Endosome trapping limits the efficiency of splicing correction by PNA-oligolysine conjugates

Splicing correction by steric-blocking oligonucleotides (ON) might lead to important clinical applications but requires efficient delivery to cell nuclei. The conjugation of short oligolysine tails has been used to deliver a correcting peptide nucleic acid (PNA) sequence in a positive readout assay in which ON hybridization to the cryptic splice site is strictly required for the expression of a luciferase reporter gene. We have investigated the mechanism of cellular uptake and the efficiency of a (Lys)₈-PNA-Lys construction in this model system. Cell uptake is temperature-dependent and leads to sequestration of the conjugate in cytoplasmic vesicles in keeping with an endocytic mechanism of internalization. Accordingly a significant and sequence-specific splicing correction is achieved only in the presence of endosome-disrupting agents as chloroquine or 0.5 M sucrose. These endosome-disrupting agents do not affect the activity of free PNA, and do not increase (Lys)₈-PNA-Lys uptake.     

Insights into the cellular trafficking of splice redirecting oligonucleotides complexed with chemically modified cell-penetrating peptides

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.     

A novel cell-penetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids.

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.     

Cell-surface Accumulation of Flock House Virus-derived Peptide Leads to Efficient Internalization via Macropinocytosis

Arginine-rich cell-penetrating peptides (CPPs), including human immunodeficiency virus type 1 (HIV-1) Tat (48–60) and oligoarginines, have been applied as carriers for delivery of cargo molecules, because of their capacity to internalize into cells and penetrate biological membranes. Despite the fact that they have been extensively studied, the factors required for the efficient internalization of CPPs are still unclear. In this report, we evaluated the internalization efficiencies of seven CPPs derived from DNA/RNA-binding peptides, and discovered that a peptide derived from the flock house virus (FHV) coat protein was internalized most efficiently into Chinese hamster ovary (CHO-K1), HeLa, and Jurkat cells. Comparison of the factors facilitating the internalization with those of the Tat peptide revealed that the FHV peptide induces macropinocytosis much more efficiently than the Tat peptide, which leads to its high cellular uptake efficiency. Additionally, the strong adsorption of the FHV peptide on cell membranes via glycosaminoglycans (GAGs) was shown to be a key factor for induction of macropinocytosis, and these steps were successfully monitored by live imaging of the peptide internalization into cells in relation to the actin organization. The remarkable methods of FHV peptide internalization thus highlighted the critical factors for internalizations of the arginine-rich CPPs.     

Cell-penetrating peptides as versatile vehicles for oligonucleotide delivery

Short regulatory oligonucleotides (ONs) have a great therapeutic potential for the modulation of gene expression due to their high specificity and low toxicity. The major obstacles for in vivo clinical applications of ONs are the poor permeability of plasma membrane to nucleic acids and the sensitivity of ONs to enzymatic degradation. Hence, various delivery vehicles have been developed to ensure the transduction of ONs into cells. Among these, the cell-penetrating peptides (CPPs) have gained quickly broadening popularity as promising nonviral transmembrane delivery vectors. For coupling of nucleic acids to CPPs, two distinct strategies may be applied-covalent and noncovalent. The majority of earlier studies have used covalent coupling of CPPs to ONs. However, the number of studies demonstrating very high therapeutic potential of noncovalent complexes of ONs with novel CPP-based delivery vehicles is explosively increasing. In this review, the recent developments in the application of CPP-mediated oligonucleotide delivery by noncovalent strategy will be discussed.     

Penetration without cells: Membrane translocation of cell-penetrating peptides in the model giant plasma membrane vesicles

The cellular internalization of cell-penetrating peptides (CPPs) is proposed to take place by both endocytic processes and by a direct translocation across the plasma membrane. So far only scarce data is available about what determines the choice between the two uptake routes, or the proportion of used pathways when both are active simultaneously. Furthermore, the mechanism(s) of membrane penetration by peptides is itself still a matter of debate. We have introduced the giant plasma membrane vesicles (GPMVs) to study the interaction of six well-described CPPs (fluorescently labeled nona-arginine, Tat peptide, Penetratin, MAP, Transportan and TP10) in a model system of native plasma membrane without the interference of endocytic processes. The membranes of GPMVs are shown to segregate into liquid-ordered and liquid-disordered phases at low temperatures and we demonstrate here by confocal microscopy that amphipathic CPPs preferentially associate with liquid-disordered membrane areas. Moreover, all tested CPPs accumulate into the lumen of GPMVs both at ambient and low temperature. The uncharged control peptide and dextran, in contrary, do not translocate from the medium into the lumen of vesicles. The absence of energy-dependent cellular processes and the impermeability to hydrophilic macromolecules makes the GPMVs a useful model to study the translocation of CPPs across the plasma membrane in conditions lacking endocytosis.     

Evaluation of cell penetrating peptides fused to elastin-like polypeptide for drug delivery.

Translocation through the plasma membrane is a major limiting step for the cellular delivery of macromolecules. Several cell penetrating peptides (CPP) have been demonstrated to efficiently internalize various molecular cargo to targets inside eukaryotic cells. In this study, the efficiency and mechanism of cellular uptake of the CPPs penetratin, Tat, and MTS fused to elastin-like polypeptides (CPP-ELP) were evaluated. Elastin-like polypeptides are biopolymers with features that make them useful as polymeric carriers for the delivery of therapeutics. Therefore, improving efficiency of cellular uptake by fusing ELPs to CPPs and understanding the mechanism of their cellular internalization could contribute to development of new therapeutic approaches. Flow cytometry and confocal fluorescence microscopy were used to elucidate the mechanism of CPP-ELP uptake. The internalization of all CPP-ELPs was impaired dramatically at 4 degrees C, under ATP depletion conditions, and with hyperosmolar sucrose, implicating involvement of an endocytic pathway for CPP-ELP internalization. Penetratin was identified as the most effective CPP for delivering ELP. Finally, in order to demonstrate the potential of CPP-ELP for drug delivery, a fusion polypeptide was made containing penetratin, ELP, and a peptide derived from the cyclin-dependent kinase inhibitor p21. This polypeptide was shown to inhibit proliferation of SKOV-3 and HeLa cells by slowing their growth rate.     

A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo

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.     

Inhibition of gene expression in Escherichia coli by antisense phosphorodiamidate morpholino oligomers

Antisense phosphorodiamidate morpholino oligomers (PMOs) were tested for the ability to inhibit gene expression in Escherichia coli. PMOs targeted to either a myc-luciferase reporter gene product or 16S rRNA did not inhibit luciferase expression or growth. However, in a strain with defective lipopolysaccharide (lpxA mutant), which has a leaky outer membrane, PMOs targeted to the myc-luciferase or acyl carrier protein (acpP) mRNA significantly inhibited their targets in a dose-dependent response. A significant improvement was made by covalently joining the peptide (KFF)(3)KC to the end of PMOs. In strains with an intact outer membrane, (KFF)(3)KC-myc PMO inhibited luciferase expression by 63%. A second (KFF)(3)KC-PMO conjugate targeted to lacI mRNA induced beta-galactosidase in a dose-dependent response. The end of the PMO to which (KFF)(3)KC is attached affected the efficiency of target inhibition but in various ways depending on the PMO. Another peptide-lacI PMO conjugate was synthesized with the cationic peptide CRRRQRRKKR and was found not to induce beta-galactosidase. We conclude that the outer membrane of E. coli inhibits entry of PMOs and that (KFF)(3)KC-PMO conjugates are transported across both membranes and specifically inhibit expression of their genetic targets.     

Nuclear localization and antisense effect of PNA internalized by ASGP-R-mediated endocytosis with protein/DNA conjugates

In order for peptide nucleic acids (PNAs) to be effective as therapeutic agents, methods for cellular delivery must be developed. Here we demonstrate spontaneous nuclear localization and antisense effects of peptide nucleic acids (PNAs) delivered to hepatic cells through asialoglycoprotein receptor-mediated endocytosis. Asialofetuin conjugates with DNA oligonucleotides (AF/DNA) complementary to the PNA of interest were designed as cell-specific delivery vectors. PNAs hybridized to the asialofetuin-oligonucleotide conjugates were internalized into murine primary hepatocytes and human HepG2 hepatocarcinoma cells effectively through receptor-mediated endocytosis in vitro. After a 4-h incubation, PNAs were largely localized in the nuclei of the cells; the mechanisms involved are still unclear. More than 70% inhibition of telomerase activity was observed when PNAs complementary to the RNA template of human telomerase were delivered to HepG2 cells using AF/DNA. The PNAs were stably associated with the AF/DNA conjugates in 50% serum at 37 °C for at least 3 h. The PNAs were spontaneously released from the conjugate through a strand exchange mechanism when complementary nucleic acid was added. The complexation of PNAs with the AF/DNA conjugates resulted in delivery of PNAs to liver after intravenous injection into mice. The present study indicates that conjugation to a natural proteinous ligand can be used as a non-toxic vector for cellular delivery of oligonucleotide analogs.     

Tat(48-60) peptide amino acid sequence is not unique in its cell penetrating properties and cell-surface glycosaminoglycans inhibit its cellular uptake

Biomolecules and drug delivery agents, such as liposomes, are often delivered intracellularly with help of cell penetrating peptides (CPPs) and, in particular, Tat peptide. Tat peptide acts as a membrane shuttle; the structural determinants of transport and the manner by which the peptide crosses the lipid bilayer are, however, still unknown. The roles of direct membrane translocation, endocytosis and cell surface proteoglycans, in particular, remain elusive. Our study aimed to explore the relationship between structure and activity of Tat peptide and its uptake mechanism. For this purpose we introduced several modifications (e.g. lipophilic, aromatic, neutral and non-natural amino acids) into the original Tat sequence. We studied the interaction of the peptides with a model lipid membrane and with three cell lines, a phagocytic cell line (human retinal pigment epithelium cell line, ARPE-19), a non-phagocytic cell line (Chinese hamster ovary cells, CHO wt) and a mutant form of the latter cell line deficient in glycosaminoglycans chondroitin sulfate and heparan sulfate (CHO-pgsB 618). The amino acid residues introduced into the original sequence of Tat peptide failed to influence cellular uptake, indicating that the cationic charge alone may be responsible for translocation. Clear discrepancy between permeation activity of the peptides into cells and their interaction with lipid bilayers of liposomes indicated the limited value of the model membrane in predicting cellular peptide delivery. Cell uptake of Tat peptide was unspecific, took place either by phagocytosis or pinocytosis, and was inhibited by cell-surface glycosaminoglycans. The internalized peptides were localized in vesicles and unable to reach the cell nuclei. In conclusion, we show that Tat-related peptides enter cells on the basis of their cationic charge following different endocytosis pathways and that glycosaminoglycans on the cell surface negatively affect their uptake. This lack of specificity should be taken into account when exploiting Tat peptide as vehicle for intracellular delivery of biomacromolecules.     

A Cell-penetrating Helical Polymer For siRNA Delivery to Mammalian Cells

Cell-penetrating peptides (CPPs) are routinely used for intracellular delivery of a variety of cargo, including drugs, genes, and short interfering RNA (siRNA). Most CPPs are active only upon exposure to acidic environments inside of late endosomes, thereby facilitating the endosomal escape of internalized vectors. Here, we describe the generation of a synthetic polypeptide—PVBLG_n-8—that is able to adopt a helical structure independent of pH. Like other CPPs, the helical structure of PVBLG_n-8 allows the polypeptide to destabilize membranes. However, since the helix is stable at all physiologically relevant pH values between pH 2 and pH 7.4, the membrane permeation properties of PVBLG_n-8 are irreversible. Given its pH-insensitive activity, our results suggest that PVBLG_n-8 is able to facilitate efficient siRNA delivery by causing pore formation in the cell membranes through which either free or complexed siRNA is able to diffuse. This nonspecific form of entry into the cell cytosol may prove useful when trying to deliver siRNA to cells which have proven to be difficult to transfect.     

Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement.

The use of membrane-permeable peptides as carrier vectors for the intracellular delivery of various proteins and macromolecules for modifying cellular function is well documented. Arginine-rich peptides, including those derived from human immunodeficiency virus 1 Tat protein, are among the representative classes of these vectors. The internalization mechanism of these vector peptides and their protein conjugates was previously regarded as separate from endocytosis, but more recent reevaluations have concluded that endocytosis is involved in their internalization. In this report, we show that the uptake of octa-arginine (R8) peptide by HeLa cells was significantly suppressed by the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) and the F-actin polymerization inhibitor cytochalasin D, suggesting a role for macropinocytosis in the uptake of the peptide. In agreement with this we observed that treatment of the cells with R8 peptide induced significant rearrangement of the actin cytoskeleton. The internalization efficiency and contribution of macropinocytosis were also observed to have a dependency on the chain length of the oligoarginine peptides. Uptake of penetratin, another representative peptide carrier, was less sensitive to EIPA and penetratin did not have such distinct effects on actin localization. The above observations suggest that penetratin and R8 peptides have distinct internalization mechanisms.     

Design of Phosphorodiamidate Morpholino Oligomers (PMOs) for the Induction of Exon Skipping of the Human DMD Gene

Duchenne muscular dystrophy (DMD) is caused by out-of-frame mutations of the human DMD gene. Antisense oligonucleotides (AOs) have previously been used to skip additional exons that border the deletions such that the reading frame is restored and internally truncated, but functional, dystrophin expressed. We have designed phosphorodiamidate morpholino oligomer (PMO) AOs to various exons of the human dystrophin gene. PMOs were designed to have their target sites overlapping areas of open RNA structure, as defined by hybridization-array analysis, and likely exonic splicing enhancer (ESE)/silencer sites on the target RNA. The ability of each PMO to produce exon skipping was tested in vitro in normal human skeletal muscle cells. Retrospective analysis of design parameters used and PMO variables revealed that active PMOs were longer, bound to their targets more strongly, had their target sites closer to the acceptor splice site of the exon, overlapped areas of open conformation (as defined by the hybridization or the RNA secondary structure prediction software), and could interfere with the binding of certain SR proteins. No other parameter appeared to show significant association to PMO-skipping efficacy. No design tool is strong enough in isolation; however, if used in conjunction with other significant parameters it can aid AO design.     

Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation

The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors.     

Inhibition of HIV-1 replication by oligonucleotide analogues directed to the packaging signal and trans-activating response region

HIV possesses a remarkable capacity for mutational escape from therapeutics that target the viral proteins and enzymes. Inhibitory strategies aimed at highly conserved nucleic acid sequences within the genome are an attractive alternative. However, it has proven difficult to achieve an effective level of therapeutic at the appropriate site within the cell. Oligonucleotide delivery is a rapidly advancing field. We have investigated oligonucleotide analogues as steric-block therapeutics against two highly conserved regions of the HIV-1 genome. In the study we show that 2'0-methyl/locked nucleic acid oligonucleotides against the packaging signal and trans-activating response regions of HIV can inhibit replication of the virus.