| 1. |
- EL Andaloussi, Samir, et al.
(author)
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Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides : a comparative study
- 2007
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In: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 407:2, s. 285-292
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Journal article (peer-reviewed)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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| 2. |
- Ezzat, Kariem, et al.
(author)
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The viral protein corona directs viral pathogenesis and amyloid aggregation
- 2019
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10
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Journal article (peer-reviewed)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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| 3. |
- Honarvar, Hadis, et al.
(author)
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Evaluation of backbone-cyclized HER2-binding 2-helix Affibody molecule for In Vivo molecular imaging
- 2013
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In: Nuclear Medicine and Biology. - : Elsevier BV. - 0969-8051 .- 1872-9614. ; 40:3, s. 378-386
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Journal article (peer-reviewed)abstract
- Introduction: Affibody molecules, small scaffold proteins, have demonstrated an appreciable potential as imaging probes. Affibody molecules are composed of three alpha-helices. Helices 1 and 2 are involved in molecular recognition, while helix 3 provides stability. The size of Affibody molecules can be reduced by omitting the third alpha-helix and cross-linking the two remaining, providing a smaller molecule with better extravasation and quicker clearance of unbound tracer. The goal of this study was to develop a novel 2-helix Affibody molecule based on backbone cyclization by native chemical ligation (NCL). Methods: The HER2-targeting NCL-cyclized Affibody molecule Z(HER2:342min) has been designed, synthesized and site-specifically conjugated with a DOTA chelator. DOTA-Z(HER2:342min) was labeled with In-111 and (68) Ga. The binding affinity of DOTA-Z(HER2:342min) was evaluated in vitro. The targeting properties of In-111- and (68) Ga-DOTA-Z(HER2:342min) were evaluated in mice bearing SKOV-3 xenografts and compared with the properties of In-111- and (68) Ga-labeled PEP09239, a DOTA-conjugated 2-helix Affibody analogue cyclized by a homocysteine disulfide bridge. Results: The dissociation constant (K-D) for DOTA-Z(HER2:342min) binding to HER2 was 18 nM according to SPR measurements. DOTA-Z(HER2:342min) was labeled with In-111 and (68) Ga. Both conjugates demonstrated bi-phasic binding kinetics to HER2-expressing cells, with K-D1 in low nanbmolar range. Both variants demonstrated specific uptake in HER2-expressing xenografts. Tumor-to-blood ratios at 2 h p.i. were 6.1 +/- 1.3 for In-111-DOTA-Z(HER2:342min) and 4.6 +/- 0.7 for (68) Ga-DOTA-Z(HER2:342min). However, the uptake of DOTA-Z(HER2:342min) in lung, liver and spleen was appreciably higher than the uptake of PEP09239-based counterparts. Conclusions: Native chemical ligation enables production of a backbone-cyclized HER2-binding 2-helix Affibody molecule (Z(HER2:342min)) with low nanomolar target affinity and specific tumor uptake.
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| 4. |
- Järver, Peter, et al.
(author)
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Applications of cell-penetrating peptides in regulation of gene expression
- 2007
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In: Biochemical Society Transactions. - 0300-5127 .- 1470-8752. ; 35:Pt 4, s. 770-774
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Journal article (peer-reviewed)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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| 5. |
- Järver, Peter, 1978-
(author)
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Cell-penetrating peptides as delivery vectors for oligonucleotides and proteins : Studies on applications and toxicity
- 2007
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Doctoral thesis (other academic/artistic)abstract
- Cell-penetrating peptides (CPPs) have for a little bit more than a decade been employed as delivery vectors for a wide range of cargoes, ranging from gold particles to entire plasmids. Although CPP are well studied and utilized in numerous publications, our knowledge about CPP mediated transport is still poor. The articles presented in this thesis all consider different aspects of CPP mediated delivery. The first two papers are evaluating and improving already known techniques. In paper I, standard polyethyleneimine (PEI) transfection is improved by conjugating the CPP TP10 to the cationic polymer. In paper II, the same CPP is employed to deliver a dsDNA decoy oligo, resulting in decreased activity of the transcription factor c-Myc. The third paper is a more general overview of the delivery efficiency of well known CPPs and how the delivered cargo influences the CPP mediated toxicity. The study shows that different CPPs are suitable for different cargos and that toxic side effects depend heavily on the cargo and coupling strategy used. In Paper IV, a novel CPP, M918, is evaluated as a delivery vector for a transposon based non-viral gene therapy system. M918 display simultaneous delivery of a plasmid carrying a selection gene and a transposase into cultured cells. This is the first study where two so vastly different molecules as a cationic protein and an anionic plasmid, are simultaneously transported into cells by a peptide vector. The method might be a first step towards a safe peptide based non-viral gene therapy platform. Taken together, the results presented in this thesis might help to improve already existing techniques, increase our understandings about CPP mediated delivery and, at the same time, develop new CPP based delivery systems.
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| 6. |
- Järver, Peter, et al.
(author)
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Chemical synthesis and evaluation of a backbone-cyclized minimized 2-helix Z-domain
- 2011
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In: Journal of Peptide Science. - : Wiley. - 1075-2617 .- 1099-1387. ; 17:6, s. 463-469
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Journal article (peer-reviewed)abstract
- The Z-molecule is a small, engineered IgG-binding affinity protein derived from the immunoglobulin-binding domain B of Staphylococcus aureus protein A. The Z-domain consists of 58 amino acids forming a well-defined antiparallel three-helix structure. Two of the three helices are involved in ligand binding, whereas the third helix provides structural support to the three-helix bundle. The small size and the stable three-helix structure are two attractive properties comprised in the Z-domain, but a further reduction in size of the protein is valuable for several reasons. Reduction in size facilitates synthetic production of any protein-based molecule, which is beneficial from an economical viewpoint. In addition, a smaller protein is easier to manipulate through chemical modifications. By omitting the third stabilizing helix from the Z-domain and joining the N- and C-termini by a native peptide bond, the affinity protein obtains the advantageous properties of a smaller scaffold and in addition becomes resistant to exoproteases. We here demonstrate the synthesis and evaluation of a novel cyclic two-helix Z-domain. The molecule has retained affinity for its target protein, is resistant to heat treatment, and lacks both N- and C-termini. Copyright (C) 2011 European Peptide Society and John Wiley & Sons, Ltd.
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| 7. |
- Järver, Peter, et al.
(author)
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Co-transduction of Sleeping Beauty Transposase and Donor Plasmid via a Cell-penetrating Peptide : A simple one-step method
- 2008
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In: International journal of peptide research and therapeutics. - : Springer Science and Business Media LLC. - 1573-3904 .- 1573-3149. ; 14:1, s. 58-63
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Journal article (peer-reviewed)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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| 8. |
- Järver, Peter, et al.
(author)
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In vivo biodistribution and efficacy of peptide mediated delivery
- 2010
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In: TIPS - Trends in Pharmacological Sciences. - : Elsevier BV. - 0165-6147 .- 1873-3735. ; 31:11, s. 528-535
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Research review (peer-reviewed)abstract
- To transverse the plasma membrane and gain access to the cellular interior is one of the major obstacles for many novel pharmaceutical molecules. Since the late 1990 s, cell-penetrating peptides (CPPs) have been utilized as transport vectors for a broad spectrum of 'biological cargoes', ranging from inert gold particles to multifaceted macromolecules such as proteins and plasmids. Numerous studies have shown that CPPs are efficient carriers for bioactive cargoes in vitro. However, even though CPPs are versatile transport vectors, this does not guarantee they can be developed into useful pharmaceutical molecules. Nevertheless, recent progress in the field has shown CPPs to be effective for in vivo delivery with retained biological activity of a wide variety of bioactive cargoes into virtually any mammalian tissue. This review will focus on recent developments and applications for CPP delivery and distribution in vivo.
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| 9. |
- Järver, Peter, et al.
(author)
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Peptide nanoparticle delivery of charge-neutral splice-switching morpholino oligonucleotides.
- 2015
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In: Nucleic Acid Therapeutics. - : Mary Ann Liebert Inc. - 2159-3337 .- 2159-3345. ; 25:2, s. 65-77
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Journal article (peer-reviewed)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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| 10. |
- Järver, Peter, et al.
(author)
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Single-Stranded Nucleic Acids Regulate TLR3/4/7 Activation through Interference with Clathrin-Mediated Endocytosis
- 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
- 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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