| 1. |
|
|
| 2. |
- Aoki, Y, et al.
(author)
-
Corrigendum
- 2018
-
In: Brain : a journal of neurology. - : Oxford University Press (OUP). - 1460-2156. ; 141:5, s. e42-
-
Journal article (peer-reviewed)
|
|
| 3. |
- Aoki, Y, et al.
(author)
-
Fine Tuning of Phosphorothioate Inclusion in 2'-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation
- 2021
-
In: Frontiers in physiology. - : Frontiers Media SA. - 1664-042X. ; 12, s. 689179-
-
Journal article (peer-reviewed)abstract
- 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.
|
|
| 4. |
|
|
| 5. |
|
|
| 6. |
|
|
| 7. |
|
|
| 8. |
- Corso, G, et al.
(author)
-
Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size exclusion chromatography
- 2017
-
In: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1, s. 11561-
-
Journal article (peer-reviewed)abstract
- Extracellular vesicles (EVs) play a pivotal role in cell-to-cell communication and have been shown to take part in several physiological and pathological processes. EVs have traditionally been purified by ultracentrifugation (UC), however UC has limitations, including resulting in, operator-dependant yields, EV aggregation and altered EV morphology, and moreover is time consuming. Here we show that commercially available bind-elute size exclusion chromatography (BE-SEC) columns purify EVs with high yield (recovery ~ 80%) in a time-efficient manner compared to current methodologies. This technique is reproducible and scalable, and surface marker analysis by bead-based flow cytometry revealed highly similar expression signatures compared with UC-purified samples. Furthermore, uptake of eGFP labelled EVs in recipient cells was comparable between BE-SEC and UC samples. Hence, the BE-SEC based EV purification method represents an important methodological advance likely to facilitate robust and reproducible studies of EV biology and therapeutic application.
|
|
| 9. |
|
|
| 10. |
- Dar, GH, et al.
(author)
-
GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain
- 2021
-
In: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 12:1, s. 6666-
-
Journal article (peer-reviewed)abstract
- Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. Here we demonstrate a role for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EV assembly and secretion. We observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif (G58), which promotes extensive EV clustering. Further studies in a Drosophila EV biogenesis model reveal that GAPDH is required for the normal generation of intraluminal vesicles in endosomal compartments, and promotes vesicle clustering. Fusion of the GAPDH-derived G58 peptide to dsRNA-binding motifs enables highly efficient loading of small interfering RNA (siRNA) onto the EV surface. Such vesicles efficiently deliver siRNA to multiple anatomical regions of the brain in a Huntington’s disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in different regions of the brain.
|
|
| 11. |
- de Jong, OG, et al.
(author)
-
A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA
- 2020
-
In: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1, s. 1113-
-
Journal article (peer-reviewed)abstract
- Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small non-coding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.
|
|
| 12. |
|
|
| 13. |
|
|
| 14. |
|
|
| 15. |
|
|
| 16. |
|
|
| 17. |
|
|
| 18. |
|
|
| 19. |
|
|
| 20. |
|
|
| 21. |
|
|
| 22. |
|
|
| 23. |
|
|
| 24. |
- Jungebluth, P, et al.
(author)
-
Retraction Statement
- 2023
-
In: Respiration. - : S. Karger AG. - 1423-0356 .- 0025-7931. ; 102:5, s. 402-404
-
Journal article (other academic/artistic)
|
|
| 25. |
|
|
| 26. |
- Lehto, T, et al.
(author)
-
Peptides for nucleic acid delivery
- 2016
-
In: Advanced drug delivery reviews. - : Elsevier BV. - 1872-8294 .- 0169-409X. ; 106:Pt A, s. 172-182
-
Journal article (peer-reviewed)
|
|
| 27. |
|
|
| 28. |
|
|
| 29. |
|
|
| 30. |
|
|
| 31. |
|
|
| 32. |
|
|
| 33. |
|
|
| 34. |
|
|
| 35. |
|
|
| 36. |
|
|
| 37. |
- Willms, E, et al.
(author)
-
Cells release subpopulations of exosomes with distinct molecular and biological properties
- 2016
-
In: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6, s. 22519-
-
Journal article (peer-reviewed)abstract
- Cells release nano-sized membrane vesicles that are involved in intercellular communication by transferring biological information between cells. It is generally accepted that cells release at least three types of extracellular vesicles (EVs): apoptotic bodies, microvesicles and exosomes. While a wide range of putative biological functions have been attributed to exosomes, they are assumed to represent a homogenous population of EVs. We hypothesized the existence of subpopulations of exosomes with defined molecular compositions and biological properties. Density gradient centrifugation of isolated exosomes revealed the presence of two distinct subpopulations, differing in biophysical properties and their proteomic and RNA repertoires. Interestingly, the subpopulations mediated differential effects on the gene expression programmes in recipient cells. In conclusion, we demonstrate that cells release distinct exosome subpopulations with unique compositions that elicit differential effects on recipient cells. Further dissection of exosome heterogeneity will advance our understanding of exosomal biology in health and disease and accelerate the development of exosome-based diagnostics and therapeutics.
|
|
