An ALS-linked mutation in TDP-43 disrupts normal protein interactions in the motor neuron response to oxidative stress.

TDP-43 pathology is a key feature of amyotrophic lateral sclerosis (ALS), but the mechanisms linking TDP-43 to altered cellular function and neurodegeneration remain unclear. We have recently described a mouse model in which human wild-type or mutant TDP-43 are expressed at low levels and where altered stress granule formation is a robust phenotype of TDP-43M337V/- expressing cells. In the present study we use this model to investigate the functional connectivity of human TDP-43 in primary motor neurons under resting conditions and in response to oxidative stress. The interactome of human TDP-43WT or TDP-43M337V was compared by mass spectrometry, and gene ontology enrichment analysis identified pathways dysregulated by the M337V mutation. We found that under normal conditions the interactome of human TDP-43WT was enriched for proteins involved in transcription, translation and poly(A)-RNA binding. In response to oxidative stress, TDP-43WT recruits proteins of the endoplasmic reticulum and endosomal-extracellular transport pathways, interactions which are reduced in the presence of the M337V mutation. Specifically, TDP-43M337V impaired protein-protein interactions involved in stress granule formation including reduced binding to the translation initiation factors Poly(A)-binding protein and Eif4a1 and the endoplasmic reticulum chaperone Grp78. The M337V mutation also affected interactions involved in endosomal-extracellular transport and this this was associated with reduced extracellular vesicle secretion in primary motor neurons from TDP-43M337V/- mice and in human iPSCs-derived motor neurons. Taken together, our analysis highlights a TDP-43 interaction network in motor neurons and demonstrates that an ALS associated mutation may alter the interactome to drive aberrant pathways involved in the pathogenesis of ALS.

CSF extracellular vesicle proteomics demonstrates altered protein homeostasis in amyotrophic lateral sclerosis.

BACKGROUND: Extracellular vesicles (EVs) released by neurons and glia reach the cerebrospinal fluid (CSF). Studying the proteome of CSF-derived EVs offers a novel perspective on the key intracellular processes associated with the pathogenesis of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and a potential source from which to develop biomarkers. METHODS: CSF EVs were extracted using ultrafiltration liquid chromatography from ALS patients and controls. EV size distribution and concentration was measured using nanoparticle tracking analysis and liquid chromatography-tandem mass spectrometry proteomic analysis performed. RESULTS: CSF EV concentration and size distribution did not differ between ALS and control groups, nor between a sub-group of ALS patients with or without an associated hexanucleotide repeat expansion (HRE) in C9orf72. Univariate proteomic analysis identified downregulation of the pentameric proteasome-like protein Bleomycin hydrolase in ALS patients, whilst Gene Ontology enrichment analysis demonstrated downregulation of proteasome core complex proteins (8/8 proteins, normalized enrichment ratio -1.77, FDR-adjusted p = 0.057) in the ALS group. The sub-group of ALS patients associated with the C9orf72 HRE showed upregulation in Ubiquitin-like modifying-activating protein 1 (UBA1) compared to non-C9orf72 cases. CONCLUSIONS: Proteomic analysis of CSF EVs in ALS detects intracellular alterations in protein homeostatic mechanisms, previously only identified in pathological tissues. This supports the wider use of CSF EVs as a source of novel biomarkers reflecting key and potentially druggable pathological intracellular pathway alterations in ALS.

Methodological Guidelines to Study Extracellular Vesicles.

Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.

Extracellular microRNAs exhibit sequence-dependent stability and cellular release kinetics.

Multiple studies have described extracellular microRNAs (ex-miRNAs) as being remarkably stable despite the hostile extracellular environment, when stored at 4ºC or lower. Here we show that many ex-miRNAs are rapidly degraded when incubated at 37ºC in the presence of serum (thereby simulating physiologically relevant conditions). Stability varied widely between miRNAs, with half-lives ranging from ~1.5 hours to more than 13 hours. Notably, ex-miRNA half-lives calculated in two different biofluids (murine serum and C2C12 mouse myotube conditioned medium) were highly similar, suggesting that intrinsic sequence properties are a determining factor in miRNA stability. By contrast, ex-miRNAs associated with extracellular vesicles (isolated by size exclusion chromatography) were highly stable. The release of ex-miRNAs from C2C12 myotubes was measured over time, and mathematical modelling revealed miRNA-specific release kinetics. While some ex-miRNAs reached the steady state in cell culture medium within 24 hours, the extracellular level of miR-16 did not reach equilibrium, even after 3 days in culture. These findings are indicative of miRNA-specific release and degradation kinetics with implications for the utility of ex-miRNAs as biomarkers, and for the potential of ex-miRNAs to transfer gene regulatory information between cells.

UFLC-Derived CSF Extracellular Vesicle Origin and Proteome.

Cerebrospinal fluid (CSF) extracellular vesicles (EVs) show promise as a source of neurological disease biomarkers, although their precise origin is poorly understood. Current extraction techniques produce disappointing yield and purity. This study describes the application of ultrafiltration LC (UFLC) to CSF-EVs, compared with ultracentrifugation (UC), and explores CSF-EV origin. EVs are extracted from human CSF by UC and UFLC and characterized using nanoparticle tracking analysis, electron microscopy, and immunoblotting. EV and CSF proteomes are analyzed by LC-MS/MS. UFLC-isolated particles have size, morphology, and marker expression characteristic of EVs. UFLC provides greater EV yield (UFLC 7.90 × 108  ± SD 1.31 × 108 EVs mL-1 CSF, UC 1.06 × 108  ± 0.57 × 108 p < 0.001). UFLC enhances purity, proteomic depth (UFLC 622 ± 49, UC 298 ± 50, p = 0.001), and consistency of quantification (CV 17% vs 23%). EVs contain more intracellular proteins (Odds ratio [OR] 2.63 p < 0.001) and fewer plasma proteins than CSF (OR 0.60, p < 0.001). CSF and EV-enriched proteomes show overrepresentation of brain-specific proteins (EV OR 3.18, p < 0.001; CSF OR 3.37, p < 0.001). Overrepresentation of cerebral white matter (OR 1.99, p = 0.015) and choroid plexus proteins (OR 1.87, p<0.001) is observed in EVs. UFLC improves yield and purity of CSF-EVs. The EV-enriched proteome better reflects the intracellular and white matter proteome than whole CSF.

Selective release of muscle-specific, extracellular microRNAs during myogenic differentiation.

MyomiRs are muscle-specific microRNAs (miRNAs) that regulate myoblast proliferation and differentiation. Extracellular myomiRs (ex-myomiRs) are highly enriched in the serum of Duchenne Muscular Dystrophy (DMD) patients and dystrophic mouse models and consequently have potential as disease biomarkers. The biological significance of miRNAs present in the extracellular space is not currently well understood. Here we demonstrate that ex-myomiR levels are elevated in perinatal muscle development, during the regenerative phase that follows exercise-induced myoinjury, and concomitant with myoblast differentiation in culture. Whereas ex-myomiRs are progressively and specifically released by differentiating human primary myoblasts and C2C12 cultures, chemical induction of apoptosis in C2C12 cells results in indiscriminate miRNA release. The selective release of myomiRs as a consequence of cellular differentiation argues against the idea that they are solely waste products of muscle breakdown, and suggests they may serve a biological function in specific physiological contexts. Ex-myomiRs in culture supernatant and serum are predominantly non-vesicular, and their release is independent of ceramide-mediated vesicle secretion. Furthermore, ex-myomiRs levels are reduced in aged dystrophic mice, likely as a consequence of chronic muscle wasting. In conclusion, we show that myomiR release accompanies periods of myogenic differentiation in cell culture and in vivo. Serum myomiR abundance is therefore a function of the regenerative/degenerative status of the muscle, overall muscle mass, and tissue expression levels. These findings have implications for the use of ex-myomiRs as biomarkers for DMD disease progression and monitoring response to therapy.

C9orf72 and RAB7L1 regulate vesicle trafficking in amyotrophic lateral sclerosis and frontotemporal dementia.

A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.

Functional Delivery of Lipid-Conjugated siRNA by Extracellular Vesicles.

Extracellular vesicles (EVs) are cell-derived, membranous nanoparticles that mediate intercellular communication by transferring biomolecules, including proteins and RNA, between cells. As a result of their suggested natural capability to functionally deliver RNA, EVs may be harnessed as therapeutic RNA carriers. One major limitation for their translation to therapeutic use is the lack of an efficient, robust, and scalable method to load EVs with RNA molecules of interest. Here, we evaluated and optimized methods to load EVs with cholesterol-conjugated small interfering RNAs (cc-siRNAs) by systematic evaluation of the influence of key parameters, including incubation time, volume, temperature, and EV:cc-siRNA ratio. EV loading under conditions that resulted in the highest siRNA retention percentage, incubating 15 molecules of cc-siRNA per EV at 37°C for 1 hr in 100 µL, facilitated concentration-dependent silencing of human antigen R (HuR), a therapeutic target in cancer, in EV-treated cells. These results may accelerate the development of EV-based therapeutics.

Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties.

Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs. FROM THE CLINICAL EDITOR: Recent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery.

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.

The role of endocytosis on the uptake kinetics of luciferin-conjugated cell-penetrating peptides.

Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides that can be used to deliver a variety of cargos into cells. However, it is still debated which routes CPPs employ to gain access to intracellular compartments. To assess this, most previously conducted studies have relied on information which is gained by using fluorescently labeled CPPs. More relevant information whether the internalized conjugates are biologically available has been gathered using end-point assays with biological readouts. Uptake kinetic studies have shed even more light on the matter because the arbitrary choice of end-point might have profound effect how the results could be interpreted. To elucidate uptake mechanisms of CPPs, here we have used a bioluminescence based assay to measure cytosolic delivery kinetics of luciferin-CPP conjugates in the presence of endocytosis inhibitors. The results suggest that these conjugates are delivered into cytosol mainly via macropinocytosis; clathrin-mediated endocytosis and caveolae/lipid raft dependent endocytosis are involved in a smaller extent. Furthermore, we demonstrate how the involved endocytic routes and internalization kinetic profiles can depend on conjugate concentration in case of certain peptides, but not in case of others. The employed internalization route, however, likely dictates the intracellular fate and subsequent trafficking of internalized ligands, therefore emphasizing the importance of our novel findings for delivery vector development.

PepFect14 peptide vector for efficient gene delivery in cell cultures.

The successful applicability of gene therapy approaches will heavily rely on the development of efficient and safe nonviral gene delivery vectors, for example, cell-penetrating peptides (CPPs). CPPs can condense oligonucleotides and plasmid DNA (pDNA) into nanoparticles, thus allowing the transfection of genetic material into cells. However, despite few promising attempts, CPP-mediated pDNA delivery has been relatively inefficient due to the unfavorable nanoparticle characteristics or the nanoparticle entrapment to endocytic compartments. In many cases, both of these drawbacks could be alleviated by modifying CPPs with a stearic acid residue, as demonstrated in the delivery of both the pDNA and the short oligonucleotides. In this study, PepFect14 (PF14) peptide, previously used for the transport of shorter oligonucleotides, is demonstrated to be suited also for the delivery of pDNA. It is shown that PF14 forms stable nanoparticles with pDNA with a negative surface charge and size of around 130-170 nm. These nanoparticles facilitate efficient gene delivery and expression in a variety of regular adherent cell lines and also in difficult-to-transfect primary cells. Uptake studies indicate that PF14/pDNA nanoparticles are utilizing class A scavenger receptors (SCARA) and caveolae-mediated endocytosis as the main route for cellular internalization. Conclusively, PF14 is an efficient nonviral vector for gene delivery.

Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo.

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.

EV-mediated promotion of myogenic differentiation is dependent on dose, collection medium, and isolation method.

Extracellular vesicles (EVs) have been implicated in the regulation of myogenic differentiation. C2C12 murine myoblast differentiation was reduced following treatment with GW4869 or heparin (to inhibit exosome biogenesis and EV uptake, respectively). Conversely, treatment with C2C12 myotube-conditioned medium enhanced myogenic differentiation. Ultrafiltration-size exclusion liquid chromatography (UF-SEC) was used to isolate EVs and non-EV extracellular protein in parallel from C2C12 myoblast- and myotube-conditioned medium. UF-SEC-purified EVs promoted myogenic differentiation at low doses (≤2 × 108 particles/mL) and were inhibitory at the highest dose tested (2 × 1011 particles/mL). Conversely, extracellular protein fractions had no effect on myogenic differentiation. While the transfer of muscle-enriched miRNAs (myomiRs) has been proposed to mediate the pro-myogenic effects of EVs, we observed that they are scarce in EVs (e.g., 1 copy of miR-133a-3p per 195 EVs). Furthermore, we observed pro-myogenic effects with undifferentiated myoblast-derived EVs, in which myomiR concentrations are even lower, suggestive of a myomiR-independent mechanism underlying the observed pro-myogenic effects. During these investigations we identified technical factors with profound confounding effects on myogenic differentiation. Specifically, co-purification of insulin (a component of Opti-MEM) in non-EV LC fractions and polymer precipitated EV preparations. These findings provide further evidence that polymer-based precipitation techniques should be avoided in EV research.

A peptide-based vector for efficient gene transfer in vitro and in vivo.

Finding suitable nonviral delivery vehicles for nucleic acid-based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice.

Choroid plexus-derived extracellular vesicles exhibit brain targeting characteristics.

The brain is protected against invading organisms and other unwanted substances by tightly regulated barriers. However, these central nervous system (CNS) barriers impede the delivery of drugs into the brain via the blood circulation and are therefore considered major hurdles in the treatment of neurological disorders. Consequently, there is a high need for efficient delivery systems that are able to cross these strict barriers. While most research focuses on the blood-brain barrier (BBB), the design of drug delivery platforms that are able to cross the blood-cerebrospinal fluid (CSF) barrier, formed by a single layer of choroid plexus epithelial cells, remains a largely unexplored domain. The discovery that extracellular vesicles (EVs) make up a natural mechanism for information transfer between cells and across cell layers, has stimulated interest in their potential use as drug delivery platform. Here, we report that choroid plexus epithelial cell-derived EVs exhibit the capacity to home to the brain after peripheral administration. Moreover, these vesicles are able to functionally deliver cargo into the brain. Our findings underline the therapeutic potential of choroid plexus-derived EVs as a brain drug delivery vehicle via targeting of the blood-CSF interface.

Assessing the uptake kinetics and internalization mechanisms of cell-penetrating peptides using a quenched fluorescence assay.

Cell-penetrating peptides (CPPs) have shown great potency for cargo delivery both in vitro and in vivo. Different biologically relevant molecules need to be delivered into appropriate cellular compartments in order to be active, for instance certain drugs/molecules, e.g. antisense oligonucleotides, peptides, and cytotoxic agents require delivery into the cytoplasm. Assessing uptake mechanisms of CPPs can help to develop novel and more potent cellular delivery vectors, especially in cases when reaching a specific intracellular target requires involvement of a specific internalization pathway. Here we measure the overall uptake kinetics, with emphasis on cytoplasmic delivery, of three cell-penetrating peptides M918, TP10 and pVec using a quenched fluorescence assay. We show that both the uptake levels and kinetic constants depend on the endocytosis inhibitors used in the experiments. In addition, in some cases only the internalization rate is affected by the endocytosis inhibitors while the total uptake level is not and vice versa, which emphasizes importance of kinetic studies when assessing the uptake mechanisms of CPPs. Also, there seems to be a correlation between lower total cellular uptake and higher first-order rate constants. Furthermore, this may indicate simultaneous involvement of different endocytic pathways with different efficacies in the internalization process, as hypothesized but not shown earlier in an uptake kinetics assay.

Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion.

The extracellular environment consists of a plethora of molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, interest in therapeutic short RNA carriers lies mainly in EVs, the vehicles conveying the great majority of the biological activity. Here, by overexpressing miRNA and shRNA sequences in parent cells and using size exclusion liquid chromatography (SEC) to separate the secretome into EV and non-EV fractions, we saw that >98% of overexpressed miRNA was secreted within the non-EV fraction. Furthermore, small RNA sequencing studies of native miRNA transcripts revealed that although the abundance of miRNAs in EVs, non-EVs and parent cells correlated well (R2 = 0.69-0.87), quantitatively an outstanding 96.2-99.9% of total miRNA was secreted in the non-EV fraction. Nevertheless, though EVs contained only a fraction of secreted miRNAs, these molecules were stable at 37 °C in a serum-containing environment, indicating that if sufficient miRNA loading is achieved, EVs can remain delivery-competent for a prolonged period of time. This study suggests that the passive endogenous EV loading strategy might be a relatively wasteful way of loading miRNA to EVs, and active miRNA loading approaches are needed for developing advanced EV miRNA therapies in the future.

Endothelial-Derived Extracellular Vesicles Induce Cerebrovascular Dysfunction in Inflammation.

Blood-brain barrier (BBB) dysfunction is a key hallmark in the pathology of many neuroinflammatory disorders. Extracellular vesicles (EVs) are lipid membrane-enclosed carriers of molecular cargo that are involved in cell-to-cell communication. Circulating endothelial EVs are increased in the plasma of patients with neurological disorders, and immune cell-derived EVs are known to modulate cerebrovascular functions. However, little is known about whether brain endothelial cell (BEC)-derived EVs themselves contribute to BBB dysfunction. Human cerebral microvascular cells (hCMEC/D3) were treated with TNFα and IFNy, and the EVs were isolated and characterised. The effect of EVs on BBB transendothelial resistance (TEER) and leukocyte adhesion in hCMEC/D3 cells was measured by electric substrate cell-substrate impedance sensing and the flow-based T-cell adhesion assay. EV-induced molecular changes in recipient hCMEC/D3 cells were analysed by RT-qPCR and Western blotting. A stimulation of naïve hCMEC/D3 cells with small EVs (sEVs) reduced the TEER and increased the shear-resistant T-cell adhesion. The levels of microRNA-155, VCAM1 and ICAM1 were increased in sEV-treated hCMEC/D3 cells. Blocking the expression of VCAM1, but not of ICAM1, prevented sEV-mediated T-cell adhesion to brain endothelia. These results suggest that sEVs derived from inflamed BECs promote cerebrovascular dysfunction. These findings may provide new insights into the mechanisms involving neuroinflammatory disorders.

Fine Tuning of Phosphorothioate Inclusion in 2'-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation.

Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2'-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood. At present, we demonstrate that the modification of PS facilitates the uptake of 2OMe in H2k-mdx myoblasts. Furthermore, we found a dependency of SSO nuclear accumulation and high splice-switching activity on PS inclusion in 2OMe (2OMePS), as tested in various reporter cell lines carrying pLuc/705. Increased exon-inclusion activity was observed in muscle, neuronal, liver, and bone cell lineages via both the gymnotic uptake and lipofection of 2OMePS. Using the photoactivatable ribonucleoside-enhanced crosslinking and a subsequent proteomic approach, we identified several 2OMePS-binding proteins, which are likely to play a role in the trafficking of 2OMePS to the nucleus. Ablation of one of them, Ncl by small-interfering RNA (siRNA) enhanced 2OMePS uptake in C2C12 myoblasts and upregulated luciferase RNA splicing in the HeLa Luc/705 reporter cell line. Overall, we demonstrate that PS inclusion increases nuclear delivery and splice switching in muscle, neuronal, liver, and bone cell lineages and that the modulation of 2OMePS-binding partners may improve SSO delivery.