital for skin homeostasis, elicits signalling sensed via cytoskeletal actin filaments to the linker of nucleoskeleton and cytoskeleton (LINC) complex (composed of nesprin and sun proteins), nuclear lamina and lamina-associated chromatin. Our hypothesis is that BMC loss during ageing leads to a deformation of the nucleocytoskeleton and disrupts chromatin organization in keratinocytes, leading to impairment of epidermal differentiation and inability to withstand mechanical stress. Methods To explore the role of BMC in mechanical stress response, stable lentiviral short hairpin (sh)RNA knockdowns of Col7 (shCol7) and Col17 (shCol17) were generated in immortalized N/TERT keratinocytes. Western blot (WB) validated knockdowns were compared with shC nontargeting control. IncuCyte assays showed shCol17 had a hyperproliferative phenotype compared with shC. Sun2 was reduced in shCol17 (P &amp;lt; 0.001) compared with shC (n = 5 experimental replicates). The mechanical stretch effect on cells on a flexible membrane was studied using immunofluorescence and WB analysis. Images taken from the IN Cell Analyzer confocal microscope were analysed to detect changes in protein expression and localization. Nuclear and cytoplasmic fractions made before and after stretch were analysed by WB. Results We observed collagen-specific responses to stretch-induced stress with changes to actin, components of the nuclear membrane, and LINC complex proteins. Additionally, work on investigating chromatin changes with BMC knockdown using Cleavage Under Targets &amp; Tagmentation and Cleavage Under Targeted Accessible Chromatin assays is ongoing. Conclusions Our data show an altered nucleocytoskeleton in the context of BMC loss and mechanotransduction in skin. These data could provide novel insights into epidermal regulation by BMC in ageing.
P11 A role for keratinocyte arginase in skin wound healing and barrier function
Authors: Denis-Cristian Szondi, Sheena Cruickshank, Leah Vardy, Jason Wong
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims Delayed cutaneous wound healing is a major problem that can result in social isolation and significant morbidity including amputation. There are many changes in cellular function in a chronic wound. Notably, the enzyme arginase (ARG)1 is markedly changed in wound healing. We have identified a role for ARG1 in keratinocyte re-epithelialization during wound closure, but the underlying mechanism driving re-epithelialization was not known. We aimed to determine the role of ARG1 in keratinocyte migration, differentiation and barrier formation. Methods Two-dimensional scratch and differentiation assays using immortalized keratinocytes (NTERT cells) treated with the ARG1 inhibitor, nor-NOHA, and short hairpin RNA-based knockdown cells were assessed. Gene and protein expression changes were highlighted. Expression of proteins was further validated using immunohistochemical profiling of human skin equivalents and human wound lesions by spatial transcriptomics. Results ARG1 was strongly expressed both 2–4h postscratching and at late stages of keratinocyte differentiation. Dysregulation of ARG1 expression was spatially highlighted in chronic lesions, further suggesting an involvement in keratinocyte migration and differentiation. ARG1 inhibition led to a significant delay in postscratch keratinocyte migration. Furthermore, upon ARG1 inhibition, a significant decrease was seen in the expression of late differentiation markers involucrin and filaggrin. ARG1 inhibition significantly decreased the expression of host defence genes β-defensin 3, lipocalin 2 and kallikrein 6. ARG1 triggers production of multiple downstream products and we confirmed a role for the pathway involving production of putrescine and urea, as the addition of these metabolites led to the rescue of these phenotypes. Conclusions ARG1 and its downstream metabolites play a major role in keratinocyte migration, differentiation, and barrier formation, which are vital functions for re-epithelialization and wound closure. Therefore, manipulation of the ARG1 pathway may be crucial in the management of chronic lesions.
P19 Defining the mechanism of skin barrier restoration by <i>Lactobacillus rhamnosus</i>
Authors: Lisa Coppock, Ryan O’Shaughnessy, Catherine O’Neill
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims We aim to investigate the lysate of Lactobacillus rhamnosus GG (LGG), a bacterium previously established as a gut probiotic, and to examine its postulated ability to enhance and restore human skin barrier function. Compromised skin barriers are implicated in various skin conditions including atopic dermatitis. Furthermore, skin barrier function decreases with age leading to dryness and sensitivity. Previous research has shown promising effects of LGG lysates on keratinocytes, including increasing transepidermal electrical resistance, increasing tight junction protein expression, cell migration promotion and mitigation of Staphylococcus aureus toxicity. This study aims to take a closer look at the molecular mechanisms behind the effect of LGG lysates on the skin, including terminal differentiation, lamin dispersal and nuclear degradation in keratinocytes. Methods Normal human epithelial keratinocytes and rat epithelial keratinocytes were grown and treated with the LGG lysate. Western blot analysis was used to determine the relative concentrations of tight junction proteins and other differentiation markers. A phosphoproteomic study was performed to identify key pathways activated by the LGG lysate. A Staphylococcus epidermidis lysate control was used to filter out any nonspecific bacterial effects. Immunostaining of nuclear proteins was used to visualize the effects of LGG lysate on lamin dispersal and nuclear degradation. short hairpin RNA filaggrin knockdown normal human keratinocytes were developed to create a ‘barrier impaired’ model to assess ability of the lysate to ‘rescue’ the skin barrier. Results The LGG lysate was shown to increase tight junction protein expression in normal human keratinocytes and increase keratinocyte terminal differentiation markers. We present Results that show decreased tight junction protein expression in filaggrin knockdown cells. Conclusions These Results show the potential for LGG lysate to enhance and restore skin barrier function and begin to suggest potential mechanisms for this effect.
P12 Transcriptional and epigenetic changes drive increased cellular plasticity in keloid fibroblasts
Authors: Stavroula Tekkela, Willow Hight-Warburton, Tanya Shaw, Mike Philpott, Edel O’Toole, Emanuel Rognoni
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims Keloids are fibroproliferative scars that progressively grow beyond the original wound site. They occur as a result of genetic, epigenetic and environmental abnormalities. Fibroblasts are best characterized for their role in depositing and remodelling the extracellular matrix (ECM) and are believed to have a crucial role in keloid formation. However, the detailed molecular signatures that promote this pathogenic phenotype are still unknown. Methods To investigate the epigenetic and genetic changes in keloid fibroblasts, we performed assay for transposase-accessible chromatin (ATAC)-Seq and RNA-Seq on a patient-matched set of keloid and normal fibroblasts. ATAC-Seq is a high-throughput sequencing method that defines chromatin accessible regions in which the binding of sites of transcription factors that regulate gene expression can be mapped. Identified candidates are validated in keloid fibroblasts via quantitative polymerase chain reaction and their functional role is analysed in various in vitro assays. Results ATAC-Seq analysis revealed more accessibility in coding regions of keloid fibroblasts compared with normal fibroblasts. Pathways enriched in these regions include transcriptional regulation, embryonic development, cell differentiation, calcium-binding and collagen remodelling. Intriguingly, several developmental epigenetic modulators were identified that may alter the transcription and chromatin landscape of keloid fibroblasts to a more plastic state and have the capacity to influence cell differentiation choices, potentially promoting chondrogenic and adipogenic cell features. Currently, we are investigating the ability of keloid fibroblasts to be more readily differentiated into other cell types including induced pluripotent stem cells, and the contribution of those candidates on fibrotic features to the cell are also being explored. Conclusions Our analysis reveals that the chromatin of keloid fibroblasts is more accessible, especially in genomic regions associated with embryonic development and keloid fibroblasts show an increased plasticity in vitro. These findings may support the development of fibroblast-targeted therapies for keloid scars and possibly other connective tissue disorders.
P17 Dissection of the molecular and cellular heterogeneity of dermal fibroblasts in skin fibrosis
Authors: Abubkr Ahmed, Thomas Kirk, Edel O’Toole, Emanuel Rognoni
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims Skin fibrosis, a common fibrotic condition that currently has no specific treatments or cures, is caused by persistent activation of skin fibroblasts after tissue injury that Results in excessive scarring, can cause severe disability and seriously affect morbidity. This study aims to uncover the specific fibroblast subsets and molecular pathways that are integral to this sustained fibroblast activation using a bleomycin-induced skin fibrosis mouse model that accurately recapitulates the human disease. Methods We have successfully shown that 2 weeks (early) and 4 weeks (advanced) of bleomycin injections Results in an increasing severity of skin fibrosis characteristics. Following this treatment, we performed single-cell RNA sequencing (scRNA-Seq) using control, early and advanced fibrotic tissue and identified fibroblast subclusters and specific gene signatures that are altered across the time course and may play a role in the fibrotic phenotype we observe. The use of advanced scRNA-Seq analysis techniques such as SCENIC and regulon analysis has allowed us to further characterize the changes in fibroblast states during fibrosis. Results Our scRNA-Seq data show a loss of fibroblast heterogeneity alongside the emergence of two main subclusters with distinct gene signatures in the advanced fibrotic state. While proliferation is minimal, RNA velocity analysis indicates that the increase in cell count is due to the convergence of neighbouring fibroblast subclusters. The pronounced profibrotic gene expression point to its key role in driving skin fibrosis and we are now exploring the therapeutic potential of chemically or genetically targeting this subcluster. Conclusions Our novel skin fibrosis time course scRNA-Seq dataset allows us to compare the cellular and molecular changes in early and advanced fibrotic lesions in order to identify the key fibroblast subpopulations, molecular regulators and connective tissue alterations promoting fibrotic tissue repair. Our findings will support the development of new fibroblast-targeted fibrosis therapies in skin and potentially in other organs.
P08 Exploring the role of cellular senescence in cutaneous repair
Authors: Alexander Johns, Holly Wilkinson
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims Chronic nonhealing wounds are a life-threatening skin disease in elderly patients and patients with diabetes that costs the National Health Service over £8 billion per year to treat. Unfortunately, current treatments are inadequate, thus there is an urgent need to develop effective therapies. Cellular senescence is emerging as a potential therapeutic target owing to its association with a wide range of age-related pathologies. Cells undergo senescence in response to ageing and stress, losing their ability to proliferate or perform normal tissue functions. Our previous work in mice provided the first mechanistic demonstration that senescent cell accumulation drives poor healing. Moreover, clinical trials currently assessing the efficacy of senescence-targeted drugs (senolytics) are providing repurposing opportunities for cutaneous pathologies. Therefore, the aim of this research is to (i) investigate the role of senescence in human chronic wounds and (ii) determine whether senolytics can be repurposed to promote pathological skin repair. Methods Skin and wounds were collected from healthy, aged and diabetic donors to quantify senescence levels (senescence-associated β-galactosidase), characterize senescent cell types (immunofluorescence) and determine key drivers of skin senescence (RNA sequencing). Additionally, we screened the wound healing potential of a panel of senolytics in healthy skin vs. skin from patients with chronic wounds. Results We found significant accumulation of senescence in pathological skin wounds, localized to stromal cells. Senescence levels also correlated substantially with clinical parameters, such as wound site, diabetic status and infection. RNA sequencing revealed key links between senescence and immunity, suggesting inflammation as a crucial driver of skin senescence. Finally, senolytic treatment significantly accelerated healing in skin from patients with chronic wounds. Conclusions Our data provide the first in-depth characterization of senescence in human chronic skin wounds, crucially identifying novel therapeutic targets of senescence, while simultaneously assessing the potential of drug repurposing for patient benefit.
P03 Anti-ageing effects of oestetrol on the skin: protection from cellular senescence and restoration of dermal architecture
Authors: Leah Cooksey, Laden Fenercioglu, Tania Bernardes, Alice Fearn, Céline Gerard, Valérie Dion, Matthew Hardman, Holly Wilkinson
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims Skin is a major endocrine organ, undergoing profound structural and functional alterations in response to hormonal changes. Postmenopause, the rapid decline in circulating 17β-oestradiol (E2) leads to skin ageing, while exogenous E2 administration restores skin function, promoting the regenerative capacity of aged skin. Nevertheless, the risk of off-target effects limits the clinical use of E2 for skin indications. Oestetrol (E4), a natural oestrogenic steroid currently undergoing clinical trials as a hormone replacement therapy, presents a favourable safety profile and could be a suitable alternative to E2. However, as no studies have investigated the effects E4 on skin ageing, the aim of this study was to evaluate the potential of E4 to overcome effects of skin ageing. Methods Micro-osmotic pumps were subcutaneously implanted into 14-month-old female mice, allowing sustained systemic delivery of E2 or E4 over 3 weeks. Reversal of skin ageing was assessed via noninvasive skin parameter testing, histological analysis and biomechanics. Aged human dermal fibroblasts were treated with E4 to measure effects on the extracellular matrix deposition (ECM) in vitro. Finally, human keratinocytes were treated with E4 prior to inducing senescence to assess potential protective effects against skin ageing. Readouts included senescence-associated beta galactosidase staining, immunofluorescence and quantitative polymerase chain reaction. Results Systemic administration of E4 reversed multiple characteristics of skin ageing. E4 treatment significantly increased skin hydration, stimulated collagen deposition and elevated elastic fibre synthesis. These structural changes improved the biomechanical function of skin, increasing breaking strength and elasticity. In vitro studies confirmed direct anti-ageing activity on multiple cell types. E4 substantially increased ECM deposition in aged human dermal fibroblasts and protected against senescence induction in human keratinocytes. Conclusions Collectively, these preclinical data demonstrate significant beneficial effects of E4 on aged skin, strongly supporting future clinical evaluation of E4 to alleviate the effects of skin ageing following menopause.
P02 Accelerated ageing of hair follicles in androgenetic alopecia
Authors: Summik Limbu, Kim Farhat, Leah Redmond, Nilofer Farjo, Bessam Farjo, Paul Kemp, Claire Higgins
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims Androgenetic alopecia (AGA) is defined by hair loss from the frontal, parietal and vertex regions of the scalp; however, occipital follicles are spared. Hair follicles in AGA undergo miniaturization, which is the transition of terminal follicles into vellus follicles. Similarly with age, the diameter of the hair shaft decreases with loss of pigmentation, which is also seen in miniaturized follicles. To investigate whether frontal follicles have accelerated ageing compared with occipital follicles, we looked at methylation levels of genes associated with ageing. As the thickness and pigmentation of hair fibre are influenced by the dermal papilla (DP), we conducted microarray analysis of frontal and occipital DP to establish whether accelerated ageing of follicles is driven by DP. Methods The biological age of frontal and occipital follicles was calculated using methylation status of ELOVL2, ASPA, EDARADD and PDE4C (n = 3). For microarray analysis, DP was microdissected from the frontal (FDP) and occipital (ODP) follicles from patients undergoing hair transplantation surgeries to treat AGA (n = 4). Results Frontal follicles were found to have significantly higher biological age compared with patient-matched occipital follicles suggesting accelerated ageing of follicles in AGA. Transcriptomic analysis revealed significant increase in expression of several genes in FDP and notably EGR1, HSPB1, DNAJB1 and APOD, which have been shown to be upregulated in the DP of aged mice compared with young mice. We found downregulation of TRPS1 in FDP, which represses EGR1. Mutations in TRPS1 underlie trichorhinophalangeal syndrome which is associated with vellus-type hair. Conclusions Our transcriptional and molecular analysis revealed that FDP and frontal follicles have an advanced biological age relative to ODP. This increased age is also reflected in the transcriptional signature of FDP. We hypothesize that these changes are in part due to regulation of signalling by the transcriptional repressor TRPS1, whose expression is decreased in FDP.
P10 Janus kinase inhibitors to restore skin barrier function in a mouse model of Harlequin ichthyosis
Authors: Elena Giusto, Eleri Jones, Florence Enjalbert, Abubkr Ahmed, Matthew Caley, Emanuel Rognoni, Edel O’Toole
Journal: British Journal of Dermatology (2024)

Abstract Introduction and aims The autosomal recessive congenital ichthyoses (ARCIs) are a group of chronic conditions where there is unmet therapeutic need. Harlequin ichthyosis (HI) is the most severe form of ARCI with aberrant differentiation of the epidermis leading to a severe barrier defect. It is caused by mutations in ABCA12, a lipid transporter involved in transport of glucosylceramides from the lamellar body to the lipid lamellae. Several Janus kinase (JAK) inhibitors inhibiting the JAK-signal transducer and activator of transcription pathway are now licensed for atopic dermatitis. Previous work from our group in an HI in vitro model showed that tofacitinib, a pan-JAK inhibitor, had a restorative effect on the lipid barrier. Methods Further selective JAKis were tested in three dimensional skin equivalents using ABCA12 CRISPR-Cas9 knockout and wildtype keratinocytes to identify the most promising JAKi for use in an HI murine model (ABCA12tm1c: Het Cre+). JAKi-A (5 mg kg−1) and vehicle (10% dimethylsulfoxide in corn oil) were tested on male and female HI-inducible and wildtype mice (N = 4). All mice were initially treated with 4-OH-Tamoxifen to induce the HI phenotype and after 1 day JAKi-A was administered via oral gavage for 9 days. All mice were killed, and skin was collected and processed for haematoxylin and eosin, Nile Red, ABCA12, glucosylceramide and immune cell marker staining. Results JAKi-A had a positive effect on HI epidermal morphology, showing a significantly reduced epidermal thickness in treated HI mice compared with controls. JAKi-A also had a positive effect on the epidermal barrier, restoring neutral and polar lipids compared with vehicle-treated mice as observed using Nile Red and glucosylceramide staining. Further analysis on immune cell infiltrates is in progress. Conclusions These Results suggest that JAKi-A restores the skin barrier in vivo and may be a promising treatment for severe ichthyosis.