Antibody-displaying extracellular vesicles for targeted cancer therapy.

Extracellular vesicles (EVs) function as natural delivery vectors and mediators of biological signals across tissues. Here, by leveraging these functionalities, we show that EVs decorated with an antibody-binding moiety specific for the fragment crystallizable (Fc) domain can be used as a modular delivery system for targeted cancer therapy. The Fc-EVs can be decorated with different types of immunoglobulin G antibody and thus be targeted to virtually any tissue of interest. Following optimization of the engineered EVs by screening Fc-binding and EV-sorting moieties, we show the targeting of EVs to cancer cells displaying the human epidermal receptor 2 or the programmed-death ligand 1, as well as lower tumour burden and extended survival of mice with subcutaneous melanoma tumours when systemically injected with EVs displaying an antibody for the programmed-death ligand 1 and loaded with the chemotherapeutic doxorubicin. EVs with Fc-binding domains may be adapted to display other Fc-fused proteins, bispecific antibodies and antibody-drug conjugates.

Identification of scaffold proteins for improved endogenous engineering of extracellular vesicles.

Extracellular vesicles (EVs) are gaining ground as next-generation drug delivery modalities. Genetic fusion of the protein of interest to a scaffold protein with high EV-sorting ability represents a robust cargo loading strategy. To address the paucity of such scaffold proteins, we leverage a simple and reliable assay that can distinguish intravesicular cargo proteins from surface- as well as non-vesicular proteins and compare the EV-sorting potential of 244 candidate proteins. We identify 24 proteins with conserved EV-sorting abilities across five types of producer cells. TSPAN2 and TSPAN3 emerge as lead candidates and outperform the well-studied CD63 scaffold. Importantly, these engineered EVs show promise as delivery vehicles in cell cultures and mice as demonstrated by efficient transfer of luminal cargo proteins as well as surface display of different functional entities. The discovery of these scaffolds provides a platform for EV-based engineering.

Surface display of functional moieties on extracellular vesicles using lipid anchors.

Extracellular vesicles (EVs) are efficient natural vehicles for intercellular communication and are under extensive investigation for the delivery of diverse therapeutics including small molecule drugs, nucleic acids, and proteins. To understand the mechanisms behind the biological activities of EVs and develop EV therapeutics, it's fundamental to track EVs and engineer EVs in a customized manner. In this study, we identified, using single-vesicle flow cytometry and microscopy, the lipid DOPE (dioleoyl phosphatidyl ethanolamine) as an efficient anchor for isolated EVs. Notably, DOPE associated with EVs quickly, and the products remained stable under several challenging conditions. Moreover, conjugating fluorophores, receptor-targeting peptides or albumin-binding molecules with DOPE enabled tracking the cellular uptake, enhanceing the cellular uptake or extending the circulation time in mice of engineered EVs , respectively. Taken together, this study reports an efficient lipid anchor for exogenous engineering of EVs and further showcases its versatility for the functionalization of EVs.

Therapeutic potential of extracellular vesicles in neurodegenerative disorders.

Neurodegenerative disorders are characterized by complex multifactorial pathogeneses, thus posing a challenge for standard therapeutic approaches that tend to focus only on one underlying disease aspect. For systemically administered drugs, the blood-brain barrier (BBB) is yet another major obstacle to overcome. In this context, naturally occurring extracellular vesicles (EVs) with intrinsic ability to cross the BBB have been investigated as therapeutics for various diseases, including Alzheimer's and Parkinson's diseases. EVs are cell-derived, lipid membrane-enclosed vesicles carrying a broad spectrum of biologically active molecules, which play a crucial role in intercellular communication. In a therapeutic context, mesenchymal stem cell (MSC)-derived EVs are in the spotlight because they reflect the therapeutic properties of their parental cells and, thus, hold promise as independent cell-free therapeutics. On the other hand, EVs can be used as drug delivery vehicles by modifying their surface or content, e.g., by decorating the surface with brain-specific ligands or loading the EVs with therapeutic RNAs or proteins, thus further enhancing the EV's targeting and therapeutic potency, respectively. Although EVs have been deemed safe for use in humans, some obstacles remain that prevent their progression into clinics. This review scrutinizes the promises and challenges of EV-based treatments for neurodegenerative disorders.

Cell-specific targeting of extracellular vesicles through engineering the glycocalyx.

Extracellular vesicles (EVs) are promising carriers for the delivery of a variety of chemical and biological drugs. However, their efficacy is limited by the lack of cellular specificity. Available methods to improve the tissue specificity of EVs predominantly rely on surface display of proteins and peptides, largely overlooking the dense glycocalyx that constitutes the outermost layer of EVs. In the present study, we report a reconfigurable glycoengineering strategy that can endogenously display glycans of interest on EV surface. Briefly, EV producer cells are genetically engineered to co-express a glycosylation domain (GD) inserted into the large extracellular loop of CD63 (a well-studied EV scaffold protein) and fucosyltransferase VII (FUT7) or IX (FUT9), so that the engineered EVs display the glycan of interest. Through this strategy, we showcase surface display of two types of glycan ligands, sialyl Lewis X (sLeX) and Lewis X, on EVs and achieve high specificity towards activated endothelial cells and dendritic cells, respectively. Moreover, the endothelial cell-targeting properties of sLeX-EVs were combined with the intrinsic therapeutic effects of mesenchymal stem cells (MSCs), leading to enhanced attenuation of endothelial damage. In summary, this study presents a reconfigurable glycoengineering strategy to produce EVs with strong cellular specificity and highlights the glycocalyx as an exploitable trait for engineering EVs.

Efficient Peptide-Mediated In Vitro Delivery of Cas9 RNP.

The toolbox for genetic engineering has quickly evolved from CRISPR/Cas9 to a myriad of different gene editors, each with promising properties and enormous clinical potential. However, a major challenge remains: delivering the CRISPR machinery to the nucleus of recipient cells in a nontoxic and efficient manner. In this article, we repurpose an RNA-delivering cell-penetrating peptide, PepFect14 (PF14), to deliver Cas9 ribonucleoprotein (RNP). The RNP-CPP complex achieved high editing rates, e.g., up to 80% in HEK293T cells, while being active at low nanomolar ranges without any apparent signs of toxicity. The editing efficiency was similar to or better compared to the commercially available reagents RNAiMAX and CRISPRMax. The efficiency was thoroughly evaluated in reporter cells and wild-type cells by restriction enzyme digest and next-generation sequencing. Furthermore, the CPP-Cas9-RNP complexes were demonstrated to withstand storage at different conditions, including freeze-thaw cycles and freeze-drying, without a loss in editing efficiency. This CPP-based delivery strategy complements existing technologies and further opens up new opportunities for Cas9 RNP delivery, which can likely be extended to other gene editors in the future.