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Role of autophagy in cell-penetrating peptide transfection model
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.ORCID-id: 0000-0002-6189-3020
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.ORCID-id: 0000-0002-4164-166X
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.ORCID-id: 0000-0001-7769-6905
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.ORCID-id: 0000-0003-1287-0495
Visa övriga samt affilieringar
Antal upphovsmän: 112017 (Engelska)Ingår i: Scientific Reports, E-ISSN 2045-2322, Vol. 7, artikel-id 12635Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Cell-penetrating peptides (CPPs) uptake mechanism is still in need of more clarification to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by PepFect14 (PF14), with or without oligonucleotide cargo on gene expression, in HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed by qPCR analysis. The gene regulations were then related to the biological processes by the study of signaling pathways that showed the induction of autophagy-related genes in early transfection. A ligand library interfering with the detected intracellular pathways showed concentration-dependent effects on the transfection efficiency of splice correction oligonucleotide complexed with PepFect14, proving that the autophagy process is induced upon the uptake of complexes. Finally, the autophagy induction and colocalization with autophagosomes have been confirmed by confocal microscopy and transmission electron microscopy. We conclude that autophagy, an inherent cellular response process, is triggered by the cellular uptake of CPP-based transfection system. This finding opens novel possibilities to use autophagy modifiers in future gene therapy.

Ort, förlag, år, upplaga, sidor
2017. Vol. 7, artikel-id 12635
Nationell ämneskategori
Biologiska vetenskaper
Forskningsämne
neurokemi med molekylär neurobiologi
Identifikatorer
URN: urn:nbn:se:su:diva-147911DOI: 10.1038/s41598-017-12747-zISI: 000412138800071PubMedID: 28974718OAI: oai:DiVA.org:su-147911DiVA, id: diva2:1149857
Tillgänglig från: 2017-10-17 Skapad: 2017-10-17 Senast uppdaterad: 2022-09-15Bibliografiskt granskad
Ingår i avhandling
1. Cell-Penetrating Peptides for Mitochondrial Targeting
Öppna denna publikation i ny flik eller fönster >>Cell-Penetrating Peptides for Mitochondrial Targeting
2018 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Mitochondria have simply been known as the cell’s powerhouse for a long time, with its vital function of producing ATP. However, substantially more attention was directed towards these organelles once they were recognized to perform several essential functions having an impact in cell biology, pharmaceutics and medicine. Dysfunctions of these organelles have been linked to several diseases such as diabetes, cancer, neurodegenerative diseases and cardiovascular disorders. Mitochondrial medicine emerged once the relationship of reactive oxygen species and mutations of the mitochondrial DNA linked to diseases was shown, referred to as mitochondrial dysfunction. This has led to the need to deliver therapeutic molecules in their active form not only to the target cells but more importantly into the targeted organelles.

In this thesis, cell-penetrating peptides (CPPs) used as mitochondrial drug delivery system and the pathways involved in the uptake mechanisms of a CPP are described. In particular, Paper I describes a novel cell-penetrating peptide targeting mitochondria with intrinsic antioxidant properties. Paper II expands upon this first finding and show that the same peptide can carry a glutathione analogue peptide with improved radical scavenging ability into cytoplasm and mitochondria. Paper III introduces mitochondrial targeting peptides for delivery of therapeutic biomolecules to modify mitochondrial gene expression. In Paper IV, the uptake mechanisms of the CPP delivery strategy has been investigated to gain a better understanding of the used transfection system.

Overall, this thesis summarizes our current effort regarding cell-penetrating peptides delivery system to target mitochondria and the progress made towards a potential gene therapy. It contributes to the field of CPPs and drug delivery with a set of peptides with radical scavenging ability, a strategy to deliver oligonucleotides to mitochondria as proof-of-concept for mitochondrial gene therapy, and to help understanding the pathways involved in CPPs uptake.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018. s. 62
Nyckelord
Mitochondrial targeting, cell-penetrating peptides, antioxidant activity, scavenging ability, oligonucleotide delivery
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-155156 (URN)978-91-7797-230-3 (ISBN)978-91-7797-231-0 (ISBN)
Disputation
2018-06-01, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Tillgänglig från: 2018-05-08 Skapad: 2018-04-13 Senast uppdaterad: 2022-02-26Bibliografiskt granskad
2. Uptake signalling of PepFect 14
Öppna denna publikation i ny flik eller fönster >>Uptake signalling of PepFect 14
2019 (Engelska)Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Cell-penetrating peptides are able to bind and carry various therapeutic agents including oligonucleotides into cells for a therapeutic effect. The aim of the cell-penetrating peptide research field is to produce a simple, safe and potent delivery platform for intracellular therapy and more especially for gene therapy. 

More than twenty five years after their discovery, numerous sequences of cell penetrating peptides have been designed based on natural substances, chimeric strategy or entirely synthetic products. The precise interactions leading to the uptake of cell-penetrating peptides is as of today still not entirely clear. Global mechanisms of direct penetration and endocytosis are proposed, but little is known about actual molecular interactions building the signalling pathway of cell-penetrating peptides.

In this thesis, with the help of the cell-penetrating peptide PepFect 14, we study the signalling of the uptake of cell-penetrating peptides either by transcriptome analysis or ligand interfering. We demonstrate the involvement of autophagy in the uptake of both PepFect 14 and the complex formed by PepFect 14 and oligonucleotides. We also present the use of a high throughput assay aimed at identifying new signalling pathways affected by the delivery of oligonucleotides using PepFect 14.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. s. 43
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-163437 (URN)
Presentation
2019-01-25, C458, Arrhenius Laboratory, Svante Arrhenius väg 16B, Stockholm, 13:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2019-01-17 Skapad: 2019-01-02 Senast uppdaterad: 2022-02-26Bibliografiskt granskad
3. PepFect14, a Versatile Cell-Penetrating Peptide
Öppna denna publikation i ny flik eller fönster >>PepFect14, a Versatile Cell-Penetrating Peptide
2020 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Cell-penetrating peptides have been discovered almost three decades ago and there are, nowadays, thousands of available sequences. They offer multiple applications in the field of drug delivery as they are able to carry therapeutic macromolecules across the plasma membrane. Throughout the years, new sequences have been developed and designed to achieve new applications such as specificity for certain kinds of cargoes, intrinsic therapeutic effects and targeted delivery.

In this thesis, we focused on a single most promising cell-penetrating peptide named PepFect14 and aimed at reaching a better understanding of the factors involved in the cellular uptake through paper I and paper II. Notably, in paper I we screened a library of small molecule drugs that influences signaling pathways and discovered that three drugs had an unreported influence on endocytosis. In paper II, After performing an RNA sequencing on cells treated with PepFect14, we demonstrated the involvement of autophagy in the intracellular trafficking of the cell-penetrating peptide. A second aim of this thesis, covered in paper III and paper IV, was to discover new applications for PepFect14 in order to broaden its potential. In paper III, we successfully used PepFect14 to mediate the intracellular delivery of heat shock protein 70kDa. This was the first protein delivery assisted by PepFect14. In paper IV, PepFect14 was covalently fused to mtCPP1, a cell-penetrating peptide that targets mitochondria and reduce the level of reactive oxygen species. The constructs showed the ability to keep the properties of both peptides and achieved a mitochondria-targeted antisense therapy.

Overall, this thesis summarizes our effort to develop and bring to their full potential already existing cell-penetrating peptides instead of developing new sequences for each new application.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2020. s. 75
Nyckelord
Cell-Penetrating Peptides, PepFect14, transfection, signaling mechanisms, intracellular targeting
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-180948 (URN)978-91-7911-110-6 (ISBN)978-91-7911-111-3 (ISBN)
Disputation
2020-06-10, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Tillgänglig från: 2020-05-18 Skapad: 2020-04-22 Senast uppdaterad: 2022-02-26Bibliografiskt granskad
4. Chimeric gene delivery vectors: Design, synthesis, and mechanisms from transcriptomics analysis
Öppna denna publikation i ny flik eller fönster >>Chimeric gene delivery vectors: Design, synthesis, and mechanisms from transcriptomics analysis
2018 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Delivery of nucleic acid is a promising approach for genetic diseases/disorders. However, gene therapy using oligonucleotides (ONs) suffers from low transfection efficacy due to negative charges, weak cellular permeability, and enzymatic degradation. Thus, cell-penetrating peptide (CPP), is a short cationic peptide, is used to improve the cell transfection. In this thesis, new strategies for gene transfection using the CPP vectors in complex with ONs without and with nanoparticles, such as magnetic nanoparticles (MNPs, Fe3O4), and graphene oxide (GO), are investigated. Furthermore, the possible CPP uptake signalling pathways are also discussed.

A fragment quantitative structure-activity relationship (FQSAR) model is applied to predict new effective peptides for plasmid DNA transfection. The best-predicted peptides were able to transfect plasmids with significant enhancement compared to the other peptides. CPPs (PeptFect220 (denoted PF220), PF221, PF222, PF223, PF224) generated from the FQSAR, and standard PF14 were able to form self-assembled complexes with MNPs and GO. The formed new hybrid vectors improved the cell transfection for plasmid (pGL3), splicing correcting oligonucleotides (SCO), and small interfering RNA (siRNA). These vectors showed high cell biocompatibility and offered high transfection efficiency (> 4-fold for MNPs, 10–25-fold for GO) compared to PF14/SCO complex, which was before reported with a higher efficacy compared to the commercial lipid-based transfection vector Lipofectamine™2000. The high transfection efficiency of the novel complexes (CPP/ON/MNPs and CPP/ON/GO) may be due to their low cytotoxicity, and the synergistic effect of MNPs, GO, and CPPs. In vivo gene delivery using PF14/pDNA/MNPs was also reported. The assembly of CPPs/ON with MNPs or GO is promising and may open new venues for potent and selective gene therapy using external stimuli. The uptake signaling pathways using CPPs vectors, the RNA expression profile for PF14, with and without ON were investigated using RNA sequencing and qPCR analysis. Data showed that the signaling pathways are due to the regulation of autophagy-related genes. Our study revealed that the autophagy regulating proteins are concentration-dependent. Confocal microscopy and transmission electron microscopy have demonstrated the autophagy initiation and colocalization of ON with autophagosomes. Results showed that the cellular uptake of CPP-based transfection activates the autophagy signaling pathway. These findings may open new opportunities to use autophagy modifiers in gene therapy.

Abstract [sv]

Genterapi med hjälp av av oligonukleotider (ON) har en enorm potential för behandling av olika genetiska sjukdomar. För att ha terapeutisk effekt måste dock oligonukleotiderna nå in i cellen och detta försvåras på grund av deras negativa laddningar och snabba nedbrytning. Cellpenetrerande peptider (CPP), är korta katjoniska peptider, som kan användas för att förbättra det cellulära upptaget (transfektionen) av oligonukleotider. I denna avhandling undersöks nya strategier för hur CPP tillsammans med magnetiska nanopartiklar, såsom MNP och Fe3O4, eller grafenoxid (GO) nanopartiklar, kan möjliggöra effektivare transfektion av ON.  Vidare studeras även de möjliga cellulära signalvägar som reglerar CPP-medierat upptag.

En så kallad ”fragment quantitative structure-activity relationship” (FQSAR) modell  användes för att förutsäga nya effektiva CPP för leverans av plasmider (ringformade DNA-molekyler med omkring 5000 nukleotidbaspar). De bäst prediktade peptiderna visade en signifikant ökad transfektionsförmåga jämfört med den tidigare använda peptiden PeptFect 14 (PF14). De nya peptiderna PF220, PF221, PF222, PF223 och PF224 som identifierades med FQSAR kunde dessutom bilda självmonterande komplex med MNP eller GO nanopartiklar. I cellulära försök uppvisade dessa nya hybridvektorer (CPP/MNP och CPP/GO) en klart förbättrad transfektionsförmåga av såväl plasmider, som splitsningskorrigerande oligonukleotider (SCO) och små interfererande RNA (siRNA), jämfört med PF14-nanopartikel hybridvektorer, såväl som den kommersiella lipidbaserade transfektionsvektorn Lipofectamine™ 2000. Den höga transfektionseffektiviteten hos dessa nya hybridvektorer beror troligen på deras låga cellulära toxicitet och en möjlig synergistisk effekt vid kombinationen av CPP och MNP/GO nanopartiklar. Förmågan hos en CPP/MNP hybridvektor att levera plasmider in vivo undersöktes också och transfektion av celler i såväl lunga och mjälte i behandlade djur kunde påvisas. Dessa nya hybridvektorer utgör således en ny lovande strategi för leverans av ON vid genterapi.

För att kartlägga de signalvägar som kontrollerar upptaget av CPP-baserade vektorer analyserades  genuttrycket hos celler som transfekterats med PF14 eller PF14-ON, med hjälp av  RNA-sekvensering och qPCR-analys. Resultaten påvisade att en ökning i uttrycket av flera autofagirelaterade gener sker tidigt vid transfektionen. Konfokal- och transmissionselektronmikroskopi demonstrerade vidare en ökad initiering av autofagi och samlokalisering av ON med autofagosomer. Detta visar att CPP-medierad transfektion aktiverar signalvägar som stryr autofagi och öppnar nya möjligheter att använda autofagimodifierare för att förbättra genterapi.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018
Nyckelord
Cell-penetrating peptides, magnetic nanoparticles, graphene oxide, autophagy, siRNA, SCO, QSAR
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-158574 (URN)978-91-7797-420-8 (ISBN)978-91-7797-421-5 (ISBN)
Disputation
2018-10-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16B, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2018-09-18 Skapad: 2018-08-27 Senast uppdaterad: 2022-02-26Bibliografiskt granskad
5. In-silico design of peptide-based transfection systems, in-vitro validation, and up-take pathways investigation
Öppna denna publikation i ny flik eller fönster >>In-silico design of peptide-based transfection systems, in-vitro validation, and up-take pathways investigation
2017 (Engelska)Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Cell-penetrating peptide-based transfection systems (PBTS) are a promising group of drug delivery vectors. Cell-penetrating peptides (CPPs) are short cationic peptides that are able of transporting cell non-permeant cargos into different cell types. Some CPPs can be used to form non-covalent complexes with oligonucleotides for gene delivery applications. For the potential use of CPPs as drug delivery tools, it is important to understand the mechanism of uptake. Here, a fragment quantitative structure–activity relationships (FQSAR) model is generated to predict novel peptides based on approved alpha helical conformers and assisted model construction with energy refinement molecular mechanics simulations of former peptides. The modeled peptides were examined for plasmid transfection efficiency and compared with their predicted biological activity. The best predicted peptides were efficient for plasmid transfection with significant enhancement compared to the former group of peptides. Our results confirm that FQSAR model refinement is an efficient method for optimizing PBTS for improved biological activity. Additionally, using RNA sequencing, we demonstrated the involvement of autophagy pathways in PBTS uptake.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Neurochemistry, Stockholm University, 2017. s. 50
Nyckelord
Cell-penetrating peptides, QSAR, PepFect
Nationell ämneskategori
Kemi Biokemi och molekylärbiologi
Forskningsämne
neurokemi med molekylär neurobiologi
Identifikatorer
urn:nbn:se:su:diva-140139 (URN)
Presentation
2017-03-15, Heilbronnsalen, C458, Svante Arrhenius väg 16B, Stockholm, 14:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2017-02-28 Skapad: 2017-02-28 Senast uppdaterad: 2022-02-28

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Dowaidar, MoatazGestin, MaximeCerrato, Carmine PasqualeJafferali, Mohammed HakimHallberg, EinarHällbrink, MattiasLangel, Ülo

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