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Splice-switching therapy with splice-switching oligonucleotides (SSOs) has recently proven to be a clinically applicable strategy for the treatment of several mis-splice disorders. Despite this, wider application of SSOs is severely limited by the inherently poor bioavailability of SSO-based therapeutic compounds. Cell-penetrating peptides (CPPs) are a class of drug delivery systems (DDSs) that have recently gained considerable attention for improving the uptake of various oligonucleotide (ON)-based compounds, including SSOs. One strategy that has been successfully applied to develop effective CPP vectors is the introduction of various lipid modifications into the peptide. Here, we repurpose hydrocarbon-modified amino acids used in peptide stapling for the orthogonal introduction of hydrophobic modifications into the CPP structure during peptide synthesis. Our data show that α,α-disubstituted alkenyl-alanines can be successfully utilized to introduce hydrophobic modifications into CPPs to improve their ability to formulate SSOs into nanoparticles (NPs), and to mediate high delivery efficacy and tolerability both in vitro and in vivo. Conclusively, our results offer a new flexible approach for the sequence-specific introduction of hydrophobicity into the structure of CPPs and for improving their delivery properties.

The toolbox for genetic engineering has quickly evolved from CRISPR/Cas9 to a myriad of different gene editors, each with promising properties and enormous clinical potential. However, a major challenge remains: delivering the CRISPR machinery to the nucleus of recipient cells in a nontoxic and efficient manner. In this article, we repurpose an RNA-delivering cell-penetrating peptide, PepFect14 (PF14), to deliver Cas9 ribonucleoprotein (RNP). The RNP-CPP complex achieved high editing rates, e.g., up to 80% in HEK293T cells, while being active at low nanomolar ranges without any apparent signs of toxicity. The editing efficiency was similar to or better compared to the commercially available reagents RNAiMAX and CRISPRMax. The efficiency was thoroughly evaluated in reporter cells and wild-type cells by restriction enzyme digest and next-generation sequencing. Furthermore, the CPP-Cas9-RNP complexes were demonstrated to withstand storage at different conditions, including freeze-thaw cycles and freeze-drying, without a loss in editing efficiency. This CPP-based delivery strategy complements existing technologies and further opens up new opportunities for Cas9 RNP delivery, which can likely be extended to other gene editors in the future.

Antisense oligonucleotides (ASOs) are a promising class of therapeutics that are starting to emerge in the clinic. Determination of intracellular concentrations required for biologic effects and identification of effective delivery vehicles are crucial for understanding the mode of action and required dosing. Here, we investigated which nuclear oligonucleotide concentration is needed for a therapeutic effect for a triplet repeat-targeting ASO in a muscle cell model of myotonic dystrophy type 1 (DM1). For cellular delivery, ASOs were complexed into nanoparticles using the cationic cell-penetrating peptides nona-arginine and PepFect14 (PF14). Although both peptides facilitated uptake, only PF14 led to a dose-dependent correction of disease-typical abnormal splicing. In line with this observation, time-lapse confocal microscopy demonstrated that only PF14 mediated translocation of the ASOs to the nucleus, which is the main site of action. Through fluorescence lifetime imaging, we could distinguish intact oligonucleotide from free fluorophore, showing that PF14 also shielded the ASOs from degradation. Finally, we employed a combination of live-cell fluorescence correlation spectroscopy and immunofluorescence microscopy and demonstrated that intranuclear blocking-type oligonucleotide concentrations in the upper nanomolar range were required to dissolve nuclear muscleblind-like protein 1 foci, a hallmark of DM1. Our findings have important implications for the clinical use of ASOs in DM1 and provide a basis for further research on other types of ASOs.-Van der Bent, M. L., Paulino da Silva Filho, O., Willemse, M., Hällbrink, M., Wansink, D. G., Brock, R. The nuclear concentration required for antisense oligonucleotide activity in myotonic dystrophy cells.

Ovarian cancer is the most lethal gynecological malignancy in the developed world. In spite of intensive research, the mortality has hardly decreased over the past twenty years. This necessitates the exploration of novel therapeutic modalities. Transient protein expression through delivery of mRNA is emerging as a highly promising option. In contrast to gene therapy there is no risk of integration into the genome. Here, we explore the expression of mRNA in models of ovarian cancer of increasing complexity. The cell-penetrating peptide (CPP) PepFect 14 (PF14) was used to formulate CPP-mRNA nanoparticles. Efficient expression of a reporter protein was achieved in two-dimensional tissue cultures and in three-dimensional cancer cell spheroids. PF14 nanoparticles greatly outperformed a lipid-based transfection agent in vivo, leading to expression in various cell types of tumor associated tissue. Protein expression was restricted to the peritoneal cavity. Messenger RNA expression across different cell types was confirmed in primary ovarian cancer explants. As ovarian cancer is confined to the peritoneal cavity in most cases, the results create the basis for applications in which the tumor microenvironment is transiently modified through protein expression., (Copyright © 2019. Published by Elsevier B.V.)

Gene-based therapies, including the delivery of oligonucleotides, offer promising methods for the treatment of cancer cells. However, they have various limitations including low efficiency. Herein, cell-penetrating peptides (CPPs)-conjugated chitosan-modified iron oxide magnetic nanoparticles (CPPs-CTS@MNPs) with high biocompatibility as well as high efficiency were tested for the delivery of oligonucleotides such as plasmid pGL3, splice correction oligonucleotides, and small-interfering RNA. A biocompatible nanocomposite, in which CTS@MNPs was incorporated in non-covalent complex with CPPs-oligonucleotide, is developed. Modifying the surface of magnetic nanoparticles with cationic chitosan-modified iron oxide improved the performance of magnetic nanoparticles-CPPs for oligonucleotide delivery. CPPs-CTS@MNPs complexes enhance oligonucleotide transfection compared to CPPs@MNPs or CPPs. The hydrophilic character of CTS@MNPs improves complexation with plasmid pGL3, splice correction oligonucleotides, and small-interfering RNA payload, which consequently resulted in not only strengthening the colloidal stability of the constructed complex but also improving their biocompatibility. Transfection using PF14-splice correction oligonucleotides-CTS@MNPs showed sixfold increase of the transfection compared to splice correction oligonucleotides-PF14 that showed higher transfection than the commercially available lipid-based vector Lipofectamine™ 2000. Nanoscaled CPPs-CTS@MNPs comprise a new family of biomaterials that can circumvent some of the limitations of CPPs or magnetic nanoparticles.

Delivery of small interfering RNA (siRNA) to suppress glioblastoma growth is a hurdle due to the critical obstacles of the blood-brain barrier and the siRNA properties of such as high negative charges and instability in serum. Therefore, the passage of siRNA to targeted cells is limited. Several siRNA carriers have been constructed using cell-penetrating peptides (CPPs) since the CPPs have shown a high potential for oligonucleotide delivery into the cells. In this study, two CPPs, PepFect 14 (PF14) and the amphipathic peptide PepFect 28 (PF28), were modified with targeting peptides by covalent conjugation and non-covalent complex formation to improve glioma-targeted specificity and gene-silencing efficiency. In conclusion, we have established an efficient non-covalently complexed carrier (PF14:TG1) for siRNA delivery to human glioblastoma cells (U87), showing a significant two-fold increase in gene-silencing efficiency compared to the parent peptide PF14 and also improved specificity to U87 cells compared to non-glioma targeted cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

A new strategy for gene transfection using the nanocarrier of cell penetrating peptides (CPPs; PepFect14 (PF14) or PepFect14 (PF14) (PF221)) in complex with graphene oxide (GO) is reported. GO complexed with CPPs and plasmid (pGL3), splice correction oligonucleotides (SCO) or small interfering RNA (siRNA) are performed. Data show adsorption of CPPs and oligonucleotides on the top of the graphenic lamellar without any observed change of the particle size of GO. GO mitigates the cytotoxicity of CPPs and improves the material biocompatibility. Complexes of GO-pGL3-CPPs (CPPs; PF14 or PF221) offer 2.1-2.5 fold increase of the cell transfection compared to pGL3-CPPs (CPPs; PF14 or PF221). GO-SCO-PF14 assemblies effectively transfect the cells with an increase of >10-25 fold compared to the transfection using PF14. The concentration of GO plays a significant role in the material nanotoxicity and the transfection efficiency. The results open a new horizon in the gene treatment using CPPs and offer a simple strategy for further investigations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Magnetic nanoparticles (MNPs, Fe 3 O 4 ) incorporated into the complexes of cell penetrating peptides (CPPs)-oligonucleotides (ONs) promoted the cell transfection for plasmid transfection, splice correction, and gene silencing efficiencies. Six types of cell penetrating peptides (CPPs; PeptFect220 (denoted PF220), PF221, PF222, PF223, PF224 and PF14) and three types of gene therapeutic agents (plasmid (pGL3), splicing correcting oligonucleotides (SCO), and small interfering RNA (siRNA) were investigated. Magnetic nanoparticles incorporated into the complexes of CPPs-pGL3, CPPs-SCO, and CPPs-siRNA showed high cell biocompatibility and efficiently transfected the investigated cells with pGL3, SCO, and siRNA, respectively. Gene transfer vectors formed among PF14, SCO, and MNPs (PF14-SCO-MNPs) showed a superior transfection efficiency (up to 4-fold) compared to the noncovalent PF14-SCO complex, which was previously reported with a higher efficiency compared to commercial vector called Lipofectamine™2000. The high transfection efficiency of the new complexes (CPPs-SCO-MNPs) may be attributed to the morphology, low cytotoxicity, and the synergistic effect of MNPs and CPPs. PF14-pDNA-MNPs is an efficient complex for in vivo gene delivery upon systemic administration. The conjugation of CPPs-ONs with inorganic magnetic nanoparticles (Fe 3 O 4 ) may open new venues for selective and efficient gene therapy.

Huntington's disease (HD) is a fatal, neurodegenerative disorder in which patients suffer from mobility, psychological and cognitive impairments. Existing therapeutics are only symptomatic and do not significantly alter the disease progression or increase life expectancy. HD is caused by expansion of the CAG trinucleotide repeat region in exon 1 of the Huntingtin gene (HTT), leading to the formation of mutant HTT transcripts (muHTT). The toxic gain-of-function of muHTT protein is a major cause of the disease. In addition, it has been suggested that the muHTT transcript contributes to the toxicity. Thus, reduction of both muHTT mRNA and protein levels would ideally be the most useful therapeutic option. We herein present a novel strategy for HD treatment using oligonucleotides (ONs) directly targeting the HTT trinucleotide repeat DNA. A partial, but significant and potentially long-term, HTT knock-down of both mRNA and protein was successfully achieved. Diminished phosphorylation of HTT gene-associated RNA-polymerase II is demonstrated, suggestive of reduced transcription downstream the ON-targeted repeat. Different backbone chemistries were found to have a strong impact on the ON efficiency. We also successfully use different delivery vehicles as well as naked uptake of the ONs, demonstrating versatility and possibly providing insights for in vivo applications., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Modifying cell-penetrating peptides (CPPs) with fatty acids has long been used to improve peptide-mediated nucleic acid delivery. In this study we have revisited this phenomenon with a systematic approach where we developed a structure-activity relationship to describe the role of the acyl chain length in the transfection process. For that we took a well-studied CPP, PepFect14, as the basis and varied its N-terminal acyl chain length from 2 to 22 carbons. To evaluate the delivery efficiency, the peptides were noncovalently complexed with a splice-correcting oligonucleotide (SCO) and tested in HeLa pLuc705 reporter cell line. Our results demonstrate that biological splice-correction activity emerges from acyl chain of 12 carbons and increases linearly with each additional carbon. To assess the underlying factors regarding how the transfection efficacy of these complexes is dependent on hydrophobicity, we used an array of different methods. For the functionally active peptides (C12-22) there was no apparent difference in their physicochemical properties, including complex formation efficiency, hydrodynamic size, and zeta potential. Moreover, membrane activity studies with peptides and their complexes with SCOs confirmed that the toxicity of the complexes at higher molar ratios is mainly caused by the free fraction of the peptide which is not incorporated into the peptide/oligonucleotide complexes. Finally, we show that the increase in splice-correcting activity correlates with the ability of the complexes to associate with the cells. Collectively these studies lay the ground work for how to design highly efficient CPPs and how to optimize their oligonucleotide complexes for lowest toxicity without losing efficiency.

A series of cell-penetrating PepFect peptide analogues was developed by substitutions of the galanin-derived N-terminal sequence. Histidine modifications were incorporated in order to make the peptides pH-responsive. The peptides were all able to form non-covalent complexes with an oligonucleotide cargo by co-incubation in buffer. The complexes were characterized by dynamic light scattering and circular dichroism, and an assay to evaluate the peptide-cargo affinity was developed. Cellular bioactivity was studied in HeLa cells using a luciferase-based splice correction assay. In addition, the membrane interactions of the peptides in large unilammelar vesicles was studied using a calcein leakage assay. The effects of substitutions were found to be dependent of the non-modified, C-terminal sequence of the peptides; for analogues of PepFect 3 we observed an increase in membrane activity and bioactivity for histidine-containing analogues, whereas the same modifications introduced to PepFect 14 lead to a decreased bioactivity. Peptides modified with a leucine/histidine sequence were found to be pH responsive, complexes formed from these peptides were small at pH 7 and grew under acidic conditions. The most promising of the novel PepFect 3 analogues, PepFect 132 has a significantly higher bioactivity and membrane activity than the parent peptide PepFect 3., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Receptor-mediated transcytosis remains a major route for drug delivery across the blood-brain barrier (BBB). PepFect 32 (PF32), a peptide-based vector modified with targeting ligand (Angiopep-2) binding to low-density lipoprotein receptor-related protein-1 (LRP-1), was previously found to be a promising vector for plasmid delivery across an in vitro model of the BBB. Cellular uptake of PF32/plasmid DNA (pDNA) complexes was speculated the internalization via LRP-1 receptor. In this study, we prove that PF32/pDNA nanocomplexes are not only transported into brain endothelial cells via LRP-1 receptor-mediated endocytosis, but also via scavenger receptor class A and B (SCARA3, SCARA5, and SR-BI)-mediated endocytosis. SCARA3, SCARA5, and SR-BI are found to be expressed in the brain endothelial cells. Inhibition of these receptors leads to a reduction of the transfection. In conclusion, this study shows that scavenger receptors also play an essential role in the cellular uptake of the PF32/pDNA nanocomplexes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.

Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs., From the Clinical Editor: Recent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Nucleic acids can be utilized in gene therapy to restore, alter, or silence gene functions. In order to reveal the biological activity nucleic acids have to reach their intracellular targets by passing through the plasma membrane, which is impermeable for these large and negatively charged molecules. Cell-penetrating peptides (CPPs) condense nucleic acids into nanoparticles using non-covalent complexation strategy and mediate their delivery into the cell, whereas the physicochemical parameters of the nanoparticles determine the interactions with the membranes, uptake mechanism, and subsequent intracellular fate. The nanoparticles are mostly internalized by endocytosis that leads to the entrapment of them in endosomal vesicles. Therefore design of new CPPs that are applicable for non-covalent complex formation strategy and harness endosomolytic properties is highly vital. Here we demonstrate that PepFects and NickFects are efficient vectors for the intracellular delivery of various nucleic acids.This chapter describes how to form CPP/pDNA nanoparticles, evaluate stable nanoparticles formation, and assess gene delivery efficacy.

The in silico methods for the prediction of the cell-penetrating peptides are reviewed. Those include the multivariate statistical methods, machine-learning methods such as the artificial neural networks and support vector machines, and molecular modeling techniques including molecular docking and molecular dynamics.The applicability of the methods is demonstrated on the basis of the exemplary cases from the literature.

Cell-penetrating peptides (CPPs) are versatile tools for the intracellular delivery of various biomolecules, including siRNA. Recently, CPPs were introduced that showed greatly enhanced delivery efficiency. However, the molecular basis of this increased activity is poorly understood. Here, we performed a detailed analysis of the molecular and physicochemical properties of seven different siRNA-CPP nanoparticles. In addition, we determined which complexes are internalized most efficiently into the leukemia cell-line SKNO-1, and subsequently inhibited the expression of a luciferase reporter gene. We demonstrated effective complexation of siRNA for all tested CPPs, and optimal encapsulation of the siRNA was achieved at very similar molar ratios independent of peptide charge. However, CPPs with an extreme high or low overall charge proved to be exceptions, suggesting an optimal range of charge for CPP-siRNA nanoparticle formation based on opposite charge. The most active CPP (PepFect6) displayed high serum resistance but also high sensitivity to decomplexation by polyanionic macromolecules, indicating the necessity for partial decomplexation for efficient uptake. Surprisingly, CPP-siRNA complexes acquired a negative ζ-potential in the presence of serum. These novel insights shed light on the observation that cell association is necessary but not sufficient for activity and motivate new research into the nature of the nanoparticle-cell interaction. Overall, our results provide a comprehensive molecular basis for the further development of peptide-based oligonucleotide transfection agents.

Cell-penetrating peptides (CPPs) promote the uptake of different cargo molecules, e.g. therapeutic compounds, making the harnessing of CPPs a promising strategy for drug design and delivery. However, the internalization mechanisms of CPPs are still under discussion, and it is not clear how cells compensate the disturbances induced by peptides in the plasma membrane. In this study, we demonstrate that the uptake of various CPPs enhances the intracellular Ca(2+) levels in Jurkat and HeLa cells. The elevated Ca(2+) concentration in turn triggers plasma membrane blebbing, lysosomal exocytosis, and membrane repair response. Our results indicate that CPPs split into two major classes: (i) amphipathic CPPs that modulate the plasma membrane integrity inducing influx of Ca(2+) and activating downstream responses starting from low concentrations; (ii) non-amphipathic CPPs that do not evoke changes at relevant concentrations. Triggering of the membrane repair response may help cells to replace distorted plasma membrane regions and cells can recover from the influx of Ca(2+) if its level is not drastically elevated.

The use of protease-resistant D-peptides is a prominent strategy for overcoming proteolytic sensitivity in the use of cell-penetrating peptides (CPPs) as delivery vectors. So far, no major differences have been reported for the uptake of L- and D-peptides. Here we report that cationic L-CPPs are taken up more efficiently than their D-counterparts in MC57 fibrosarcoma and HeLa cells but not in Jurkat T leukemia cells. Reduced uptake of D-peptides co-occurred with persistent binding to heparan sulfates (HS) at the plasma membrane. In vitro binding studies of L- and D-peptides with HS indicated similar binding affinities. Our results identify two key events in the uptake of CPPs: binding to HS chains and the initiation of internalization. Only the second event depends on the chirality of the CPP. This knowledge may be exploited for a stereochemistry-dependent preferential targeting of cells., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

As more and more studies utilize cell-penetrating peptides to deliver pharmacologically interesting substances, there is a growing need to understand their effect on the plasma membrane. If a cell-penetrating peptide together with its cargo is to be used as a drug, it is necessary to understand how the conjugate interacts with the plasma membrane to enter the cell. A key regulator of the transportation network in the cell is calcium. This chapter describes five methods that can be employed for understanding how the plasma membrane reacts to the presence of cell-penetrating peptides and the involvement of calcium.

Despite increasing interest in cell-penetrating peptides (CPPs) as carriers for drugs and in gene therapy, the current understanding of their exact internalization mechanism is still far from complete. The cellular translocation of CPPs and their payloads has been mostly described by fluorescence- and activity-based methods, leaving the more detailed characterization at the ultrastructural level almost out of attention. Herein, we used transmission electron microscopy to characterize the membrane interaction and internalization of a cell-penetrating peptide S4(13)-PV. We demonstrate that S4(13)-PV peptide forms spherical nanoparticle-like regular structures upon association with cell surface glycosaminoglycans on the plasma membrane. Insertion of S4(13)-PV particles into plasma membrane induces disturbances and leads to the vesicular uptake of peptides by cells. We propose that for efficient cellular translocation S4(13)-PV peptides have to assemble into particles of specific size and shape. The spherical peptide particles are not dissociated in intracellular vesicles but often retain their organization and remain associated with the membrane of vesicles, destabilizing them and promoting the escape of peptides into cytosol. Lowering the temperature and inhibition of dynamins' activity reduce the internalization of S4(13)-PV peptides, but do not block it completely. Our results provide an ultrastructural insight into the interaction mode of CPPs with the plasma membrane and the distribution in cells, which might help to better understand how CPPs cross the biological membranes and gain access into cells.

Although cell-penetrating peptides are able to deliver cargo into cells, their uptake mechanism is still not fully understood and needs to be elucidated to improve their delivery efficiency. Herein, we present evidence of a new mechanism involved in uptake, the membrane repair response. Recent studies have suggested that there might be a direct penetration of peptides in parallel with different forms of endocytosis. The direct penetration of hydrophilic peptides through the hydrophobic plasma membrane, however, is highly controversial. Three proteins involved in target cell apoptosis--perforin, granulysin, and granzymes--share many features common in uptake of cell-penetrating peptides (e.g., they bind proteoglycans). During perforin uptake, the protein activates the membrane repair response, a resealing mechanism triggered in cells with injured plasma membrane, because of extracellular calcium influx. On activation of the membrane repair response, internal vesicles are mobilized to the site of the disrupted plasma membrane, resealing it within seconds. In this study, we have used flow cytometry, fluorescence, and electron microscopy, together with high-performance liquid chromatography and mass spectrometry, to present evidence that the membrane repair response is able to mask damages caused during cell-penetrating peptide uptake, thus preventing leakage of endogenous molecules out of the cell.

The production of bacterial ghosts involves the lysis gene E plasmid in order to lyse and empty the bacteria of their cytoplasmic contents. After lysis the ghosts can either be loaded with new desired DNA and used for delivery to mammalian cells or used in vaccination. Cell-penetrating peptides have been used as delivery vehicles of drugs and oligonucleotides. Although many of them show low toxicity they have been compared to antimicrobial peptides involved in innate immunity. Recently we showed that cell-penetrating peptides also could be antimicrobial. In this study we take advantage of the antimicrobial effect of one cell-penetrating peptide, namely MAP, which is a model amphipathic peptide and treat bacteria with the peptide to produce bacterial ghosts. This new peptide based strategy is not dependent on the lysis gene E plasmid thus; several tiresome steps are removed in the production of ghosts. In addition the ghosts can be preloaded with a desired plasmid or DNA further removing time consuming reprocessing steps. To our knowledge this is the first study that uses a cell-penetrating peptide based strategy to produce bacterial ghosts to be used in plasmid delivery.

Cell-penetrating peptide mediated uptake of labels appears to follow an equilibrium-like process. However, this assumption is only valid if the peptides are stabile. Hence, in this study we investigate intracellular and extracellular peptide degradation kinetics of two fluorescein labeled cell-penetrating peptides, namely MAP and penetratin, in Chinese hamster ovarian cells. The degradation and uptake kinetics were assessed by RP-HPLC equipped with a fluorescence detector. We show that MAP and penetratin are rapidly degraded both extracellularly and intracellularly giving rise to several degradation products. Kinetics indicates that intracellularly, the peptides exist in (at least) two distinct pools: one that is immediately degraded and one that is stabile. Moreover, the degradation could be decreased by treating the peptides with BSA and phenanthroline and the uptake was significantly reduced by cytochalasin B, chloroquine and energy depletion. The results indicate that the extracellular degradation determines the intracellular peptide concentration in this system and therefore the stability of cell-penetrating peptides needs to be evaluated.

Cell-penetrating peptides (CPPs) are carriers developed to improve mammalian cell uptake of important research tools such as antisense oligonucleotides and short interfering RNAs. However, the data on CPP uptake into non-mammalian cells are limited. We have studied the uptake and antimicrobial effects of the three representative peptides penetratin (derived from a non-mammalian protein), MAP (artificial peptide) and pVEC (derived from a mammalian protein) using fluorescence HPLC in four common model systems: insect cells (Sf9), gram-positive bacteria (Bacillus megaterium), gram-negative bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). We demonstrate that non-mammalian cells internalize CPPs and a comparison of the uptake of the peptides show that the intracellular concentration and degradation of the peptides varies widely among organisms. In addition, these CPPs showed antimicrobial activity.

Resistance to chemotherapy limits the effectiveness of anti-cancer drug treatment. Here, we present a new approach to overcome the setback of drug resistance by designing a conjugate of a cell-penetrating peptide and the cytostatic agent methotrexate (MTX). Two different peptides, YTA2 and YTA4, were designed and their intracellular delivery efficiency was characterized by fluorescence microscopy and quantified by fluorometry. MTX was conjugated to the transport peptides and the ability of the peptide-MTX conjugates to inhibit dihydrofolate reductase, the target enzyme of MTX, was found to be 15 and 20 times less potent than MTX. In addition, in vitro studies were performed in a drug resistant cell model using the 100-fold MTX resistant breast cancer cells MDA-MB-231. At a concentration of 1 microM, the peptide-MTX conjugates were shown to overcome MTX resistance and kill the cells more efficiently than MTX alone. Estimated EC50's were determined for MTX, MTX-YTA2 and YTA2 to be 18.5, 3.8 and 20 microM, respectively. In summary, cell-penetrating peptide conjugation of MTX is a new way of increasing delivery, and thereby, the potency of already well-characterized therapeutic molecules into drug resistant tumour cells.

The uptake of different cell-penetrating peptides (CPPs) in two yeast species, Saccharomyces cerevisiae and Candida albicans, was studied using fluorescence HPLC-analyses of cell content. Comparison of the ability of penetratin, pVEC and (KFF)(3)K to traverse the yeast cell envelope shows that the cellular uptake of the peptides varies widely. Moreover, the intracellular degradation of the CPPs studied varies from complete stability to complete degradation. We show that intracellular degradation into membrane impermeable products can significantly contribute to the fluorescence signal. pVEC displayed highest internalizing capacity, and considering its stability in both yeast species, it is an attractive candidate for further studies.

The influence of the peptide-to-cell ratio and energy depletion on uptake and degradation of the cell-penetrating peptides (CPPs) MAP (model amphipathic peptide) was investigated. The intracellular concentration of the CPPs, MAP and penetratin was monitored while varying the number of cells at fixed peptide concentration and incubation volume, or changing the concentration and incubation volume at fixed cell number. The uptake of CPPs was shown to be dependent on the peptide/cell ratio. At given peptide concentration and incubation volume, the intracellular concentration of peptide increased with lower cell number. At given cell number, doubling of the incubation volume increased intracellular peptide concentration to a similar extent as the doubling in incubation concentration. From a practical view, this means that the peptide/cell ratio has at least the same importance for the uptake of CPPs as the used peptide concentration. No influence of the peptide/cell ratio was found for the cellular uptake of peptide nucleic acid (PNA), or a non-amphipathic MAP analogue, investigated in parallel for comparison purposes. Energy depletion resulted in significantly reduced quantities of intracellular fluorescence label. Moreover, we show that this difference is mainly due to a membrane-impermeable fluorescent-labelled degradation product, which is lacking in energy-depleted cells. The mechanism of its generation is not likely to be endosomal degradation of endocytosed material, as it is not chloroquine- or brefeldin-sensitive.

Antimicrobial drug action is limited by both microbial and host cell membranes. Microbes stringently exclude the entry of most drugs, and mammalian membranes limit drug distribution and access to intracellular pathogens. Recently, cell-penetrating peptides (CPPs) have been developed as carriers to improve mammalian cell uptake. Given that CPPs are cationic and often amphipathic, similar to membrane active antimicrobial peptides, it may be possible to use CPP activity to improve drug delivery to microbes. Here, two CPPs, TP10 and pVEC, were found to enter a range of bacteria and fungi. The uptake route involves rapid surface accumulation within minutes followed by cell entry. TP10 inhibited Candida albicans and Staphylococcus aureus growth, and pVEC inhibited Mycobacterium smegmatis growth at low micromolar doses, below the levels that harmed human HeLa cells. Therefore, although TP10 and pVEC entered all cell types tested, they preferentially damage microbes, and this effect was sufficient to clear HeLa cell cultures from noninvasive S. aureus infection. Also, conversion of the cytotoxicity indicator dye SYTOX Green showed that TP10 causes rapid and lethal permeabilization of S. aureus and pVEC permeabilizes M. smegmatis, but not HeLa cells. Therefore, TP10 and pVEC can enter both mammalian and microbial cells and preferentially permeabilize and kill microbes.

Previous studies revealed the importance of the third intracellular loop of glucagon-like peptide-1 receptor (GLP-1R) in coupling to G(s) and G(i1) proteins. In order to further study the signaling mechanisms of GLP-1R, we tested three peptides, corresponding to the sequences of the first (IC(1)), the second (IC(2)), and the third (IC(3)) intracellular loop of GLP-1R, for their interactions with heterotrimeric G-proteins of different types (G(alphas), G(alphao), G(alphai1), and G(alpha11) plus G(beta1gamma2)) overexpressed in sf9 cells. IC(3) peptide powerfully stimulates all types of tested G-proteins, whereas IC(1) and IC(2) peptides show differential effects on G-proteins. Both IC(1) and IC(2) peptides activate G(s) and cooperate with IC(3) peptide in its stimulation. G(o) is not affected by IC(1) and IC(2). G(i1) and G(11) are not affected by IC(1), but are activated by IC(2), which in activation cooperates with IC(3). We suggest that GLP-1R is not coupled only to G(s) and G(i1), as shown previously, but also to G(o) and G(11). IC(3) loop is the main switch that mediates signaling via GLP-1R to G-proteins, while IC(1) and IC(2) loops are important in discrimination between different types of G-proteins., (Copyright 2002 Elsevier Science B.V.)

The impaired glucose-induced insulin release in type 2 diabetes mellitus may be accounted for by reduced B-cell ATP/ADP ratio or decreased phosphorylation of proteins regulating exocytosis of insulin. This, in turn, could be due to enhanced phosphatase activity. Using in situ hybridization techniques to assess the expression of 11 different phosphotyrosine phosphatases (PTPases), known to be present in the B-cells, overexpression by approximately 60% of PTP sigma (also known as LAR-PTP2 or PTP NE-3) was demonstrated in pancreatic islets and liver of spontaneously type 2 diabetic Goto-Kakizaki (GK) rats. In agreement with these findings Western blot of islet lysates, using a polyclonal PTP sigma antiserum, showed increased amounts of the protein in GK relative to control rat islets. Exposure of isolated islets for 20 h to 5 muM antisense to PTP sigma, composed of an antisense PNA sequence of 15 bases linked to the cell penetrating peptide transportan, increased glucose-induced insulin secretion from GK rat islets, but not from control islets. In parallel, the amounts of the phosphatase decreased. In conclusion, increased expression of PTP sigma may be of pathogenetic significance for the defective insulin secretion in GK rat islets.

A diversity of cell-penetrating peptides (CPPs), is known, but so far the only common denominator for these peptides is the ability to gain cell entry in an energy-independent manner. The mechanism used by CPPs for cell entry is largely unknown, and data comparing the different peptides are lacking. In order to gain more information about the cell-penetrating process, as well as to quantitatively compare the uptake efficiency of different CPPs, we have studied the cellular uptake and cargo delivery kinetics of penetratin, transportan, Tat (48-60) and MAP (KLAL). The respective CPPs (labelled with the fluorescence quencher, 3-nitrotyrosine) are coupled to small a pentapeptide cargo (labelled with the 2-amino benzoic acid fluorophore) via a disulfide bond. The cellular uptake of the cargo is registered as an increase in fluorescence intensity when the disulfide bond of the CPP-S-S-cargo construct is reduced in the intracellular milieu. Our data show that MAP has the fastest uptake, followed by transportan, Tat(48-60) and, last, penetratin. Similarly, MAP has the highest cargo delivery efficiency, followed by transportan, Tat (48-60) and, last, penetratin. Since some CPPs have been found to be toxic at high concentration, we characterized the influence of CPPs on cellular 2-[(3)H]deoxyglucose-6-phosphate leakage. Measurements on this system show that the membrane-disturbing potential appears to be correlated with the hydrophobic moment of the peptides. In summary, the yield and kinetics of cellular cargo delivery for four different CPPs has been quantitatively characterized.

Novel analogues of the cell-penetrating peptides penetratin and transportan were synthesized. The distribution of the biotin-labeled peptides in Bowes melanoma cell line has been investigated by indirect fluorescence with fluorescein-streptavidin detection. The time course of uptake of (125)I-labeled transportan analogues has been characterized in the same cell line. Molecular modeling was used to analyze the penetration and the orientation of molecules in a simulated biological membrane. The results, both from molecular modeling and fluorescence studies, imply that penetratin and transportan do not enter the cells by related mechanisms and that they do not belong to the same family of translocating peptides.

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.

The established view in cellular biology dictates that the cellular internalization of hydrophilic macromolecules can only be achieved through the classical endocytosis pathway. However, in the past five years several peptides have been demonstrated to translocate across the plasma membrane of eukaryotic cells by a seemingly energy-independent pathway. These peptides have been used successfully for the intracellular delivery of macromolecules with molecular weights several times greater than their own. Cellular delivery using these cell-penetrating peptides offers several advantages over conventional techniques because it is efficient for a range of cell types, can be applied to cells en masse and has a potential therapeutic application.

PNA is a nucleic acid analog with an achiral polyamide backbone consisting of N-(2-aminoethyl)glycine units (figure 1). The purine or pyrimidine bases are linked to the each unit via a methylene carbonyl linker (1-3) to target the complementary nucleic acid (4). PNA binds to complementary RNA or DNA in a parallel or antiparallel orientation following the Watson-Crick base-pairing rules (5-7). The uncharged nature of the PNA oligomers enhances the stability of the hybrid PNA/DNA(RNA) duplexes as compared to the natural homoduplexes. The non-natural character of the PNA makes PNA oligomers highly resistant to protease and nuclease attacks (8). These properties of PNA oligomers suggest that they could potentially serve as efficient antisense or antigene reagents. Indeed, peptide nucleic acids have been applied to block protein expression on the transcriptional (9) and translational level (10,11), and microinjected PNA oligomers demonstrate a strong antisense effect in intact cells (12). However, contrary to the "normal" nucleic acid analogs, PNA oligomers are not efficiently delivered into the cytoplasm of the cell, and until recently this has hindered the application of PNA oligomers as antisense reagents. In this work we summarize some recent achievements on PNA antisense application, especially these concerned with whole cell or tissue delivery of the PNA.

Two chimeric peptides, consisting of the linear vasopressin receptor V1 antagonist PhAc-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr, in the N-terminus and mastoparan in the C-terminus connected directly (M375) or via 6-aminohexanoic acid (M391), have been synthesised. At 10 microM concentration, these novel peptides increased insulin secretion from isolated rat pancreatic islet cells 18-26-fold at 3.3 mM glucose and 3.5-5-fold at 16.7 mM glucose. PTX pretreatment of the islets decreased the peptide-induced insulin release. M375 and M391 bind to V1a vasopressin receptors with affinities lower than the unmodified vasopressin antagonist, but with K(D) values of 3.76 nM and 9.02 nM, respectively, both chimeras are high affinity ligands. The GTPase activity and GTPgammaS binding in the presence of these peptides has been characterised in Rin m5F cells. Comparison of the influence of the peptides M375 and M391 on GTPase activity in native and pertussis toxin-treated cells suggests a selective activation of G alpha(i)/G alpha(o) subunits, combined with a suppression of other GTPases, primarily G alpha(s). However, the GTPgammaS binding data show that the peptides retain some of the activating property even in PTX-treated cell membranes. In conclusion, the conjugation of mastoparan with the V1a receptor antagonists produce peptides with properties different from the parent peptides that could be used to elucidate the role of different G proteins in insulin release.