Biology subjects

Mastrobattista, E.

Publications and source records attributed to Mastrobattista, E..

4 recordsLinked to original sources

bioRxiv2025

Detailed splenic single-cell biodistribution of phosphatidylglycerol-containing liposomes

Antigen-specific tolerance induction is a promising therapeutic strategy for autoimmune and chronic inflammatory diseases. This can be achieved by targeted activation of regulatory T and B cells via antigen-presenting cells (APCs) in a tolerogenic context. Anionic antigen-carrying liposomes have shown potential, however, their efficacy is highly dependent on the administration route and liposomal rigidity. Here, we investigate the biodistribution and splenic APC subset-specific uptake of rigid DSPC:DSPG:CHOL liposomes compared to flexible DOPC:DOPG:CHOL liposomes using high-parameter flow cytometry. We developed a panel enabling identification of rare splenic APC subsets involved in immune tolerance, including CD169+ and MARCO+ marginal zone macrophages, red pulp macrophages, and conventional/plasmacytoid dendritic cells. Our findings confirm that rigid liposomes predominantly accumulate in the liver and spleen following IV injection, with negligible uptake in lymph nodes or lungs. Importantly, systemic distribution is significantly inhibited by subcutaneous administration, which is essential for tolerance induction. Among splenic APCs, macrophage subsets are major contributors to liposome uptake, though the liver remains the primary site of accumulation and may play a more dominant role in tolerance induction. This study underscores the importance of both liposomal design and delivery route in optimizing nanoparticle-based immune modulation strategies.

immunology↗

bioRxiv2025

Generation of a Three-dimensional Human Neurovascular Unit Model in a Microfluidic Chip

BackgroundThe well-functioning of the neurovascular unit (NVU) is supported by the 3D brain physiological microenvironment that allows for extensive neural-neural and neural-vascular interactions. This microenvironment is normally hard to create in traditional in-vitro models such as the transwell model. Organ-on-a-chip (OOC) emerges as advanced model systems by providing better physiological microenvironments. However, NVU modeling in many chip platforms has not met a full 3D condition for neural cultures. MethodsHere, we describe a novel NVU model generated in a microfluidic chip that reproduces the neural-neural and neural-vascular interactions in a full-3D format. The model features an extracellular matrix (ECM) environment that supports both a perfused brain endothelial vessel and 3D cultured neural cells (astrocytes and neurons) beside the tube. Culture conditions were comprehensively optimized for better endothelial tube integrity as well as ECM gel longevity. The model was used to model neuroinflammation-induced brain tube disruption and immune cell extravasation. Furthermore, as a drug testing platform, the model was explored for brain endothelial transcytosis of the heparin-binding EGF-like growth factor (HB-EGF) targeted nanobodies and the data was compared to a parallel transwell model. ResultsImmunofluorescent staining confirmed the expression of endothelial junctional proteins, as well as astrocytic and neuronal markers. The perfused brain endothelial tube exhibited resistance to paracellular leakage of 20 kDa FITC-dextran. Astrocytes and neurons growing in ECM gel developed extensive neural network and showed spontaneous neuronal firing. The neural-vascular interactions were formed through astrocyte migration and axonal outgrowth in the ECM gel towards the tube. Exposure to neuroinflammatory cytokines disrupted the tube barrier, resulting in increased barrier leakage and the recruitment of peripheral blood mononuclear cells (PBMCs) as well as their extravasation. Owing to full-3D model design, endothelial transcytosis and abluminal distribution of the fluorescently labeled HB-EGF targeting Nbs can be clearly visualized in situ. Compared to a transwell model counterpart, the NVU chip model performed better in revealing the binding and transcytosis specificity of the targeted nanobodies. ConclusionsWe demonstrate improved physiological relevance in this full-3D NVU-on-a-chip model. The model could become a faithful platform for NVU research under both healthy and diseased conditions, and can be used as a reliable drug testing platform that aims at developing novel brain-targeted therapeutics.

bioengineering↗

bioRxiv2024

A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release

CRISPR-Cas9 gene editing technology offers the potential to permanently repair genes containing pathological mutations. However, efficient intracellular delivery of the Cas9 ribonucleoprotein complex remains one of the major hurdles in its therapeutic application. Extracellular vesicles (EVs) are biological nanosized membrane vesicles released by cells, that play an important role in intercellular communication. Due to their innate capability of intercellular transfer of proteins, RNA, and various other biological cargos, EVs have emerged as a novel promising strategy for the delivery of macromolecular biotherapeutics, including CRISPR-Cas9 ribonucleoproteins. Here, we present a versatile, modular strategy for the loading and delivery of Cas9. We leverage the high affinity binding of MS2 coat proteins (MCPs) fused to EV-enriched proteins to MS2 aptamers incorporated into single guide RNAs (sgRNAs), in combination with a UV-activated photocleavable linker domain, PhoCl. Combined with the Vesicular stomatitis virus G (VSV-G) protein this modular platform enables efficient loading and subsequent delivery of the Cas9 ribonucleoprotein complex, which shows critical dependence on the incorporation and activation of the photocleavable linker domain. As this approach does not require any direct fusion of Cas9 to EV-enriched proteins, we demonstrate that Cas9 can readily be exchanged for other variants, including transcriptional activator dCas9-VPR and adenine base editor ABE8e, as confirmed by various sensitive fluorescent reporter assays. Taken together, we describe a robust and modular strategy for successful Cas9 delivery, which can be applied for CRISPR-Cas9-based genetic engineering as well as transcriptional regulation, underlining the potential of EV-mediated strategies for the treatment of genetic diseases.

molecular biology↗

bioRxiv2023

Anti-cancer compound screening identifies Aurora Kinase A inhibition as a means to favor CRISPR/Cas9 gene correction over knock-out

CRISPR gene therapy holds the potential to cure a variety of genetic diseases by targeting causative mutations and introducing double stranded DNA breaks, subsequently allowing the host DNA repair mechanisms to introduce mutations. One option to introduce precise gene corrections is via the homology-directed repair (HDR) pathway. HDR can introduce a range of desired mutations dictated by a DNA template which holds a corrected DNA sequence which is written into the targeted gene. The problem in utilizing this pathway is that CRISPR-induced double stranded DNA breaks are repaired more often through the non-homologous end joining (NHEJ) pathway, which does not use a designed template and introduces random DNA damage in the form of insertions and deletions at the cut site. Since HDR activation depends on many interconnected processes in the cell, we aimed to screen a small library of drug compounds in clinical use or clinical development for cancer, to steer the DNA repair process towards preferential HDR activation. We included compounds in our screen based on three relevant mechanisms in CRISPR gene editing: the cell cycle, DNA repair processing and chromosomal packing. We included forty compounds, based on these criteria, screened their toxicity and dosed them in sub-toxic concentrations in cells during genome editing. Of these forty compounds we identified nine potential hits to have an effect on preferential activation of the HDR pathway over NHEJ. Alisertib, rucaparib and belinostat revealed a significant and major effect on gene editing pathway selection in further validation. Alisertib, an Aurora kinase A inhibitor, showed a particularly strong effect towards improving HDR over NHEJ. We subsequently investigated this effect at the genetic level and in a murine hepatoma cell line, which corroborated the initial findings. Alisertib led to an over 4-fold increase in preferential gene correction over gene knock-out, at a dose of 0.3 micromolar. However, the observations that Aurora kinase A inhibitors show considerable cytotoxicity (<50% cell viability) and can induce morphological changes at this concentration pose a limitation for the direct use of these inhibitors as HDR enhancers. However these findings do implicate that the pathways mediated by Aurora kinase A strongly influence HDR outcomes, which warrants further investigation into the downstream pathways driving this effect.

pharmacology and toxicology↗