Biology subjects

Nazareth, D.

Publications and source records attributed to Nazareth, D..

4 recordsLinked to original sources

bioRxiv2025

AAV2 Bypasses Direct Endosomal Escape by Using AAVR to Access the Trans-Golgi Network en Route to the Nucleus.

Vectors based on the adeno-associated virus are widely used as delivery methods in gene therapy applications, yet understanding of the mechanisms governing its intracellular trafficking remains incomplete. Traditional models suggest that AAV escapes from endosomes via membrane disruption, but direct evidence for this process are lacking. Here, we show that AAVR, the essential AAV cell entry receptor, functions as a bona fide retromer cargo. Using in vitro reconstitution assays, we demonstrate that AAVRs cytosolic tail is sufficient to engage the SNX3-retromer complex and drive membrane tubulation, a hallmark of retrograde trafficking. In AAVR-knockout HuH-7 cells, AAV2 particles are internalized but fail to reach the trans-Golgi network (TGN) and support transgene expression. Galectin-8 recruitment assays reveal no evidence of endosomal membrane rupture during productive transduction, distinguishing AAV2 from lytic vectors such as lipid nanoparticles. Moreover, VP1u-deficient and PLA2-mutant AAV2 capsids accumulate at the TGN, indicating that VP1u is dispensable for early trafficking but required for post-TGN progression toward productive transduction. These findings challenge the prevailing model of direct endosomal escape and position AAV transduction as a vesicle-guided, receptor-mediated process.

cell biology↗

bioRxiv2024

Sputum Proteomics reveals unique signatures linked to key outcomes in cystic fibrosis trials.

RationaleLung function (FEV1) and quality of life (QoL) are key outcomes in most interventional clinical trials conducted in people living with cystic fibrosis. However, no robust pre-clinical surrogates for FEV1 and QoL exist. The precise physiological mechanisms leading to treatment-related improvements in these outcomes are incompletely understood. In this post-hoc analysis we explored the relationship between changes in the sputum proteome and these outcomes with the aim of identifying translational biomarkers. MethodsPaired sputum samples collected during the AZTEC-CF study (NCT02894684) pre and post 14 days of antibiotic treatment for an acute pulmonary exacerbation were included. Samples were analysed using in vitro Mesoscale Discovery (MSD) assays and by nano LC-MS/MS. Peptide identification and quantification was performed and the log-fold change for individual proteins and relationships between protein change and changes in FEV1 and QoL were evaluated. ResultsDistinct patterns were found between proteins that correlated with FEV1 and those that correlated with QoL improvements. FEV1 improvement was characterised by increases in bacterially-derived proteins accompanied by decreases in proteins relating to neutrophil degranulation. Conversely, changes in QoL were associated with increases in antiprotease and antioxidant proteins. MSD analysis revealed changes in some neutrophil-associated markers significantly correlated with FEV1 improvements, but no markers significantly correlated with QoL improvements. ConclusionsThese results suggest changes in two key CF clinical trial outcomes (FEV1 and QoL) may be underpinned by different physiological mechanisms. Understanding these divergent mechanisms is vital to fortify optimal clinical trial design in CF and panels of biomarkers may be needed to improve translational confidence.

immunology↗

bioRxiv2024

Mechanism by which Aurora B inhibitors promotes RB and p53-dependent senescence.

Polyploidy is a common outcome of chemotherapies, but there is conflicting evidence as to whether this is a source of increased chemotherapy resistance and aggressive disease, or a benign or even favorable outcome. We have used Aurora B kinase (AURKB) inhibitors that efficiently promote polyploidy in many cell types to investigate the fate of polyploid cells. We demonstrate AURKB inhibitor treatment of cells that have loss of RB and p53 function causes them to become hyper-polyploid, undergoing continuous rounds of growth, replication and failed mitosis/cytokinesis (endomitosis), whereas RB and p53 functional cells will eventually exit the cell cycle. These hyper-polyploid cells (>4n DNA content) are viable and undergo continuous endomitotic cycles, but have lost the ability to form viable colonies in vitro or form tumours in vivo. Investigation of mitosis in these cells revealed that centrosome duplication remained coupled to DNA replication, with the hyper-polyploid cells containing high numbers of centrosome that were capable of supporting functional mitotic spindle poles, but these failed to progress to anaphase/telophase structures even when AURKB inhibitor was removed after 2-3 days. However, when AURKB inhibitor was removed after 1 day and cells had failed a single cytokinesis to become tetraploid, they retained long term colony forming ability. Collectively, these findings demonstrate that tetraploidy is well tolerated by tumour cells but higher ploidy states are incompatible with long term proliferative potential.

cell biology↗

bioRxiv2023

Harnessing the power of whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation

Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8-derived variants performed efficiently. This study establishes the use of normothermic liver perfusion as an invaluable preclinical model for evaluating liver-targeted gene therapies and providing guidance for making essential decisions that promote the most effective translational programs.

molecular biology↗