| 1. |
- Amin, Risul, et al.
(författare)
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The kidney injury caused by the onset of acute graft-versus-host disease is associated with down-regulation of alpha Klotho
- 2020
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Ingår i: International Immunopharmacology. - : Elsevier BV. - 1567-5769 .- 1878-1705. ; 78
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Tidskriftsartikel (refereegranskat)abstract
- Acute graft-versus-host disease (aGVHD) and kidney injury are the major complications after allogeneic hematopoietic stem cell transplantation (HSCT). Although the underlying mechanisms for the development of these complications are not yet fully understood, it has been proposed that emergence of aGVHD contributes to the development of kidney injury after HSCT. We have shown previously that aGVHD targets the kidney in a biphasic manner: at the onset, inflammatory genes are up-regulated, while when aGVHD becomes established, donor lymphocytes infiltrate the kidney. Here, we characterize renal manifestations at the onset of aGVHD. Mice receiving allogeneic bone marrow and spleen cells displayed symptoms of aGVHD and elevated serum levels of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) within 4 days. There was concurrent kidney injury with the following characteristics: (1) elevated expression of the kidney injury biomarker, neutrophil gelatinase-associated lipocalin (NGAL), (2) accumulation of hetero-lysosomes in proximal tubule epithelial cells, and (3) reductions in alpha Klotho mRNA and protein and increased serum levels of fibroblast growth factor 23 (Fgf23), phosphate and urea. This situation resembled acute renal injury caused by bacterial lipopolysaccharide. We conclude that the onset of aGVHD is associated with kidney injury involving down-regulation of alpha Klotho, a sight that may inspire novel therapeutic approaches.
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| 2. |
- Bestas, Burcu, et al.
(författare)
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Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model
- 2014
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Ingår i: Journal of Clinical Investigation. - 0021-9738 .- 1558-8238. ; 124:9, s. 4067-4081
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Tidskriftsartikel (refereegranskat)abstract
- X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTKtranscripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.
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| 3. |
- Bost, Jeremy P., et al.
(författare)
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Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides
- 2022
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 5:1
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Tidskriftsartikel (refereegranskat)abstract
- The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.
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| 4. |
- Cavallaro, Sara, et al.
(författare)
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Label-Free Surface Protein Profiling of Extracellular Vesicles by an Electrokinetic Sensor
- 2019
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Ingår i: ACS Sensors. - : AMER CHEMICAL SOC. - 2379-3694. ; 4:5, s. 1399-1408
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Tidskriftsartikel (refereegranskat)abstract
- Small extracellular vesicles (sEVs) generated from the endolysosomal system, often referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics, as they carry valuable biological information and reflect their cells of origin. Herein, we propose a simple and inexpensive electrical method for label-free detection and profiling of sEVs in the size range of exosomes. The detection method is based on the electrokinetic principle, where the change in the streaming current is monitored as the surface markers of the sEVs interact with the affinity reagents immobilized on the inner surface of a silica microcapillary. As a proof-of-concept, we detected sEVs derived from the non-small-cell lung cancer (NSCLC) cell line H1975 for a set of representative surface markers, such as epidermal growth factor receptor (EGFR), CD9, and CD63. The detection sensitivity was estimated to be similar to 175000 sEVs, which represents a sensor surface coverage of only 0.04%. We further validated the ability of the sensor to measure the expression level of a membrane protein by using sEVs displaying artificially altered expressions of EGFR and CD63, which were derived from NSCLC and human embryonic kidney (HEK) 293T cells, respectively. The analysis revealed that the changes in EGFR and CD63 expressions in sEVs can be detected with a sensitivity in the order of 10% and 3%, respectively, of their parental cell expressions. The method can be easily parallelized and combined with existing microfluidic-based EV isolation technologies, allowing for rapid detection and monitoring of sEVs for cancer diagnosis.
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| 5. |
- Cavallaro, Sara, et al.
(författare)
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Multiparametric Profiling of Single Nanoscale Extracellular Vesicles by Combined Atomic Force and Fluorescence Microscopy : Correlation and Heterogeneity in Their Molecular and Biophysical Features
- 2021
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Ingår i: Small. - : Wiley. - 1613-6810 .- 1613-6829. ; 17:14
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Tidskriftsartikel (refereegranskat)abstract
- Being a key player in intercellular communications, nanoscale extracellular vesicles (EVs) offer unique opportunities for both diagnostics and therapeutics. However, their cellular origin and functional identity remain elusive due to the high heterogeneity in their molecular and physical features. Here, for the first time, multiple EV parameters involving membrane protein composition, size and mechanical properties on single small EVs (sEVs) are simultaneously studied by combined fluorescence and atomic force microscopy. Furthermore, their correlation and heterogeneity in different cellular sources are investigated. The study, performed on sEVs derived from human embryonic kidney 293, cord blood mesenchymal stromal and human acute monocytic leukemia cell lines, identifies both common and cell line-specific sEV subpopulations bearing distinct distributions of the common tetraspanins (CD9, CD63, and CD81) and biophysical properties. Although the tetraspanin abundances of individual sEVs are independent of their sizes, the expression levels of CD9 and CD63 are strongly correlated. A sEV population co-expressing all the three tetraspanins in relatively high abundance, however, having average diameters of <100 nm and relatively low Young moduli, is also found in all cell lines. Such a multiparametric approach is expected to provide new insights regarding EV biology and functions, potentially deciphering unsolved questions in this field.
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| 6. |
- Coenen-Stass, Anna M. L., et al.
(författare)
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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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Ingår i: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 13, s. 1-15
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Tidskriftsartikel (refereegranskat)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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| 7. |
- Eiríksdóttir, Emelía, et al.
(författare)
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Cellular Internalization Kinetics of (Luciferin-)Cell-Penetrating Peptide Conjugates
- 2010
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 21:9, s. 1662-1672
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) belong to a class of delivery vectors that have been extensively used for the cellular delivery of various, otherwise impermeable, macromolecules. However, results on the cellular internalization efficacy of CPPs obtained from various laboratories are sometimes challenging to compare because of differences in the experimental setups. Here, for the first time, the cellular uptake kinetics of eight well-established CPPs is compared in HeLa pLuc 705 cells using a recently published releasable luciferin assay. Using this assay, the kinetic behavior of cytosolic entry of these luciferin-CPP conjugates are registered in real time. Our data reveal that the uptake rate of CPPs reaches its maximum either in seconds or in tens of minutes, depending on the CPP used. Tat and higher concentrations of MAP and TP10 display fast internalization profiles that resemble the kinetic profile of membrane-permeable free luciferin. The uptake of the other peptides, pVec, penetratin, M918, and EB I, is much slower and is consistent with the reported observations of endocytosis being the predominant internalization mechanism. Additionally, to some extent, the latter CPPs can be clustered into subgroups which are based on time points when the most pronounced uptake rates are observed. This may indicate once more involvement of various (concentration dependent) mechanisms in the uptake of CPPs. In summary, the variances in the internalization profiles for the CPPs demonstrate the importance of measuring kinetics instead of only relying on simple end-point studies, and with the luciferin CPP assay, more lucid information can be retrieved when studying the internalization mechanisms of CPPs.
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| 8. |
- EL Andaloussi, Samir, et al.
(författare)
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A novel cell-penetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids
- 2007
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Ingår i: Molecular Therapy. - : Elsevier BV. - 1525-0016 .- 1525-0024. ; 15:10, s. 1820-1826
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) have attracted increasing attention in the past decade as a result of their high potential to convey various, otherwise impermeable, bioactive agents across cellular plasma membranes. Albeit different CPPs have proven potent in delivery of different cargoes, there is generally a correlation between high efficacy and cytotoxicity for these peptides. Hence, it is of great importance to find new, non-toxic CPPs with more widespread delivery properties. We present a novel CPP, M918, that efficiently translocates various cells in a non-toxic fashion. In line with most other CPPs, the peptide is internalized mainly via endocytosis, and in particular macropinocytosis, but independent of glycosaminoglycans on the cell surface. In addition, in a splice correction assay using antisense peptide nucleic acid (PNA) conjugated via a disulphide bridge to M918 (M918-PNA), we observed a dose-dependent increase in correct splicing, exceeding the effect of other CPPs. Our data demonstrate that M918 is a novel CPP that can be used to translocate different cargoes inside various cells efficiently.
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| 9. |
- EL Andaloussi, Samir, et al.
(författare)
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Application of PepFect peptides for the delivery of splice-correcting oligonucleotides
- 2011
-
Ingår i: Cell-penetrating peptides. - New York : Humana Press. - 9781607619185 ; , s. 361-373
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- One oligonucleotide-based approach that appear very promising for the treatment of different genetic disorders are based on so-called splice-correcting oligonucleotides (SCOs) that are exploited to manipulate splicing patterns. In order to increase the bioavailability, cell-penetrating peptides (CPPs) have readily been covalently conjugated to SCOs to facilitate cellular internalization. While being a successful strategy for the delivery of uncharged oligonucleotides (ONs), it is extremely difficult to generate covalent conjugates between commonly used negatively charged ON analogs and cationic CPPs. Furthermore, high concentrations of ONs in the micromolar range are often needed to obtain biological responses, most likely as a result of endosomal entrapment of material. Therefore, exploring other vectorization methods using CPPs with endosomolytic properties are highly desired. A method of using stearyl modified CPP (i.e., TP10) analogs, named PepFect3 and PepFect4, are being described for the transfection of antisense SCOs using a simple one-step co-incubation procedure. These peptides form complexes with SCOs and efficiently promote cellular uptake by facilitating endosomal escape. This chapter describes the methods of how to form and characterize these nanoparticles and the cellular assay used to address the delivery.
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| 10. |
- EL Andaloussi, Samir, et al.
(författare)
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Assessing the delivery efficacy and internalization route of cell-penetrating peptides
- 2007
-
Ingår i: Nature Protocols. - : Springer Science and Business Media LLC. - 1754-2189 .- 1750-2799. ; 2:8, s. 2043-2047
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Tidskriftsartikel (refereegranskat)abstract
- Developing efficient delivery vectors for bioactive molecules is of great importance within both traditional and novel drug development, such as oligonucleotide (ON)-based therapeutics. To address delivery efficiency using cell-penetrating peptides (CPPs), we here present a protocol based on splice correction utilizing both neutral and anionic antisense ONs, either covalently conjugated via a disulfide bridge or non-covalently complexed, respectively, that generates positive readout in the form of luciferase expression. The decisive advantage of using splice correction for evaluation of CPPs is that the ON induces a biological response in contrast to traditionally used methods, for example, fluorescently labeled peptides. An emerging number of studies emphasize the role of endocytosis in translocation of CPPs, and this protocol is also utilized to determine the relative contribution of different endocytic pathways in the uptake of CPPs, which provides valuable information for future design of novel, more potent CPPs for bioactive cargoes.
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| 11. |
- EL Andaloussi, Samir, et al.
(författare)
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Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides : a comparative study
- 2007
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 407:2, s. 285-292
-
Tidskriftsartikel (refereegranskat)abstract
- The use of CPPs (cell-penetrating peptides) as delivery vectors for bioactive molecules has been an emerging field since 1994 when the first CPP, penetratin, was discovered. Since then, several CPPs, including the widely used Tat (transactivator of transcription) peptide, have been developed and utilized to translocate a wide range of compounds across the plasma membrane of cells both in vivo and in vitro. Although the field has emerged as a possible future candidate for drug delivery, little attention has been given to the potential toxic side effects that these peptides might exhibit in cargo delivery. Also, no comprehensive study has been performed to evaluate the relative efficacy of single CPPs to convey different cargos. Therefore we selected three of the major CPPs, penetratin, Tat and transportan 10, and evaluated their ability to deliver commonly used cargos, including fluoresceinyl moiety, double-stranded DNA and proteins (i.e. avidin and streptavidin), and studied their effect on membrane integrity and cell viability. Our results demonstrate the unfeasibility to use the translocation efficacy of fluorescein moiety as a gauge for CPP efficiency, since the delivery properties are dependent on the cargo used. Furthermore, and no less importantly, the toxicity of CPPs depends heavily on peptide concentration, cargo molecule and coupling strategy.
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| 12. |
- EL Andaloussi, Samir, et al.
(författare)
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Cell-penetrating peptides-based strategies for the delivery of splice redirecting antisense oligonucleotides
- 2011
-
Ingår i: Therapeutic Oligonucleotides. - New York : Humana Press. ; 764, s. 75-89
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Progress in our understanding of the molecular pathogenesis of human malignancies has provided therapeutic targets amenable to oligonucleotide (ON)-based strategies. Antisense ON-mediated splicing regulation in particular offers promising prospects since the majority of human genes undergo alternative splicing and since splicing defects have been found in many diseases. However, their implementation has been hampered so far by the poor bioavailability of nucleic acids-based drugs. Cell-penetrating peptides (CPPs) now appear as promising non-viral delivery vector for non-permeant biomolecules. We describe here new CPPs allowing the delivery of splice redirecting steric-block ON using either chemical conjugation or non-covalent complexation. We also describe a convenient and robust splice redirecting assay which allows the quantitative assessment of ON nuclear delivery.
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| 13. |
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| 14. |
- EL Andaloussi, Samir, et al.
(författare)
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Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo
- 2011
-
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:9, s. 3972-3987
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Tidskriftsartikel (refereegranskat)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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| 15. |
- EL Andaloussi, Samir, et al.
(författare)
-
Induction of splice correction by cell-penetrating peptide nucleic acids
- 2006
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Ingår i: Journal of Gene Medicine. - : Wiley. - 1099-498X .- 1521-2254. ; 8:10, s. 1262-1273
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundDirecting splicing using oligonucleotides constitutes a promising therapeutic tool for a variety of diseases such as β-thalassemia, cystic fibrosis, and certain cancers. The rationale is to block aberrant splice sites, thus directing the splicing of the pre-mRNA towards the desired protein product. One of the difficulties in this setup is the poor bioavailability of oligonucleotides, as the most frequently used transfection agents are unsuitable for in vivo use. Here we present splice-correcting peptide nucleic acids (PNAs), tethered to a variety of cell-penetrating peptides (CPPs), evaluating their mechanism of uptake and ability to correct aberrant splicing.MethodsHeLa cells stably expressing luciferase containing an aberrant splice site were used. A previously described PNA sequence, capable of correcting the aberrant splicing, was conjugated to the CPPs, Tat, penetratin and transportan, via a disulfide bridge. The ability of the CPP-PNA conjugates to correct splicing was measured, and membrane disturbance and cell viability were evaluated using LDH leakage and WST-1 assays. Lysosomotropic agents, inhibition of endocytosis at 4 °C and confocal microscopy were used to investigate the importance of endocytosis in the uptake of the cell-penetrating PNAs.ResultsAll the three CPPs were able to promote PNA translocation across the plasma membrane and induce splice correction. Transportan (TP) was the most potent vector and significantly restored splicing in a concentration-dependent manner. Interestingly, TP also rendered a concentration-dependent splice correction in serum, in contrast to Tat and penetratin. Addition of the lysosomotrophic agent chloroquine increases the splice correction efficacy of the CPP-PNA conjugates up to 4-fold, which together with experiments at 4 °C and the visual information from confocal microscopy, indicate that the mechanism of uptake responsible for internalization of CPP-PNA conjugates is mainly endocytic. Finally, co-localization studies with dextran further indicate that conjugates, at least in the case of TP, internalize via endocytosis and in particular macropinocytosis.ConclusionsThese data demonstrate that CPPs can be used for the delivery of splice-correcting PNAs, with potential to be used as a therapeutic approach for regulating splicing in a variety of diseases. Transportan presents itself as the overall most suitable vector in this study, generating the most efficient conjugates for splice correction.
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| 16. |
- EL Andaloussi, Samir
(författare)
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Vectorization of oligonucleotides with cell-penetrating peptides : Characterization of uptake mechanisms and cytotoxicity
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The hydrophobic plasma membrane constitutes an indispensable barrier for cells in living animals. Albeit being pivotal for the maintenance of cells, the inability to cross the plasma membrane is still one of the major obstacles to overcome in order to progress current drug development. A group of substances, with restricted access to the interior of cells, which has shown great promise for future clinical use is oligonucleotides that are exploited to interfere with gene expression. Short interfering RNAs that are utilized to confer gene silencing and splice correcting oligonucleotides, applied for the manipulation of splicing patterns, are two classes of oligonucleotides that have been explored in this thesis. Cell-penetrating peptides (CPPs) are a class of peptides that has gained increasing focus in last years. This ensues as a result of their remarkable ability to convey various, otherwise impermeable, macromolecules across the plasma membrane of cells in a relatively non-toxic fashion. This thesis aims at further characterizing well-established, and newly designed, CPPs in terms of toxicity, delivery efficacy, and internalization mechanism. Our results demonstrate that different CPPs display different toxic profiles and that cargo conjugation alters the toxicity and uptake levels. Furthermore, we confirm the involvement of endocytosis in translocation of CPPs, and in particular the importance of macropinocytosis. All tested peptides facilitate the delivery of splice correcting oligonucleotides with varying efficacy, the newly designed CPP, M918, being the most potent. Finally we conclude that by promoting endosomolysis, by exploring new CPPs with improved endosomolytic properties, the biological response increases significantly. In conclusion, we believe that these results will facilitate the development of new CPPs with improved delivery properties that could be used for transportation of oligonucleotides in clinical settings.
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| 17. |
- Ezzat, Kariem, et al.
(författare)
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PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation
- 2011
-
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:12, s. 5284-5298
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Tidskriftsartikel (refereegranskat)abstract
- Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne's muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine™ 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.
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| 18. |
- Ezzat, Kariem, et al.
(författare)
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Peptide-based matrices as drug delivery vehicles
- 2010
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Ingår i: Current pharmaceutical design. - 1381-6128 .- 1873-4286. ; 16:9, s. 1167-1178
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Forskningsöversikt (refereegranskat)abstract
- Peptides, polypeptides and proteins have been extensively studied for their various structural and functional roles in living organisms. However, breakthrough discoveries in the last decades identified some peptide-based matrices that posses the ability to traverse biological membranes, and many peptides, polypeptides and even complete proteins have been shown to have such properties. Hence, these matrices have been successfully used for the intracellular delivery of many therapeutic cargos including small molecules, proteins, peptides, oligonucleutides, plasmids and nanoparticles both in vitro and in vivo. Being neither toxic nor carcinogenic and meanwhile efficient in delivery, they are recognized as very promising vectors to overcome the shortcomings of the available technologies. The characteristics of these peptide-based matrices and their applications in drug delivery are here briefly illustrated together with current challenges and future prospects.
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| 19. |
- Ezzat, Kariem, et al.
(författare)
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Scavenger receptor-mediated uptake of cell-penetrating peptide nanoparticles with oligonucleotides
- 2012
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 26:3, s. 1172-1180
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) are shortcationic peptides that penetrate cells by interacting withthe negatively charged plasma membrane; however, thedetailed uptake mechanism is not clear. In contrary to theconventional mode of action of CPPs, we show here thata CPP, PepFect14 (PF14), forms negatively charged nanocomplexeswith oligonucleotides and their uptake is mediatedby class-A scavenger receptors (SCARAs). Specificinhibitory ligands of SCARAs, such as fucoidin, polyinosinicacid, and dextran sulfate, totally inhibit the activityof PF14-oligonucleotide nanocomplexes in the HeLapLuc705 splice-correction cell model, while nonspecific,chemically related molecules do not. Furthermore, RNAinterference (RNAi) knockdown of SCARA subtypes(SCARA3 and SCARA5) that are expressed in this cell lineled to a significant reduction of the activity to <50%. Inline with this, immunostaining shows prevalent colocalizationof the nanocomplexes with the receptors, and electronmicroscopy images show no binding or internalizationof the nanocomplexes in the presence of theinhibitory ligands. Interestingly, naked oligonucleotidesalso colocalize with SCARAs when used at high concentrations.These results demonstrate the involvement ofSCARA3 and SCARA5 in the uptake of PF14-oligonucleotidenanocomplexes and suggest for the first time thatsome CPP-based systems function through scavenger receptors,which could yield novel possibilities to understandand improve the transfection by CPPs.
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| 20. |
- Ezzat, Kariem, et al.
(författare)
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Solid formulation of cell-penetrating peptide nanoparticles with siRNA and their stability in simulated gastric conditions
- 2012
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Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 162:1, s. 1-8
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) are short cationic peptides that have been extensively studied as drug delivery vehicles for proteins, nucleic acids and nanoparticles. However, the formulation of CPP-based therapeutics into different pharmaceutical formulations and their stability in relevant biological environments have not been given the same attention. Here, we show that a newly developed CPP, PepFect 14 (PF14), forms non-covalent nanocomplexes with short interfering RNA (siRNA), which are able to elicit efficient RNA-interference (RNAi) response in different cell-lines. RNAi effect was obtained at low siRNA doses with a unique kinetic profile. Furthermore, we utilized the solid dispersion technique to formulate PF14/siRNA nanocomplexes into solid formulations that were as active as the freshly prepared nanocomplexes in solution. Importantly, the freshly prepared nanocomplexes and solid formulations were stable after incubation with simulated gastric fluid having a pH of 1.2 and containing proteolytic enzymes. These results demonstrate the activity of PF14 in delivering and protecting siRNA in different pharmaceutical forms and biological environments.
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| 21. |
- Ezzat, Kariem, et al.
(författare)
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The viral protein corona directs viral pathogenesis and amyloid aggregation
- 2019
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Artificial nanoparticles accumulate a protein corona layer in biological fluids, which significantly influences their bioactivity. As nanosized obligate intracellular parasites, viruses share many biophysical properties with artificial nanoparticles in extracellular environments and here we show that respiratory syncytial virus (RSV) and herpes simplex virus type 1 (HSV-1) accumulate a rich and distinctive protein corona in different biological fluids. Moreover, we show that corona pre-coating differentially affects viral infectivity and immune cell activation. In addition, we demonstrate that viruses bind amyloidogenic peptides in their corona and catalyze amyloid formation via surface-assisted heterogeneous nucleation. Importantly, we show that HSV-1 catalyzes the aggregation of the amyloid beta-peptide (A beta(42)), a major constituent of amyloid plaques in Alzheimer's disease, in vitro and in animal models. Our results highlight the viral protein corona as an acquired structural layer that is critical for viral-host interactions and illustrate a mechanistic convergence between viral and amyloid pathologies.
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| 22. |
- Gupta, Dhanu, et al.
(författare)
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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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Ingår i: Nature Biomedical Engineering. - Stockholm : Karolinska Institutet, Dept of Laboratory Medicine. - 2157-846X.
-
Tidskriftsartikel (refereegranskat)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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| 23. |
- Guterstam, Peter, et al.
(författare)
-
Characterization of cellular internalization pathways for CPP-mediated oligonucleotide delivery
- 2011
-
Ingår i: Cell-penetrating peptides. - New York : Humana Press. - 9781607619185 ; , s. 219-230
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- The methods for evaluating internalization pathways of cellular CPP-mediated ON delivery utilizing a pre-mRNA splice correction assay and fluorescence-based quantification are described. Examples for characterization of CPP uptake routes, employing various endocytosis inhibitors, and special treatment conditions are demonstrated. The methods are developed to characterize cellular delivery of pre-mRNA splice switching peptide nucleic acids conjugated to CPPs by disulfide bond.
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| 24. |
- Guterstam, Peter, 1977-, et al.
(författare)
-
Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate
- 2009
-
Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1788:12, s. 2509-2517
-
Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) are membrane permeable vectors recognized for their intrinsic ability to gain access to the cell interior. The hydrophobic counter-anion, pyrenebutyrate, enhances cellular uptake of oligoarginine CPPs. To elucidate CPP uptake mechanisms, the effect of pyrenebutyrate on well-recognized CPPs with various hydrophobicity and arginine content is investigated. The cellular CPP-uptake and CPP-mediated oligonucleotide delivery is analyzed by fluorescence activated cell sorting, confocal microscopy, and a cell based splice-switching assay. The splice-switching oligonucleotide is a mixmer of 2’-O-methyl RNA and locked nucleic acids delivered as a non-covalent complex with 10-fold molar CPP excess. CPP-induced membrane perturbation on large unilamellar vesicles is investigated in calcein release experiments. We observed that pyrenebutyrate facilitates cellular uptake and translocation of oligonucleotide mediated by oligoarginine nonamer while limited effect of pyrenebutyrate on more hydrophobic CPPs was observed. By combining the different experimental results we conclude that the pathway for cellular uptake of oligoarginine is dominated by direct membrane translocation, whereas the pathway for oligoarginine-mediated oligonucleotide translocation is dominated by endocytosis. Both mechanisms are promoted by pyrenebutyrate and we suggest that pyrenebutyrate has different sites of action for the two uptake and translocation mechanisms.
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| 25. |
- Guterstam, Peter, et al.
(författare)
-
Splice-switching efficiency and specificity for oligonucleotides with locked nucleic acid monomers
- 2008
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 412, s. 307-313
-
Tidskriftsartikel (refereegranskat)abstract
- The use of antisense oligonucleotides to modulate splicing patterns has gained increasing attention as a therapeutic platform and, hence, the mechanisms of splice-switching oligonucleotides are of interest. Cells expressing luciferase pre-mRNA interrupted by an aberrantly spliced beta-globin intron, HeLa pLuc705, were used to monitor the splice-switching activity of modified oligonucleotides by detection of the expression of functional luciferase. It was observed that phosphorothioate 2'-O-methyl RNA oligonucleotides containing locked nucleic acid monomers provide outstanding splice-switching activity. However, similar oligonucleotides with several mismatches do not impede splice-switching activity which indicates a risk for off-target effects. The splice-switching activity is abolished when mismatches are introduced at several positions with locked nucleic acid monomers suggesting that it is the locked nucleic acid monomers that give rise to low mismatch discrimination to target pre-mRNA. The results highlight the importance of rational sequence design to allow for high efficiency with simultaneous high mismatch discrimination for splice-switching oligonucleotides and suggest that splice-switching activity is tunable by utilizing locked nucleic acid monomers.
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| 26. |
- Hassane, Fatouma Said, et al.
(författare)
-
Insights into the cellular trafficking of splice redirecting oligonucleotides complexed with chemically modified cell-penetrating peptides
- 2011
-
Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 153:2, s. 163-172
-
Tidskriftsartikel (refereegranskat)abstract
- Conjugates of cell-penetrating peptides (CPP) and splice redirecting oligonucleotides (ON) display clinical potential as attested by in vivo experimentation in murine models of Duchenne muscular dystrophy. However, micromolar concentrations of these conjugates are required to obtain biologically relevant responses as a consequence of extensive endosomal sequestration following endocytosis. Recent work from our group has demonstrated that appending stearic acid to CPPs increases their efficiency and that the inclusion of pH titrable entities leads to further improvement. Moreover, these modified CPPs form non covalent complexes with charged ON analogs or siRNAs, which allows decreasing the concentrations of ONs by nearly one log. These modified CPPs and the parent peptides have been compared here in the same in vitro model in terms of cell uptake, trafficking and splicing redirection activity. The increased splicing redirection activity of our modified CPPs cannot be explained by differences in cell uptake but rather by their enhanced ability to escape from endocytic vesicles. Accordingly, a clear correlation between membrane destabilizing activity and splicing redirection was observed using a liposome leakage assay. Studies of cellular trafficking for the most active PF6:ON complexes indicate uptake by clathrin-mediated endocytosis using either FACS cell uptake or a splicing redirection functional assay. Acidification of intracellular vesicles and membrane potential were found important for splicing redirection but not for cell uptake. These results do confirm that the increased potency of PF6:ON complexes is not due to the use of a non endocytic route of cell internalization as proposed for some CPPs.
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| 27. |
- Holm, Tina, et al.
(författare)
-
Comparison of CPP uptake methods
- 2011
-
Ingår i: Cell-penetrating peptides. - New York : Humana Press. - 9781607619185 ; , s. 207-217
-
Bokkapitel (refereegranskat)abstract
- In the last 15 years, an ever expanding pool of cell-penetrating peptides (CPPs) has been discovered and recently focus has shifted towards improving already existing CPPs by different modifications. Since the number of published peptide sequences with cell-penetrating ability is now reaching several hundreds, the consensus methods to compare the efficacy of these is clearly needed. Many research groups are evaluating the applicability of CPPs as drug delivery vectors, all having their preferred methods of assessing uptake and intracellular distribution. Even when applying the same method, the use of different cell lines, peptide concentrations, exposure conditions, etc. are complicating comparison of data between different groups. This book is a welcome contribution to the CPP research field, hopefully paving the way for standardized protocols to be used in the future. Some of the most common methods used to this date are presented and compared in this chapter.
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| 28. |
- Holm, Tina, et al.
(författare)
-
Retro-inversion of certain cell-penetrating peptides causes severe cellular toxicity
- 2011
-
Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0005-2736. ; 1808:6, s. 1544-1551
-
Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) are a promising group of delivery vectors for various therapeutic agents but their application is often hampered by poor stability in the presence of serum. Different strategies to improve peptide stability have been exploited, one of them being "retro-inversion" (RI) of natural peptides. With this approach the stability of CPPs has been increased, thereby making them more efficient transporters. Several RI-CPPs were here assessed and compared to the corresponding parent peptides in different cell-lines. Surprisingly, treatment of cells with these peptides induced trypsin insensitivity and rapid severe toxicity in contrast to l-peptides. This was measured as reduced metabolic activity and condensed cell nuclei, in parity with the apoptosis inducing agent staurosporine. Furthermore, effects on mitochondrial network, focal adhesions, actin cytoskeleton and caspase-3 activation were analyzed and adverse effects were evident at 20μM peptide concentration within 4h while parent l-peptides had negligible effects. To our knowledge this is the first time RI peptides are reported to cause cellular toxicity, displayed by decreased metabolic activity, morphological changes and induction of apoptosis. Considering the wide range of research areas that involves the use of RI-peptides, this finding is of major importance and needs to be taken under consideration in applications of RI-peptides.
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| 29. |
- Hyvonen, Maija, et al.
(författare)
-
Novel Target for Peptide-Based Imaging and Treatment of Brain Tumors
- 2014
-
Ingår i: Molecular Cancer Therapeutics. - 1535-7163 .- 1538-8514. ; 13:4, s. 996-1007
-
Tidskriftsartikel (refereegranskat)abstract
- Malignant gliomas are associated with high mortality due to infiltrative growth, recurrence, and malignant progression. Even with the most efficient therapy combinations, median survival of the glioblastoma multiforme (grade 4) patients is less than 15 months. Therefore, new treatment approaches are urgently needed. We describe here identification of a novel homing peptide that recognizes tumor vessels and invasive tumor satellites in glioblastomas. We demonstrate successful brain tumor imaging using radiolabeled peptide in whole-body SPECT/CT imaging. Peptide-targeted delivery of chemotherapeutics prolonged the lifespan of mice bearing invasive brain tumors and significantly reduced the number of tumor satellites compared with the free drug. Moreover, we identified mammary-derived growth inhibitor (MDGI/H-FABP/FABP3) as the interacting partner for our peptide on brain tumor tissue. MDGI was expressed in human brain tumor specimens in a grade-dependent manner and its expression positively correlated with the histologic grade of the tumor, suggesting MDGI as a novel marker for malignant gliomas. Mol Cancer Ther; 13(4); 996-1007. (C)2014 AACR.
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| 30. |
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| 31. |
- Johansson, Henrik J., et al.
(författare)
-
Mimicry of protein function with cell-penetrating peptides
- 2011
-
Ingår i: Cell-penetrating peptides. - Totowa, NJ : Humana Press. - 9781607619185 - 9781607619192 ; 683, s. 233-47
-
Bokkapitel (refereegranskat)abstract
- Proteins are essential components of cellular processes inside cells, and their interactions between each other and with genes are important for the normal physiological functioning of cells as well as for disease states. Modulating protein interactions by different means can potentially control these interactions and restore normal function to diseased cells. The ways to do so are multiple, and such efforts often begin with knowledge of potential target proteins in order to devise mediators that retain the function of the original protein, i.e., mimic the protein functions. An alternative strategy is to utilize protein mimics to inhibit target proteins rather than restoring the activity of a protein. The vast majority of protein mimics exploited to date have been designed to inhibit the activity of oncogenes or activate tumor suppressors for the purpose of tumor therapy. These protein mimics are usually based on small organic compounds or peptides, derived from interaction surfaces of the proteins, and in some cases, full proteins have been exploited. Although peptides and proteins are naturally highly specific and efficient inside cells, they suffer from low bioavailability resulting from their inability to enter cells. One strategy increasingly employed to facilitate the internalization of peptides and proteins has been to chemically conjugate them to cell-penetrating peptides (CPP) or to recombinantly express protein–CPP fusion constructs.This chapter provides an overview of some of the aspects of perturbing and mimicking protein interactions using peptides and proteins and CPP as transport vectors.
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| 32. |
- Järver, Peter, et al.
(författare)
-
Applications of cell-penetrating peptides in regulation of gene expression
- 2007
-
Ingår i: Biochemical Society Transactions. - 0300-5127 .- 1470-8752. ; 35:Pt 4, s. 770-774
-
Tidskriftsartikel (refereegranskat)abstract
- CPPs (cell-penetrating peptides) can be defined as short peptides that are able to efficiently penetrate cellular lipid bilayers. Because of this remarkable feature, they are excellent candidates regarding alterations in gene expression. CPPs have been utilized in in vivo and in vitro experiments as delivery vectors for different bioactive cargoes. This review focuses on the experiments performed in recent years where CPPs have been used as vectors for multiple effectors of gene expression such as oligonucleotides for antisense, siRNA (small interfering RNA) and decoy dsDNA (double-stranded DNA) applications, and as transfection agents for plasmid delivery.
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| 33. |
- Järver, Peter, et al.
(författare)
-
Co-transduction of Sleeping Beauty Transposase and Donor Plasmid via a Cell-penetrating Peptide : A simple one-step method
- 2008
-
Ingår i: International journal of peptide research and therapeutics. - : Springer Science and Business Media LLC. - 1573-3904 .- 1573-3149. ; 14:1, s. 58-63
-
Tidskriftsartikel (refereegranskat)abstract
- Transposable elements have emerged as a promising candidate for human non-viral gene-therapy. The Tc1/mariner transposon Sleeping Beauty is to date one of the most efficient transposons in mammals. Sleeping Beauty transposase has so far mostly been delivered to cells via a DNA source. This might cause spontaneous integration of the transposase gene and cause fatal damage to the affected cell. Hence, it would be advantageous to employ a non-genetic source for the transposase. We here show that a novel Cell-penetrating peptide, M918, has the ability to facilitate cellular delivery of both the transposase Sleeping Beauty as a protein and a transposon donor-plasmid carrying an antibiotic resistance gene in vitro. The technique is a simple and straightforward one-step method that might render a safe and efficient delivery platform for Sleeping Beauty mediated gene therapy.
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| 34. |
- Järver, Peter, et al.
(författare)
-
Peptide nanoparticle delivery of charge-neutral splice-switching morpholino oligonucleotides.
- 2015
-
Ingår i: Nucleic Acid Therapeutics. - : Mary Ann Liebert Inc. - 2159-3337 .- 2159-3345. ; 25:2, s. 65-77
-
Tidskriftsartikel (refereegranskat)abstract
- Oligonucleotide analogs have provided novel therapeutics targeting various disorders. However, their poor cellular uptake remains a major obstacle for their clinical development. Negatively charged oligonucleotides, such as 2'-O-Methyl RNA and locked nucleic acids have in recent years been delivered successfully into cells through complex formation with cationic polymers, peptides, liposomes, or similar nanoparticle delivery systems. However, due to the lack of electrostatic interactions, this promising delivery method has been unsuccessful to date using charge-neutral oligonucleotide analogs. We show here that lipid-functionalized cell-penetrating peptides can be efficiently exploited for cellular transfection of the charge-neutral oligonucleotide analog phosphorodiamidate morpholino. The lipopeptides form complexes with splice-switching phosphorodiamidate morpholino oligonucleotide and can be delivered into clinically relevant cell lines that are otherwise difficult to transfect while retaining biological activity. To our knowledge, this is the first study to show delivery through complex formation of biologically active charge-neutral oligonucleotides by cationic peptides.
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| 35. |
- Järver, Peter, et al.
(författare)
-
Single-Stranded Nucleic Acids Regulate TLR3/4/7 Activation through Interference with Clathrin-Mediated Endocytosis
- 2018
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
-
Tidskriftsartikel (refereegranskat)abstract
- Recognition of nucleic acids by endosomal Toll-like receptors (TLR) is essential to combat pathogens, but requires strict control to limit inflammatory responses. The mechanisms governing this tight regulation are unclear. We found that single-stranded oligonucleotides (ssON) inhibit endocytic pathways used by cargo destined for TLR3/4/7 signaling endosomes. Both ssDNA and ssRNA conferred the endocytic inhibition, it was concentration dependent, and required a certain ssON length. The ssON-mediated inhibition modulated signaling downstream ofTLRs that localized within the affected endosomal pathway. We further show that injection of ssON dampens dsRNA-mediated inflammatory responses in the skin of non-human primates. These studies reveal a regulatory role for extracellular ssON in the endocytic uptake of TLR ligands and provide a mechanistic explanation of their immunomodulation. The identified ssON-mediated interference of endocytosis (SOMIE) is a regulatory process that temporarily dampens TLR3/4/7 signaling, thereby averting excessive immune responses.
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| 36. |
- Katayama, Mutsumi, et al.
(författare)
-
Circulating Exosomal miR-20b-5p is Elevated in Type 2 Diabetes and Could Impair Insulin Action in Human Skeletal Muscle.
- 2019
-
Ingår i: Diabetes. - : American Diabetes Association. - 1939-327X .- 0012-1797. ; 68:3, s. 515-526
-
Tidskriftsartikel (refereegranskat)abstract
- MicroRNAs (miRNAs) are noncoding RNAs representing an important class of gene expression modulators. Extracellular circulating miRNAs are both candidate biomarkers for disease pathogenesis and mediators of cell-to-cell communication. We examined the miRNA expression profile of total serum and serum derived exosome-enriched extracellular vesicles in people with normal glucose tolerance or type 2 diabetes. In contrast to total serum miRNA, which did not reveal any differences in miRNA expression, we identified differentially abundant miRNAs in type 2 diabetes patients using miRNA expression profiles of exosome RNA (exoRNA). To validate the role of these differentially abundant miRNAs on glucose metabolism, we transfected miR-20b-5p, a highly abundant exoRNA in type 2 diabetic patients, into primary human skeletal muscle cells. miR-20b-5p overexpression increased basal glycogen synthesis in human skeletal muscle cells. We identified AKTIP and STAT3 as miR-20b-5p targets. miR-20b-5p overexpression reduced AKTIP abundance and insulin-stimulated glycogen accumulation. In conclusion, exosome derived extracellular miR-20b-5p is a circulating biomarker associated with type 2 diabetes, which plays an intracellular role in modulating insulin-stimulated glucose metabolism via AKT signaling.
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| 37. |
- Kilk, Kalle, et al.
(författare)
-
Evaluation of transportan 10 in PEI mediated plasmid delivery assay
- 2005
-
Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 103:2, s. 511-23
-
Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) are novel high-capacity delivery vectors for different bioactive cargoes. We have evaluated the CPP transportan 10 (TP10) as a delivery vector in different in vitro plasmid delivery assays. Tested methods include: TP10 crosslinked to a plasmid via a peptide nucleic acid (PNA) oligomer, TP10 conjugation with polyethyleneimine (PEI), and addition of unconjugated TP10 to standard PEI transfection assay. We found that without additional DNA condensing agents, TP10 has poor transfection abilities. However, the presence of TP10 increases the transfection efficiency several folds compared to PEI alone. At as low concentrations as 0.6 nM, TP10–PNA constructs were found to enhance plasmid delivery up to 3.7-fold in Neuro-2a cells. Interestingly, the transfection efficiency was most significant at low PEI concentrations, allowing reduced PEI concentration without loss of gene delivery. No increase in cytotoxicity due to TP10 was observed and the uptake mechanism was determined to be endocytosis, as previously reported for PEI mediated transfection. In conclusion, TP10 can enhance PEI mediated transfection at relatively low concentrations and may help to develop future gene delivery systems with reduced toxicity.
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| 38. |
- Last, Torben, et al.
(författare)
-
Micro Swirl-Nozzle Enables Portable Delivery ofLarge-Molecule Biopharmaceuticals to the Lung
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Large-molecule pharmaceuticals offer new treatment options for severe lung disease. However, delivering these drugs to the lung is challenging due to the elevated shear rates during the aerosolization process. So far, this has prevented an application in portable inhalers, holding back the portable use of biopharmaceuticals for drug delivery. We demonstrate that a micro swirl nozzle can aerosolize fragile biopharmaceuticals in an aqueous solution. Shear rate simulations of the nozzle unit indicate orders of magnitude in shear rate reduction compared with conventional aqueous aerosolization units. Catalase protein can survive the aerosolization process at up to 50 bar without significant degradation. The protein further remains enzymatically active after the spray event.Using an in-vitro model, we present the delivery of more complex and fragile mRNA structures (Nanoluc mRNA) at high concentrations when encapsulated in solid lipid nanoparticles (LNPs) or Extracellular vesicles (EVs). These vesicles maintain their capability to pass the cell wall in in-vitro cell cultures, leading to an expression of the encapsulated protein structure within the celll. Micro swirl nozzles can enable the portable delivery of large molecule pharmaceuticals and bring new treatment options to patients who have so far had to rely on stationary devices.
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| 39. |
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| 40. |
- Lehto, Taavi, et al.
(författare)
-
A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo
- 2011
-
Ingår i: Molecular Therapy. - : Elsevier BV. - 1525-0016 .- 1525-0024. ; 19:8, s. 1457-1467
-
Tidskriftsartikel (refereegranskat)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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| 41. |
- Lehto, Taavi, et al.
(författare)
-
Delivery of nucleic acids with a stearylated (RxR)4 peptide using a non-covalent co-incubation strategy
- 2010
-
Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 141:1, s. 42-51
-
Tidskriftsartikel (refereegranskat)abstract
- In recent years, oligonucleotide-based molecules have been intensely used to modulate gene expression. All these molecules share the common feature of being essentially impermeable over cellular membranes and they therefore require efficient delivery vectors. Cell-penetrating peptides are a group of delivery peptides that has been readily used for nucleic acid delivery. In particular, polyarginine and derivates thereof, i.e. the (RxR)4 peptide, have been applied with success both in vitro and in vivo. A major problem, however, with these arginine-rich peptides is that they frequently remain trapped in endosomal compartments following internalization. The activity of polyarginine has previously been improved by conjugation to a stearyl moiety. Therefore, we sought to investigate what impact such modification would have on the pre-clinically used (RxR)4 peptide for non-covalent delivery of plasmids and splice-correcting oligonucleotides (SCOs) and compare it with stearylated Arg9 and Lipofectamine™ 2000. We show that stearyl-(RxR)4 mediates efficient plasmid transfections in several cell lines and the expression levels are significantly higher than when using unmodified (RxR)4 or stearylated Arg9. Although the transfection efficiency is lower than with Lipofectamine™ 2000, we show that stearyl-(RxR)4 is substantially less toxic. Furthermore, using a functional splice-correction assay, we show that stearyl-(RxR)4 complexed with 2′-OMe SCOs promotes significant splice correction whereas stearyl-Arg9 fails to do so. Moreover, stearyl-(RxR)4 promotes dose-dependent splice correction in parity with (RxR)4-PMO covalent conjugates, but at least 10-times lower concentration. These features make this stearic acid modified analog of (RxR)4 an intriguing vector for future in vivo experiments.
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| 42. |
- Lener, Thomas, et al.
(författare)
-
Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper.
- 2015
-
Ingår i: Journal of extracellular vesicles. - : Wiley. - 2001-3078. ; 4
-
Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.
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| 43. |
- Li, Chen, et al.
(författare)
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Cutaneous squamous cell carcinoma-derived extracellular vesicles exert an oncogenic role by activating cancer-associated fibroblasts
- 2023
-
Ingår i: Cell Death Discovery. - : Springer Nature. - 2058-7716. ; 9:1
-
Tidskriftsartikel (refereegranskat)abstract
- Cutaneous squamous cell carcinoma (cSCC) is a fast-increasing cancer with metastatic potential. Extracellular vesicles (EVs) are small membrane-bound vesicles that play important roles in intercellular communication, particularly in the tumor microenvironment (TME). Here we report that cSCC cells secrete an increased number of EVs relative to normal human epidermal keratinocytes (NHEKs) and that interfering with the capacity of cSCC to secrete EVs inhibits tumor growth in vivo in a xenograft model of human cSCC. Transcriptome analysis of tumor xenografts by RNA-sequencing enabling the simultaneous quantification of both the human and the mouse transcripts revealed that impaired EV-production of cSCC cells prominently altered the phenotype of stromal cells, in particular genes related to extracellular matrix (ECM)-formation and epithelial-mesenchymal transition (EMT). In line with these results, co-culturing of human dermal fibroblasts (HDFs) with cSCC cells, but not with normal keratinocytes in vitro resulted in acquisition of cancer-associated fibroblast (CAF) phenotype. Interestingly, EVs derived from metastatic cSCC cells, but not primary cSCCs or NHEKs, were efficient in converting HDFs to CAFs. Multiplex bead-based flow cytometry assay and mass-spectrometry (MS)-based proteomic analyses revealed the heterogenous cargo of cSCC-derived EVs and that especially EVs derived from metastatic cSCCs carry proteins associated with EV-biogenesis, EMT, and cell migration. Mechanistically, EVs from metastatic cSCC cells result in the activation of TGFβ signaling in HDFs. Altogether, our study suggests that cSCC-derived EVs mediate cancer-stroma communication, in particular the conversion of fibroblasts to CAFs, which eventually contribute to cSCC progression.
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| 44. |
- Lundberg, Pontus, et al.
(författare)
-
Delivery of short interfering RNA using endosomolytic cell-penetrating peptides
- 2007
-
Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 21:11, s. 2664-2671
-
Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) are peptides able to promote uptake of various cargos, including proteins and plasmids. Advances in recent years imply the uptake to be endocytic, where the current hurdle for efficient intracellular delivery is material being retained in the endosomes. In this study we wanted to compare the ability of various established CPPs to deliver siRNA and induce gene silencing of luciferase, with a novel designed penetratin analog having endosomolytic properties, using a noncovalent strategy. In principal, the penetratin analog EB1 will, upon protonation in the early-late endosomes, be able to form an amphipathic alpha helix resulting in permeabilization of the endosomal membrane. We demonstrate that even though all CPPs evaluated in this study can form complexes with siRNA, there is not a direct relationship between the complex formation ability and delivery efficacy. More important, although all CPPs significantly promote siRNA uptake, in some cases no gene silencing effect can be observed unless endosomal escape is induced. We find the designed endosomolytic peptide EB1 to be far more effective both in forming complexes and transporting biologically active siRNA than its parent peptide penetratin. We believe that developing CPPs with increased endosomolytical properties is a necessary step toward achieving biological effects at low concentrations for future in vivo applications.—Lundberg, P., El-Andaloussi, S., Sütlü, T., Johansson, H., Langel, Ü. Delivery of short interfering RNA using endosomolytic cell-penetrating peptides.
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| 45. |
- Lundin, Per, et al.
(författare)
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Distinct Uptake Routes of Cell-Penetrating Peptide Conjugates
- 2008
-
Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 19:12, s. 2535-2542
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) are a growing family of peptides that have opened a new avenue in drug delivery, allowing various hydrophilic macromolecules to enter cells. In accordance with most other cationic delivery vectors, CPPs seem to rely mostly on endocytosis for internalization. However, due to conflicting results the exact endocytic pathways for CPP uptake have not yet been resolved. Here, we evaluated the ability of seven CPPs, with different chemical properties, to convey peptide nucleic acids (PNAs) inside cells. Assays based on both splice correction, generating biologically active read-out, and on traditional fluorescence measurements were utilized. The same assays were employed to assess different endocytic pathways and the dependence on extracellular heparan sulfates for internalization. Both highly cationic CPPs (M918, penetratin, and Tat) and amphipathic peptides (transportan, TP10, MAP, and pVEC) were investigated in this study. Conjugate uptake relied on endocytosis for all seven peptides but splice-correcting activity varied greatly for the investigated CPPs. The exact endocytic internalization routes were evaluated through the use of well-known endocytosis inhibitors and tracers. In summary, the different chemical properties of CPPs have little correlation with their ability to efficiently deliver splice-correcting PNA. However, conjugates of polycationic and amphipathic peptides appear to utilize different internalization routes.
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| 46. |
- Lundin, Per, et al.
(författare)
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Toxicity methods for CPPs
- 2011
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Ingår i: Cell-penetrating peptides. - New York : Humana Press. - 9781607619185 ; , s. 195-205
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- CPPs have for numerous years been utilized as delivery vectors of various pharmaceutically interesting cargoes, both in vitro and in vivo. As CPPs are gradually approaching the bedsides, investigating toxicity associated with these highly interesting peptides becomes increasingly important and thorough initial assessment of cytotoxicity in vitro is a first step towards advancing these delivery vehicles in to the clinics. The present chapter describes protocols for four cytotoxicity assays in order to provide a toolbox for toxicity assessment of CPPs. The foci lie on membrane integrity (deoxyglucose leakage and propidium iodide assays) and cell viability (the MTT assay), but the chapter also provides a protocol for assessing an important parameter for future clinical applications, namely the hemolytic properties of CPPs.
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| 47. |
- Muñoz-Alarcón, Andrés, et al.
(författare)
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Modulating Anti-MicroRNA-21 Activity and Specificity Using Oligonucleotide Derivatives and Length Optimization
- 2012
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Ingår i: ISRN Pharmaceutics. - : Hindawi Limited. - 2090-6145 .- 2090-6153. ; 2012
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Tidskriftsartikel (refereegranskat)abstract
- MicroRNAs are short, endogenous RNAs that direct posttranscriptional regulation of gene expression vital for many developmental and cellular functions. Implicated in the pathogenesis of several human diseases, this group of RNAs provides interesting targets for therapeutic intervention. Anti-microRNA oligonucleotides constitute a class of synthetic antisense oligonucleotides used to interfere with microRNAs. In this study, we investigate the effects of chemical modifications and truncations on activity and specificity of anti-microRNA oligonucleotides targeting microRNA-21. We observed an increased activity but reduced specificity when incorporating locked nucleic acid monomers, whereas the opposite was observed when introducing unlocked nucleic acid monomers. Our data suggest that phosphorothioate anti-microRNA oligonucleotides yield a greater activity than their phosphodiester counterparts and that a moderate truncation of the anti-microRNA oligonucleotide improves specificity without significantly losing activity. These results provide useful insights for design of anti-microRNA oligonucleotides to achieve both high activity as well as efficient mismatch discrimination.
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| 48. |
- Muthukrishnan, Uma, 1984-, et al.
(författare)
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The exosome membrane localization of histones is independent of DNA and upregulated in response to stress
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Extracellular histones contribute to many acute and chronic diseases but also populate the secretomes of healthy cells and biofluids. However, a secretory pathway for histones has not been described. Here we report that core and linker histones localize to multivesicular bodies and are secreted via exosomes. Histones are tightly associated with the exosome membrane, with N-terminal domains exposed, in a DNA-independent manner. Furthermore, rapid upregulation of exosomal histones occurs following heat stress, accompanied by enhanced vesicle secretion and a shift towards a population of smaller vesicles. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) complex as a possible mechanism underlying increased histone secretion.We show for the first time that membrane-associated histones are actively secreted from intact cells via the multivesicular body/exosomal pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease.
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| 49. |
- Mäe, Maarja, et al.
(författare)
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A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy
- 2009
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Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 134:3, s. 221-227
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Tidskriftsartikel (refereegranskat)abstract
- Aberrations in splicing patterns play a significant role in several diseases, and splice correction, together with other forms of gene regulation, is consequently an emerging therapeutic target. In order to achieve successful oligonucleotide transfection, efficient delivery vectors are generally necessary. In this study we present one such vector, the chemically modified cell-penetrating peptide (CPP) TP10, for efficient delivery of a splice-correcting 2'-OMe RNA oligonucleotide. Utilizing a functional splice correction assay, we assessed the transfection efficiency of non-covalent complexes of oligonucleotides and stearylated or cysteamidated CPPs. Stearylation of the CPPs Arg9 and penetratin, as well as cysteamidation of MPG and TP10, did not improve transfection, whereas the presence of an N-terminal stearyl group on TP10 improved delivery efficiency remarkably compared to the unmodified peptide. The splice correction levels observed with stearyl-TP10 are in fact in parity with the effects seen with the commercially available transfection agent Lipofectamine (TM) 2000. However, the inherent toxicity associated with cationic lipid-based transfections can be completely eliminated when using the stearylated TP10, making this vector highly promising for non-covalent delivery of negatively charged oligonucleotides.
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| 50. |
- Mäe, Maarja, et al.
(författare)
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Chemically modified cell-penetrating peptides for the delivery of nucleic acids
- 2009
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Ingår i: Expert Opinion on Drug Delivery. - : Informa Healthcare. - 1742-5247 .- 1744-7593. ; 6:11, s. 1195-1205
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Tidskriftsartikel (refereegranskat)abstract
- Short nucleic acids targeting biologically important RNAs and plasmids have been shown to be promising future therapeutics; however, their hydrophilic nature greatly limits their utility in clinics and therefore efficient delivery vectors are greatly needed. Cell-penetrating peptides (CPPs) are relatively short amphipathic and/or cationic peptides that are able to transport various biologically active molecules inside mammalian cells, both in vitro and in vivo, in a seemingly non-toxic fashion. Although CPPs have proved to be appealing drug delivery vehicles, their major limitation in nucleic acid delivery is that most of the internalized peptide-cargo is entrapped in endosomal compartments following endocytosis and the bioavailability is therefore severely reduced. Several groups are working towards overcoming this obstacle and this review highlights the evidence that by introducing chemical modification in CPPs, the bioavailability of delivered nucleic acids increases significantly.
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