| 1. |
- Coenen-Stass, Anna M. L., et al.
(author)
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Comprehensive RNA-Sequencing Analysis in Serum and Muscle Reveals Novel Small RNA Signatures with Biomarker Potential for DMD
- 2018
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In: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 13, s. 1-15
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Journal article (peer-reviewed)abstract
- Extracellular small RNAs (sRNAs), including microRNAs (miRNAs), are promising biomarkers for diseases such as Duchenne muscular dystrophy (DMD), although their biological relevance is largely unknown. To investigate the relationship between intracellular and extracellular sRNA levels on a global scale, we performed sRNA sequencing in four muscle types and serum from wild-type, dystrophic mdx, and mdx mice in which dystrophin protein expression was restored by exon skipping. Differentially abundant sRNAs were identified in serum (mapping to miRNA, small nuclear RNA [snRNA], and PIWI-interacting RNA [piRNA] loci). One novel candidate biomarker, miR-483, was increased in both mdx serum and muscle, and also elevated in DMD patient sera. Dystrophin restoration induced global shifts in miRNA (including miR-483) and snRNA-fragment abundance toward wild-type levels. Specific serum piRNA-like sRNAs also responded to exon skipping therapy. Absolute miRNA expression in muscle was positively correlated with abundance in the circulation, although multiple highly expressed miRNAs in muscle were not elevated in mdx serum, suggesting that both passive and selective release mechanisms contribute to serum miRNA levels. In conclusion, this study has revealed new insights into the sRNA biology of dystrophin deficiency and identified novel DMD biomarkers.
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| 2. |
- EL Andaloussi, Samir, et al.
(author)
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Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo
- 2011
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:9, s. 3972-3987
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Journal article (peer-reviewed)abstract
- 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.
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| 3. |
- Gupta, Dhanu, et al.
(author)
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Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles
- 2021
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In: Nature Biomedical Engineering. - Stockholm : Karolinska Institutet, Dept of Laboratory Medicine. - 2157-846X.
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Journal article (peer-reviewed)abstract
- 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.
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| 4. |
- Lehto, Taavi, et al.
(author)
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A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo
- 2011
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In: Molecular Therapy. - : Elsevier BV. - 1525-0016 .- 1525-0024. ; 19:8, s. 1457-1467
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Journal article (peer-reviewed)abstract
- 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.
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| 5. |
- Sork, Helena, et al.
(author)
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Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome
- 2018
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
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Journal article (peer-reviewed)abstract
- 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.
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| 6. |
- Sork, Helena, et al.
(author)
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Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency
- 2016
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In: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 5
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Journal article (peer-reviewed)abstract
- The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.
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| 7. |
- Sork, Helena
(author)
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Profiling and exploiting lipid-based nanoparticles in vitro and in vivo
- 2018
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Doctoral thesis (other academic/artistic)abstract
- One of the major hurdles for therapeutic applications is the efficient delivery of bioactive molecules to the site of action. The high flexibility and biosafety of lipid-based nanoparticles has greatly enhanced their employment as delivery systems not only for synthetic but also for natural molecules such as proteins and nucleic acids. This thesis was brought about to investigate the nucleic acid delivery potential of synthetic lipid-based nanoparticles as well as to look into the composition and delivery patterns of their natural counterparts, extracellular vesicles (EVs), in order to set ground for future lipid-based therapeutic interventions. Firstly, in Paper I we explored the potency of a lipid-based delivery agent, Lipofectamine 2000 which after being frozen and thawed showed orders of magnitude higher nucleic acid delivery efficiency in vitro and in vivo than the non-frozen counterpart. This effect was consistent across different cryo-manipulations, cell lines and also various types of nucleic acid. Further analysis with different methodologies revealed that the underlying potency plausibly relies on the elevated sedimentation and spreading of the complexes and/or relates to the specific structure or composition of the carrier. These findings illustrate that a simple freeze-thawing procedure allows to drastically reduce the amount of transfection reagent for cellular nucleic acid delivery, whilst not losing the desired activity. Secondly, we shifted our focus to natural lipid-based carriers, EVs in order to shed light on the vesicular and non-vesicular (non-EV) small RNA patterns and their relation to the EV proteome (Paper II and III). Though the studies exploited different EV enrichment methods the relative depletion of vesicular small RNAs was confirmed in both instances. A detailed analysis of the secretory repertoire of small RNAs showed a significant depletion of microRNA (miRNA) sequences, matching well with the depletion of “miRNA related” proteins in EVs. The relative expression level of cellular, EV and non-EV miRNAs correlated well and though some differentially expressed (DE) miRNAs were detected, these had a relatively low expression in both the source cells as well as in the secretory fractions. We also quantified the total level of selected miRNAs in EVs and non-EV fraction investigating both the basal as well as overexpressed levels and could verify that the vast majority of mature miRNA is secreted to the non-EV portion of the secretome. Paper IV was brought about to gain a comprehensive overview of the biodistribution of exogenous EVs. This study confirmed that fluorescent lipophilic dyes are suitable for membrane labelling and in vivo tracking of EVs. The general biodistribution pattern of EVs was seen to follow a common mononuclear phagocytic system (MPS) uptake pattern with the majority of EVs accumulating in the liver, spleen and lungs. Nevertheless, depending on the cell source, administration route, dose and the presence of targeting moieties this distribution could be altered. The present findings are important to gain a thorough understanding of the nucleic acid delivery capacity of lipid-based nanoparticles, especially EVs and thereby progress their employment as therapeutic nucleic acid carriers.
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| 8. |
- Sork, Helena, et al.
(author)
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Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion
- 2021
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In: Cells. - : MDPI AG. - 2073-4409. ; 10:6
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Journal article (peer-reviewed)abstract
- 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 (R-2 = 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 degrees 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.
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