Biology subjects

Ambrosetti, E.

Publications and source records attributed to Ambrosetti, E..

3 recordsLinked to original sources

bioRxiv2026

Dual Ligand Cooperation at the Plasma Membrane Drives Transport of Engineered Small Extracellular Vesicles Across Brain Endothelial Cells

The natural delivery properties of small extracellular vesicles (sEVs) can be harnessed and enhanced through engineering to create a new class of biotherapeutics, particularly for central nervous system (CNS) disorders. While evidence supports the ability of sEVs to cross biological barriers and deliver functional cargo to target cells, a limited understanding of their uptake and transport across the brain hinders their translational potential. In this study, we investigated either native and engineered sEVs, developed by us, using a novel modular engineering platform that employs a dual-targeting strategy to facilitate uptake and transport through human brain endothelial cells (BECs). By utilizing super-resolution microscopy, we provided direct insights into the mechanisms of docking, intracellular sorting, and transport of engineered sEVs. The engineered sEVs formulation demonstrated significantly enhanced uptake, intracellular trafficking across BECs, and the ability to bypass degradative pathways. In vivo, the engineered sEVs exhibited preferential accumulation in the brain choroid plexus, a structure located within the lateral and fourth ventricles, thereby effectively targeting the blood-cerebrospinal fluid (CSF) barrier. These findings highlight the potential of combining advanced targeting strategies with high-resolution imaging to study sEV interactions with the brain biological barriers and develop more effective CNS therapies.

bioengineering↗

bioRxiv2024

Engineering multivalent Fc display for FcγR blockade

Autoimmune diseases, driven by Fc{gamma} receptor (Fc{gamma}R) activation through autoantibody immune complexes (IC), present a complex therapeutic challenge of achieving pharmacological blockade of Fc{gamma}R without triggering receptor activation. The assembly of ICs into polydisperse, higher-order structures is required for Fc{gamma}R activation. However, engineered multimeric, monodisperse Fc assemblies have been reported to prevent Fc{gamma}R activation, suggesting that Fc spatial organization determines Fc{gamma}R activation. In this study, we engineered a functional single-chain Fc domain protein (scFc) for unidirectional, multivalent presentation by virus-like particles (VLPs), used as a display platform. We found that the multivalent display of scFc on the VLPs elicited distinct cellular responses compared with monovalent scFc, highlighting the importance of the structural context of scFc on its function. scFc-VLPs had minimal impact on the nanoscale spatial organization of Fc{gamma}R at the cell membrane and caused limited receptor activation and internalization. In contrast, the monovalent scFc acted as an Fc{gamma}R agonist, inducing receptor clustering, activation, and internalization. Increasing scFc valency in scFc-VLPs was associated with increased binding to monocytes, reaching a plateau at high valencies. Notably, the ability of scFc-VLPs to block IC-mediated phagocytosis in vitro increased with scFc valency. In a murine model of passive immune thrombocytopenia (ITP), a high valency scFc-VLP variant with a desirable immunogenicity profile induced attenuation of thrombocytopenia. Here we show that multivalent presentation of an engineered scFc on a display platform can be tailored to promote suppression of IC-mediated phagocytosis while preventing Fc{gamma}R activation. This work introduces a new paradigm that can contribute to the development of therapies for autoimmune diseases.

bioengineering↗

bioRxiv2019

A DNA nanoassembly-based approach to map membrane protein nanoenvironments

Super-resolution imaging has revealed that most proteins at the plasma membrane are not uniformly distributed but localize to dynamic domains of nanoscale dimensions. To investigate their functional relevance, there is a need for methods that enable comprehensive mapping of the compositions and spatial organizations of membrane protein nanodomains in cell populations. However, current superresolution methods are limited to analysing small, preselected subsets of proteins, at very low sampling fractions. Here we describe the development of a non-microscopy based super-resolution method for unbiased ensemble analysis of membrane protein nanodomains. The method, termed NANOscale DEciphEring of membrane Protein nanodomains (NanoDeep), is based on the use of DNA nanoassemblies to translate membrane protein organization information into a DNA sequencing readout. Using NanoDeep, we characterized the nanoenvironments of Her2, a membrane receptor of critical relevance in cancer. We found that the occupancies of Her2, Her3 and EGFR in the nanoenvironments surrounding Her2 were similar in two cell lines with vastly different expression levels of Her2. Further, we found that adding Heregulin-{beta}1 to cancer cells led to increased occupancy of Her2 and Her3, and to a lesser extent EGFR, in Her2 nanoenvironments. NanoDeep has the potential to provide new insights into the roles of the composition and spatial organization of protein nanoenvironments in the regulation of membrane protein function.

bioengineering↗