Biology subjects

Waters, S. A.

Publications and source records attributed to Waters, S. A..

5 recordsLinked to original sources

bioRxiv2025

Gene-Corrected Basal Cells Restore CFTR In Vitro; Transplants Regenerate Epithelium in a Preclinical Sinus Model

BackgroundCystic fibrosis (CF) is caused by mutations in the CFTR gene, leading to epithelial dysfunction and progressive lung disease. Although CFTR modulators have transformed care, [~]10% of people with CF remain without effective therapy. Durable, mutation-agnostic approaches are urgently needed. MethodWe used a lentiviral (LV) vector to deliver wild-type CFTR to airway basal cells derived from 13 paediatric CF participants with a range of genotypes. Transduced cells were assessed for transgene expression, epithelial differentiation, and CFTR function using air-liquid interface (ALI) cultures. Separately, to evaluate regenerative capacity in vivo, LVGFP-transduced rabbit airway basal cells were transplanted into the denuded nasal septum of healthy New Zealand white rabbits using a biocompatible scaffold. ResultsTransduced basal cells retained multilineage differentiation capacity, forming well-organized, pseudostratified epithelium with intact barrier function and ciliary activity. CFTR channel activity was restored to levels comparable to or exceeding those achieved with elexacaftor/tezacaftor/ivacaftor (ETI), including in individuals with nonsense mutations. Combined CFTR transduction plus ETI treatment showed additive benefit. In vivo, transplanted rabbit basal cells engrafted and differentiated to regenerate a mucociliary epithelium, with improved nasal potential difference and mucociliary clearance compared to scaffold-only controls. ConclusionOur study demonstrates that LV-mediated CFTR gene addition restores CFTR function in vitro across genotypes and supports epithelial regeneration in a clinically relevant animal airway model. This two-part platform offers a scalable path toward cell therapies for all people with CF and may have broader applications in upper airway epithelial repair.

cell biology↗

bioRxiv2022

Assessment of pre-clinical liver models based on their ability to predict the liver-tropism of AAV vectors

The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors (rAAV). Multiple clinical trials have been undertaken for this target in the past 15 years, however we are still to see market approval of the first liver-targeted AAV-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically- and clinically-predictive preclinical models. To this end, this study reports findings of a functional evaluation of six AAV vectors in twelve preclinical models of the human liver, with the aim to uncover which model is the most relevant for the selection of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.

bioengineering↗

bioRxiv2021

Ageing impairs the airway epithelium defence response to SARS-CoV-2

Age-dependent differences in the clinical response to SARS-CoV-2 infection is well-documented1-3 however the underlying molecular mechanisms involved are poorly understood. We infected fully differentiated human nasal epithelium cultures derived from healthy children (1-12 years old), young adults (26-34 years old) and older adults (56-62 years old) with SARS-COV-2 to identify age-related cell-intrinsic differences that may influence viral entry, replication and host defence response. We integrated imaging, transcriptomics, proteomics and biochemical assays revealing age-related changes in transcriptional regulation that impact viral replication, effectiveness of host responses and therapeutic drug targets. Viral load was lowest in infected older adult cultures despite the highest expression of SARS-CoV-2 entry and detection factors. We showed this was likely due to lower expression of hijacked host machinery essential for viral replication. Unlike the nasal epithelium of young adults and children, global host response and induction of the interferon signalling was profoundly impaired in older adults, which preferentially expressed proinflammatory cytokines mirroring the "cytokine storm" seen in severe COVID-194,5. In silico screening of our virus-host-drug network identified drug classes with higher efficacy in older adults. Collectively, our data suggests that cellular alterations that occur during ageing impact the ability for the host nasal epithelium to respond to SARS-CoV-2 infection which could guide future therapeutic strategies.

immunology↗

bioRxiv2020

Novel antioxidant therapy with the immediate precursor to glutathione, γ-glutamylcysteine (GGC), ameliorates LPS-induced cellular stress in an in vitro cystic fibrosis model

IntroductionGlutathione deficiency and chronic bacterial inflammation exacerbates the oxidative stress damage to airways in cystic fibrosis. Improvements to current antioxidant therapeutic strategies are needed. Dietary supplement, {gamma}-glutamylcysteine (GGC), the immediate precursor to glutathione, rapidly boosts cellular glutathione levels following a single dose in healthy individuals. Efficacy of GGC against Pseudomonas aeruginosa derived lipopolysaccharide (LPS), a prominent factor in mediating both bacterial virulence and host responses, in CF remains unassessed. MethodsPrimary F508del/F508del mucociliary differentiated bronchial and nasal epithelial cells were created to model LPS-induced oxidative stress and inflammation of CF. The proteomic signature of GGC treated cells was resolved by qLC-MS/MS. Parameters including cell redox state (glutathione, ROS), anti-inflammatory mediators (IL-8, IDO-1) and cellular health (membrane integrity, stress granule formation and cell viability) were assayed. ResultsProteomic analysis identified perturbation of several pathways related to cellular respiration and stress responses upon LPS challenge. Most of these were resolved when cells were treated with GGC. While GGC did not resolve LPS-induced IL-8 and IDO-1 activity, it effectively attenuated LPS-induced ROS and stress granule formation, while significantly increasing intracellular glutathione levels and improving epithelial cell barrier integrity. Moreover, we compared the effect of GGC with thiols NAC and glutathione on cell viability. GGC was the only thiol that increased cell viability; protecting cells against LPS induced cell death. Both therapeutic and prophylactic treatments were successful. ConclusionTogether, these findings indicate that GGC has therapeutic potential for treatment and prevention of oxidative stress related damage to airways in Cystic Fibrosis.

cell biology↗

bioRxiv2019

Non-linear sequence similarity between the Xist and Rsx long noncoding RNAs suggests shared functions of tandem repeat domains

The marsupial inactive X chromosome expresses a long noncoding RNA (lncRNA) called Rsx that has been proposed to be the functional analogue of eutherian Xist. Despite the possibility that Xist and Rsx encode related functions, the two lncRNAs harbor no linear sequence similarity. However, both lncRNAs harbor domains of tandemly repeated sequence. In Xist, these repeat domains are known to be critical for function. Using k-mer based comparison, we show that the repeat domains of Xist and Rsx unexpectedly partition into two major clusters that each harbor substantial levels of non-linear sequence similarity. Xist Repeats B, C and D were most similar to each other and to Rsx Repeat 1, whereas Xist Repeats A and E were most similar to each other and to Rsx Repeats 2, 3, and 4. Similarities at the level of k-mers corresponded to domain-specific enrichment of protein-binding motifs. Within individual domains, protein-binding motifs were often enriched to extreme levels. Our data support the hypothesis that Xist and Rsx encode similar functions through different spatial arrangements of functionally analogous protein-binding domains. We propose that the two clusters of repeat domains in Xist and Rsx function in part to cooperatively recruit PRC1 and PRC2 to chromatin. The physical manner in which these domains engage with protein cofactors may be just as critical to the function of the domains as the protein cofactors themselves. The general approaches we outline in this report should prove useful in the study of any set of RNAs.

genetics↗