32 research outputs found
Purification and biodistribution of extracellular vesicles
Extracellular vesicles (EVs) are nano-sized vesicles that contain bioactive lipids, RNAs and
proteins, which can be transferred to recipient cells. EVs are important for physiological as
well as pathological processes, such as coagulation and immune homeostasis, aiding cancer
metastasis and spread of infectious diseases. Owing to their relatively small size the
purification of EVs is a challenge, hence we have established and optimised workflows
consisting of ultrafiltration with subsequent size exclusion liquid chromatography (UFLC)(
Paper I) and bind-elute combined with size exclusion (BE-SEC) columns (Paper III) for
EV purification.
UF-LC allowed for purification of biophysically intact EVs with better yield and purity
compared to ultracentrifugation (UC), which is the gold standard purification method in the
field. The biodistribution of UF-LC EVs was different compared to vesicles isolated using
UC, despite having highly similar protein composition according to proteomics analysis. We
found that UF-LC vesicles accumulated less in lung, possibly owing to their higher integrity.
Indeed, fluorescence correlation spectroscopy and transmission electron microscopy
indicated that the high gravitational forces in UC lead to aggregation and disruption of the
vesicles. The BE-SEC method is a similar method to UF-LC, however protein impurities less
than 700 kDa in size are bound in the interior of the beads, thus improving simple size-based
exclusion. The BE-SEC method is scalable, produces samples with better purity than UC,
displaying yields exceeding 70% and demonstrates a good reproducibility between samples.
Moreover, vesicles purified by BE-SEC display the same EV surface markers as UC purified
EVs, and CD63-eGFP positive vesicles are taken up in recipient cells to the same extent. In
summary, the BE-SEC method is a reproducible and fast alternative to UF-LC for large
media volumes.
Reliable purification methods are important for the implementation of therapeutically active
EVs, however knowledge regarding their eventual organotropism and biodistribution is
equally important. Thus, in article II we evaluated the biodistribution of EVs specifically
labeled with a near-infrared dye. The main sites of accumulation of exogenously injected EVs
were liver, spleen and lungs. Biodistribution profile of EVs depended strongly on injection
route, and to certain extent, on EV cell type source, as dendritic cell derived EVs exhibited a
more pronounced uptake in spleen compared to the other cell sources tested. We further
showed that small alterations of EV surface proteins could significantly affect biodistribution
as well, since EVs equipped with a brain targeting peptide on their surface increased the
uptake of targeted EVs in brain. This study highlights that the biodistribution of EVs follows
other nano-sized particles with uptake mainly in liver. Administration route, cell source and a
targeting peptide influence the distribution, however the overall distribution is unaltered with
the highest signal originating from liver.
To summarise, this thesis has resulted in improvements of the EV field by systematically
enhancing EV isolation workflows to achieve greater sample purity and at the same time
preserving EV biophysical characteristics. Furthermore, it has laid groundwork for studying
in vivo effects of exogenous vesicles. Both these aspects are particularly important for
understanding EV biology more clearly and with increased detail
Supramolecular Assembly of AminoethyleneâLipopeptide PMO Conjugates into RNA SpliceâSwitching Nanomicelles
Phosphorodiamidate morpholino oligomers (PMOs) are oligonucleotide analogs that can be used for therapeutic modulation of preâmRNA splicing. Similar to other classes of nucleic acidâbased therapeutics, PMOs require delivery systems for efficient transport to the intracellular target sites. Here, artificial peptides based on the oligo(ethylenamino) acid succinylâtetraethylenpentamine (Stp), hydrophobic modifications, and an azide group are presented, which are used for strainâpromoted azideâalkyne cycloaddition conjugation with spliceâswitching PMOs. By systematically varying the lead structure and formulation, it is determined that the type of contained fatty acid and supramolecular assembly have a critical impact on the delivery efficacy. A compound containing linolenic acid with three cis double bonds exhibits the highest spliceâswitching activity and significantly increases functional protein expression in pLuc/705 reporter cells in vitro and after local administration in vivo. Structural and mechanistic studies reveal that the lipopeptide PMO conjugates form nanoparticles, which accelerate cellular uptake and that the content of unsaturated fatty acids enhances endosomal escape. In an in vitro Duchenne muscular dystrophy exon skipping model using H2Kâmdx52 dystrophic skeletal myotubes, the highly potent PMO conjugates mediate significant spliceâswitching at very low nanomolar concentrations. The presented aminoethyleneâlipopeptides are thus a promising platform for the generation of PMOâtherapeutics with a favorable activity/toxicity profile
Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome
Extracellular vesicles (EVs) mediate cell-to-cell communication by delivering or displaying macromolecules to their recipient cells. While certain broad-spectrum EV effects reflect their protein cargo composition, others have been attributed to individual EV-loaded molecules such as specific miRNAs. In this work, we have investigated the contents of vesicular cargo using small RNA sequencing of cells and EVs from HEK293T, RD4, C2C12, Neuro2a and C17.2. The majority of RNA content in EVs (49-96%) corresponded to rRNA-, coding-and tRNA fragments, corroborating with our proteomic analysis of HEK293T and C2C12 EVs which showed an enrichment of ribosome and translation-related proteins. On the other hand, the overall proportion of vesicular small RNA was relatively low and variable (2-39%) and mostly comprised of miRNAs and sequences mapping to piRNA loci. Importantly, this is one of the few studies, which systematically links vesicular RNA and protein cargo of vesicles. Our data is particularly useful for future work in unravelling the biological mechanisms underlying vesicular RNA and protein sorting and serves as an important guide in developing EVs as carriers for RNA therapeutics.Peer reviewe
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
Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles
Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.International Society for Advancement of Cytometry Marylou Ingram Scholar 2019-2023H2020 EXPERTSwedish foundation of Strategic Research (SSF-IRC; FormulaEx)ERC CoG (DELIVER)Swedish Medical Research CouncilAccepte
Schizophrenia-associated somatic copy-number variants from 12,834 cases reveal recurrent NRXN1 and ABCB11 disruptions
While germline copy-number variants (CNVs) contribute to schizophrenia (SCZ) risk, the contribution of somatic CNVs (sCNVs)âpresent in some but not all cellsâremains unknown. We identified sCNVs using blood-derived genotype arrays from 12,834 SCZ cases and 11,648 controls, filtering sCNVs at loci recurrently mutated in clonal blood disorders. Likely early-developmental sCNVs were more common in cases (0.91%) than controls (0.51%, p = 2.68eâ4), with recurrent somatic deletions of exons 1â5 of the NRXN1 gene in five SCZ cases. Hi-C maps revealed ectopic, allele-specific loops forming between a potential cryptic promoter and non-coding cis-regulatory elements upon 5âČ deletions in NRXN1. We also observed recurrent intragenic deletions of ABCB11, encoding a transporter implicated in anti-psychotic response, in five treatment-resistant SCZ cases and showed that ABCB11 is specifically enriched in neurons forming mesocortical and mesolimbic dopaminergic projections. Our results indicate potential roles of sCNVs in SCZ risk
Genomic Dissection of Bipolar Disorder and Schizophrenia, Including 28 Subphenotypes
publisher: Elsevier articletitle: Genomic Dissection of Bipolar Disorder and Schizophrenia, Including 28 Subphenotypes journaltitle: Cell articlelink: https://doi.org/10.1016/j.cell.2018.05.046 content_type: article copyright: © 2018 Elsevier Inc
Preparation and isolation of siRNA-loaded extracellular vesicles
RNA interference (RNAi) has tremendous potential for specific silencing of disease-causing genes. Its clinical usage however critically depends on the development of carrier systems that can transport the RNAimediating small interfering RNA (siRNA) molecules to the cytosol of target cells. Recent reports have suggested that extracellular vesicles (EVs) form a natural transport system through which biomolecules, including RNA, is exchanged between cells. Therefore, EVs are increasingly being considered as potential therapeutic siRNA delivery systems. In this chapter we describe a method for preparing siRNA-loaded EVs, including a robust, scalable method to isolate them from cell culture supernatants
Preparation and isolation of siRNA-loaded extracellular vesicles
RNA interference (RNAi) has tremendous potential for specific silencing of disease-causing genes. Its clinical usage however critically depends on the development of carrier systems that can transport the RNAimediating small interfering RNA (siRNA) molecules to the cytosol of target cells. Recent reports have suggested that extracellular vesicles (EVs) form a natural transport system through which biomolecules, including RNA, is exchanged between cells. Therefore, EVs are increasingly being considered as potential therapeutic siRNA delivery systems. In this chapter we describe a method for preparing siRNA-loaded EVs, including a robust, scalable method to isolate them from cell culture supernatants
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