Biology subjects

Aubert, M.

Publications and source records attributed to Aubert, M..

5 recordsLinked to original sources

bioRxiv2026

Cell-specific isolation and functional analysis of primary cardiomyocyte-derived large extracellular vesicles from infarcted mouse hearts

Extracellular vesicles (EVs) are membrane-bound particles secreted by all cells and serve as critical mediators of intercellular communication in both physiological and pathological contexts. However, elucidating the precise functions of endogenous EVs remains challenged by a fundamental technical bottleneck: current isolation methodologies depend on shared physical properties or generic surface markers, preventing the selective enrichment of cell-type-specific EVs within complex tissues. Consequently, EVs recovered from biofluids or tissue extracts represent heterogeneous populations, confounding the direct attribution of biochemical signatures and biological activities to distinct cellular origins. Here, we describe a versatile genetic-labeling and affinity-based purification strategy that enables the selective isolation of cell-specific primary EV. Using cardiomyocyte-derived EV (CM-EVs) as a proof-of-concept, we crossed Myh6-Cre mice with the ROSA26-mTmG reporter line to restrict membrane-targeted GFP expression to cardiomyocytes. Following myocardial infarction, cardiac EV were recovered and GFP-positive CM-EVs were selectively purified by anti-GFP nanobody-based magnetic pulldown. This approach yields highly enriched, functional cardiomyocyte-derived extracellular vesicles (CM-EVs). To accommodate diverse analytical needs, we established two distinct elution workflows: (i) a denaturing protocol optimized for downstream proteomics and immunoblotting, and (ii) a gentle, non-denaturing protocol that preserves vesicle integrity and biological activity for downstream in vitro and in vivo functional assays. Using this method, we characterized CM-EVs size distribution and proteomic signature, and demonstrated their capacity to transfer functional cargo to target cells in vitro. Because the purification approach relies solely on targeting a genetically restricted membrane tag rather than assumed surface markers, it is immediately adaptable to virtually any cell type for which a validated Cre driver exists. This method removes a long-standing bottleneck in EV research and provides a broadly applicable platform for deciphering the biochemical composition and in-vivo functions of cell-type-specific primary EV in physiological and pathological settings.

cell biology↗

bioRxiv2025

Minimization of gene editing off-target effects by tissue restriction of expression

Therapeutic in vivo gene editing with highly specific nucleases has the potential to revolutionize treatment for a wide range of human diseases, including genetic disorders and latent viral infections like herpes simplex virus (HSV). However, challenges regarding specificity, efficiency, delivery, and safety must be addressed before its clinical application. A key concern is the risk of off-target effects, which can cause unintended and potentially harmful genetic changes. We previously developed a curative in vivo gene editing approach to eliminate latent HSV using HSV-specific meganuclease delivered by an AAV vector. In this study, we investigate off-target effects of meganuclease by identifying potential off-target sites through GUIDE-tag analysis and assessing genetic alterations using amplicon deep sequencing in tissues from meganuclease treated mice. Our results show that meganuclease expression driven by a ubiquitous promoter leads to high off-target gene editing in the mouse liver, a non-relevant target tissue. However, restricting the meganuclease expression with a neuron-specific promoter and/or a liver-specific miRNA target sequence efficiently reduces off-target effects in both liver and trigeminal ganglia. These findings suggest that incorporation of regulatory DNA elements for tissue-specific expression in viral vectors can reduce off-target effects and improve the safety of therapeutic in vivo gene editing.

molecular biology↗

bioRxiv2022

AAV-delivered gene editing for latent genital or orofacial herpes simplex virus infection reduces ganglionic viral load and minimizes subsequent viral shedding in mice

Herpes simplex virus (HSV) establishes latency in ganglionic neurons of the peripheral nervous system, from which it can reactivate, causing recurrent disease and possible transmission to a new host. Current anti-HSV therapy does not eliminate latent HSV, and thus is only suppressive rather than curative. We developed a potentially curative approach to latent HSV infection and pathogenesis, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Our results demonstrated that a dual meganuclease therapy, composed of two anti-HSV-1 meganucleases delivered by a triple AAV serotype combination (AAV9, AAV-Dj/8, AAV-Rh10), can eliminate up to 97% of latent HSV DNA from ganglia in both ocular and vaginal mouse models of latent HSV infection. Using a novel pharmacological approach to reactivate latent HSV-1 in mice with the bromodomain inhibitor JQ-1, we demonstrated that this reduction in ganglionic viral load leads to a significant reduction of viral shedding from treated vs. control mice, with many treated mice showing no detectable virus shedding. In general, therapy was well tolerated, although dose-ranging studies showed hepatotoxicity at high AAV doses, consistent with previous observations in animals and humans. Also in agreement with previous literature, we observed subtle histological evidence of neuronal injury in some experimental mice, although none of the mice demonstrated observable neurological signs or deficits. These results reinforce the curative potential of gene editing for latent orofacial and genital HSV disease, and provide a framework for additional safety studies before human trials can begin.

neuroscience↗

bioRxiv2022

Liver humanized NSG-PiZ mice support the study of chronic hepatitis B virus infection and antiviral therapies

Hepatitis B virus (HBV) is a pathogen of major public health importance that is largely incurable once a chronic hepatitis B (CHB) infection is established. Only humans and great apes are fully permissive to HBV replication, and this species restriction has impacted HBV research by limiting the utility of small animal models of HBV. To combat the species restriction of HBV and enable more HBV studies in vivo, liver-humanized mouse models have been developed that harbor primary human hepatocytes (PHH) and are fully permissive to HBV infection and replication. Unfortunately, these models can be difficult to establish and are expensive commercially, which has limited their academic use. As an alternative mouse model to study HBV, we evaluated liver-humanized NSG-PiZ mice and showed that they are fully permissive to HBV and can develop CHB. Mice were infected with a precore mutant clinical isolate that has now been serially passaged through 3 generations of mice without loss of fitness. HBV selectively replicates in hCK18+ human hepatocytes within chimeric livers, and HBV+ mice secrete infectious virions and HBsAg into blood, while also harboring covalently closed circular DNA (cccDNA). HBV+ mice remain viremic for at least 169 days, which should enable the study of new curative therapies targeting CHB and respond to antiviral entecavir therapy. The extended duration of viremia is sufficient to enable the study of established and new therapeutic approaches targeting CHB. Furthermore, HBV+ PHH in NSG-PiZ mice can be transduced by the hepatotropic AAV3b and AAV.LK03 vector capsids, which should enable the study of curative gene therapies that target CHB. In summary, our data demonstrates that liver humanized NSG-PiZ mice can be used as a robust and cost-effective alternative to existing CHB models and may enable more academic research labs to study HBV disease pathogenesis and antiviral therapy in a setting that is fully permissive to ongoing replication.

microbiology↗

bioRxiv2020

Cytoplasmic aggregation of uranium in human dopaminergic cells after continuous exposure to soluble uranyl at non-cytotoxic concentrations

Uranium exposure can lead to neurobehavioral alterations in particular of the monoaminergic system, even at non-cytotoxic concentrations. However, the mechanisms of uranium neurotoxicity after non-cytotoxic exposure are still poorly understood. In particular, imaging uranium in neurons at low intracellular concentration is still very challenging. We investigated uranium intracellular localization by means of synchrotron X-ray fluorescence imaging with high spatial resolution (< 300 nm) and high analytical sensitivity (< 1 g.g-1 per 300 nm pixel). Neuron-like SH-SY5Y human cells differentiated into a dopaminergic phenotype were continuously exposed, for seven days, to a non-cytotoxic concentration (10 M) of soluble natural uranyl. Cytoplasmic submicron uranium aggregates were observed accounting on average for 62% of the intracellular uranium content. In some aggregates, uranium and iron were co-localized suggesting common metabolic pathways between uranium and iron storage. Uranium aggregates contained no calcium or phosphorous indicating that detoxification mechanisms in neuron-like cells are different from those described in bone or kidney cells. Uranium intracellular distribution was compared to fluorescently labeled organelles (lysosomes, early and late endosomes) and to fetuin-A, a high affinity uranium-binding protein. A strict correlation could not be evidenced between uranium and the labelled organelles, or with vesicles containing fetuin-A. Our results indicate a new mechanism of uranium cytoplasmic aggregation after non-cytotoxic uranyl exposure that could be involved in neuronal defense through uranium sequestration into less reactive species. The remaining soluble fraction of uranium would be responsible for protein binding and the resulting neurotoxic effects.

pharmacology and toxicology↗