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Abstract Background Mesenchymal stromal cell (MSC) therapy holds great potential yet efficacy and safety concerns with cell therapy persist. The beneficial effects of MSCs are often attributed to their secretome that includes extracellular vesicles (EVs). EVs carry biologically active molecules, protected by a lipid bilayer. However, several barriers hinder large-scale MSC EV production. A serum-free culturing approach is preferred for producing clinical-grade MSC-derived EVs but this can affect both yield and purity. Consequently, new strategies have been explored, including genetically engineering MSCs to alter EV compositions to enhance potency, increase circulation time or mediate targeting. However, efficient transfection of MSCs is challenging. Typical sources of MSC include adipose tissue and bone marrow, which both require invasive extraction procedures. Here, we investigate the use of urine-derived stem cells (USCs) as a non-invasive and inexhaustible source of MSCs for EV production. Methods We isolated, expanded, and characterized urine-derived stem cells (USCs) harvested from eight healthy donors at three different time points during the day. We evaluated the number of clones per urination, proliferation capacity and conducted flow cytometry to establish expression of surface markers. EVs were produced in chemically defined media and characterized. PEI/DNA transfection was used to genetically engineer USCs using transposon technology. Results There were no differences between time points for clone number, doubling time or viability. USCs showed immunophenotypic characteristics of MSCs, such as expression of CD73, CD90 and CD105, with no difference at the assessed time points, however, male donors had reduced CD73 + cells. Expanded USCs were incubated without growth factors or serum for 72 h without a loss in viability and EVs were isolated. USCs were transfected with high efficiency and after 10 days of selection, pure engineered cell cultures were established. Conclusions Isolation and expansion of MSCs from urine is non-invasive, robust, and without apparent sex-related differences. The sampling time point did not affect any measured markers or USC isolation potential. USCs offer an attractive production platform for EVs, both native and engineered.

Extracellular vesicles (EVs) are promising therapeutic delivery vehicles, although their potential is limited by a lack of efficient engineering strategies to enhance loading and functional cargo delivery. Using an in-house bioinformatics analysis, we identified N-glycosylation as a putative EV-sorting feature. PTTG1IP (a small, N-glycosylated, single-spanning transmembrane protein) was found to be a suitable scaffold for EV loading of therapeutic cargoes, with loading dependent on its N-glycosylation at two arginine residues. Chimeric proteins consisting of PTTG1IP fused with various cargo proteins, and separated by self-cleaving sequences (to promote cargo release), were shown to enable highly efficient functional delivery of Cre protein to recipient cell cultures and mouse xenograft tumors, and delivery of Cas9-sgRNA complexes to recipient reporter cells. The favorable membrane topology of PTTG1IP enabled facile engineering of further variants with improved properties, highlighting its versatility and potential as a platform for EV-based therapeutics.

Abstract Messenger RNA (mRNA) has emerged as an attractive therapeutic molecule for a plethora of clinical applications. For in vivo functionality, mRNA therapeutics require encapsulation into effective, stable, and safe delivery systems to protect the cargo from degradation and reduce immunogenicity. Here, a bioengineering platform for efficient mRNA loading and functional delivery using bionormal nanoparticles, extracellular vesicles (EVs), is established by expressing a highly specific RNA‐binding domain fused to CD63 in EV producer cells stably expressing the target mRNA. The additional combination with a fusogenic endosomal escape moiety, Vesicular Stomatitis Virus Glycoprotein, enables functional mRNA delivery in vivo at doses substantially lower than currently used clinically with synthetic lipid‐based nanoparticles. Importantly, the application of EVs loaded with effective cancer immunotherapy proves highly effective in an aggressive melanoma mouse model. This technology addresses substantial drawbacks currently associated with EV‐based nucleic acid delivery systems and is a leap forward to clinical EV applications.

Abstract Extracellular vesicles (EVs) are lipid nanoparticles and play an important role in cell‐cell communications, making them potential therapeutic agents and allowing to engineer for targeted drug delivery. The expanding applications of EVs in next generation medicine is still limited by existing tools for scaling standardized EV production, single EV tracing and analytics, and thus provide only a snapshot of tissue‐specific EV cargo information. Here, we present the Snorkel‐tag, for which we have genetically fused the EV surface marker protein CD81, to a series of tags with an additional transmembrane domain to be displayed on the EV surface, resembling a snorkel. This system enables the affinity purification of EVs from complex matrices in a non‐destructive form while maintaining EV characteristics in terms of surface protein profiles, associated miRNA patterns and uptake into a model cell line. Therefore, we consider the Snorkel‐tag to be a widely applicable tool in EV research, allowing for efficient preparation of EV standards and reference materials, or dissecting EVs with different surface markers when fusing to other tetraspanins in vitro or in vivo.

Abstract 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.

Two new endosomolytic small compounds increase delivery of splice-switching oligonucleotides by interfering with their trafficking to the LAMP1-positive endosomal compartments, inducing endosomal membrane rupture and concurrent oligonucleotide escape into the cytosol.

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a crucial role in maintaining normal homeostatic processes under the pathogenesis of various inflammatory and autoimmune diseases. This context-dependent effect from a cytokine is due to two distinctive forms of signaling: cis-signaling and trans-signaling. IL-6 cis-signaling involves binding IL-6 to the membrane-bound IL-6 receptor and Glycoprotein 130 (GP130) signal-transducing subunit. By contrast, in IL-6 trans-signaling, complexes of IL-6 and the soluble form of the IL-6 receptor (sIL-6R) signal via membrane-bound GP130. Various strategies have been employed in the past decade to target the pro-inflammatory effect of IL-6 in numerous inflammatory disorders. However, their development has been hindered since these approaches generally target global IL-6 signaling, also affecting the anti-inflammatory effects of IL-6 signaling too. Therefore, novel strategies explicitly targeting the pro-inflammatory IL-6 trans-signaling without affecting the IL-6 cis-signaling are required and carry immense therapeutic potential. Here, we have developed a novel approach to specifically decoy IL-6-mediated trans-signaling by modulating alternative splicing in GP130, an IL-6 signal transducer, by employing splice switching oligonucleotides (SSO), to induce the expression of truncated soluble isoforms of the protein GP130. This isoform is devoid of signaling domains but allows for specifically sequestering the IL-6/sIL-6R receptor complex with high affinity in serum and thereby suppressing inflammation. Using the state-of-the-art Pip6a cell-penetrating peptide conjugated to PMO-based SSO targeting GP130 for efficient in vivo delivery, reduced disease phenotypes in two different inflammatory mouse models of systemic and intestinal inflammation were observed. Overall, this novel gene therapy platform holds great potential as a refined therapeutic intervention for chronic inflammatory diseases.

Abstract Extracellular vesicles (EVs) have shown promise as potential therapeutics for the treatment of various diseases. However, their rapid clearance after administration could be a limitation in certain therapeutic settings. To solve this, an engineering strategy is employed to decorate albumin onto the surface of the EVs through surface display of albumin binding domains (ABDs). ABDs were either included in the extracellular loops of select EV‐enriched tetraspanins (CD63, CD9 and CD81) or directly fused to the extracellular terminal of single transmembrane EV‐sorting domains, such as Lamp2B. These engineered EVs exert robust binding capacity to human serum albumins (HSA) in vitro and mouse serum albumins (MSA) after injection in mice. By binding to MSA, circulating time of EVs dramatically increases after different routes of injection in different strains of mice. Moreover, these engineered EVs show considerable lymph node (LN) and solid tumour accumulation, which can be utilized when using EVs for immunomodulation, cancer‐ and/or immunotherapy. The increased circulation time of EVs may also be important when combined with tissue‐specific targeting ligands and could provide significant benefit for their therapeutic use in a variety of disease indications.

We have determined whether orange juice-derived nanovesicles (ONVs) could be used for the treatment of obesity-associated intestinal complications. ONVs were characterized by lipidomic, metabolomic, electron microscopy. In vitro, intestinal barriers (IBs = Caco-2+HT-29-MTX) were treated with ONVs and co-cultured with adipocytes to monitor IB fat release. In vivo, obesity was induced with a high-fat, high-sucrose diet (HFHSD mice) for 12 weeks. Then, half of HFHSD mice were gavaged with ONVs. One-month ONV treatment did not modify HFHSD-induced insulin resistance but reversed diet-induced gut modifications. In the jejunum, ONVs increased villi size, reduced triglyceride content, and modulated mRNA levels of genes involved in immune response (tumor necrosis factor [TNF]-α and interleukin [IL]-1β), barrier permeability (CLDN1, OCLN, ZO1), fat absorption, and chylomicron release. ONVs targeted microsomal triglyceride transfer protein (MTP) and angiopoietin-like protein-4 (ANGPTL4), two therapeutic targets to reduce plasma lipids and inflammation in gastrointestinal diseases. Interestingly, ONV treatment did not aggravate liver steatosis, as MTP mRNA was increased in the liver. Therefore, ONVs protected both intestine and the liver from fat overload associated with the HFHSD. As ONVs concentrated amino acids and bioactive lipids versus orange juice, which are deficient in obese patients, the use of ONVs as a dietary supplement could bring physiological relevant compounds in the jejunum to accelerate the restoration of intestinal functions during weight loss in obese patients.

Extracellular vesicles (EV) facilitate intercellular transfer of biological material including RNA, but the regulatory mechanisms for their formation and transfer are incompletely known. Here the authors develop a CRISPR-based reporting system to detect the transfer of guide RNAs and identify genes not previously linked to EV-mediated RNA delivery.

Extracellular vesicles (EVs), comprising exosomes, microvesicles, and apoptotic bodies, are released by all cells into the extracellular matrix and body fluids, where they play important roles in intercellular communication and matrix remodeling in various pathological conditions. Malignant pleural mesothelioma (MPM) is a primary tumor of mesothelial origin, predominantly related to asbestos exposure. The detection of MPM at an early stage and distinguishing it from benign conditions and metastatic adenocarcinomas (AD) is sometimes challenging. Pleural effusion is often the first available biological material and an ideal source for characterizing diagnostic and prognostic factors. Specific proteins have previously been identified as diagnostic markers in effusion, but it is not currently known whether these are associated with vesicles or released in soluble form. Here, we study and characterize tumor heterogeneity and extracellular vesicle diversity in pleural effusion as diagnostic or prognostic markers for MPM. We analyzed extracellular vesicles and soluble proteins from 27 pleural effusions, which were collected and processed at the department of pathology and cytology at Karolinska University Hospital, representing three different patient groups, MPM (n = 9), benign (n = 6), and AD (n = 12). The vesicles were fractionated into apoptotic bodies, microvesicles, and exosomes by differential centrifugation and characterized by nanoparticle tracking analysis and Western blotting. Multiplex bead-based flow cytometry analysis showed that exosomal markers were expressed differently on EVs present in different fractions. Further characterization of exosomes by a multiplex immunoassay (Luminex) showed that all soluble proteins studied were also present in exosomes, though the ratio of protein concentration present in supernatant versus exosomes varied. The proportion of Angiopoietin-1 present in exosomes was generally higher in benign compared to malignant samples. The corresponding ratios of Mesothelin, Galectin-1, Osteopontin, and VEGF were higher in MPM effusions compared to those in the benign group. These findings demonstrate that relevant diagnostic markers can be recovered from exosomes.

Extracellular vesicles (EVs) are naturally occurring nano-sized carriers that are secreted by cells and facilitate cell-to-cell communication by their unique ability to transfer biologically active cargo. Despite the pronounced increase in our understanding of EVs over the last decade, from disease pathophysiology to therapeutic drug delivery, improved molecular tools to track their therapeutic delivery are still needed. Unfortunately, the present catalogue of tools utilised for EV labelling lacks sensitivity or are not sufficiently specific. Here, we have explored the bioluminescent labelling of EVs using different luciferase enzymes tethered to CD63 to achieve a highly sensitive system for in vitro and in vivo tracking of EVs. Using tetraspanin fusions to either NanoLuc or ThermoLuc permits performing highly sensitive in vivo quantification of EVs or real-time imaging, respectively, at low cost and in a semi-high throughput manner. We find that the in vivo distribution pattern of EVs is determined by the route of injection, but that different EV subpopulations display differences in biodistribution patterns. By applying this technology for real-time non-invasive in vivo imaging of EVs, we show that their distribution to different internal organs occurs just minutes after administration.

Splice-switching therapy with splice-switching oligonucleotides (SSOs) has recently proven to be a clinically applicable strategy for the treatment of several mis-splice disorders. Despite this, wider application of SSOs is severely limited by the inherently poor bioavailability of SSO-based therapeutic compounds. Cell-penetrating peptides (CPPs) are a class of drug delivery systems (DDSs) that have recently gained considerable attention for improving the uptake of various oligonucleotide (ON)-based compounds, including SSOs. One strategy that has been successfully applied to develop effective CPP vectors is the introduction of various lipid modifications into the peptide. Here, we repurpose hydrocarbon-modified amino acids used in peptide stapling for the orthogonal introduction of hydrophobic modifications into the CPP structure during peptide synthesis. Our data show that α,α-disubstituted alkenyl-alanines can be successfully utilized to introduce hydrophobic modifications into CPPs to improve their ability to formulate SSOs into nanoparticles (NPs), and to mediate high delivery efficacy and tolerability both in vitro and in vivo. Conclusively, our results offer a new flexible approach for the sequence-specific introduction of hydrophobicity into the structure of CPPs and for improving their delivery properties.

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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell’s activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.