Cellular trafficking determines the exon skipping activity of Pip6a-PMO in mdx skeletal and cardiac muscle cells.

Cell-penetrating peptide-mediated delivery of phosphorodiamidate morpholino oligomers (PMOs) has shown great promise for exon-skipping therapy of Duchenne Muscular Dystrophy (DMD). Pip6a-PMO, a recently developed conjugate, is particularly efficient in a murine DMD model, although mechanisms responsible for its increased biological activity have not been studied. Here, we evaluate the cellular trafficking and the biological activity of Pip6a-PMO in skeletal muscle cells and primary cardiomyocytes. Our results indicate that Pip6a-PMO is taken up in the skeletal muscle cells by an energy- and caveolae-mediated endocytosis. Interestingly, its cellular distribution is different in undifferentiated and differentiated skeletal muscle cells (vesicular versus nuclear). Likewise, Pip6a-PMO mainly accumulates in cytoplasmic vesicles in primary cardiomyocytes, in which clathrin-mediated endocytosis seems to be the pre-dominant uptake pathway. These differences in cellular trafficking correspond well with the exon-skipping data, with higher activity in myotubes than in myoblasts or cardiomyocytes. These differences in cellular trafficking thus provide a possible mechanistic explanation for the variations in exon-skipping activity and restoration of dystrophin protein in heart muscle compared with skeletal muscle tissues in DMD models. Overall, Pip6a-PMO appears as the most efficient conjugate to date (low nanomolar EC50), even if limitations remain from endosomal escape.

Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo.

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.

Cell penetrating peptides: overview and applications to the delivery of oligonucleotides.

Crossing biological barriers represents a major limitation for clinical applications of biomolecules such as nucleic acids, peptides or proteins. Cell penetrating peptides (CPP), also named protein transduction domains, comprise short and usually basic amino acids-rich peptides originating from proteins able to cross biological barriers, such as the viral Tat protein, or are rationally designed. They have emerged as a new class of non-viral vectors allowing the delivery of various biomolecules across biological barriers from low molecular weight drugs to nanosized particles. Encouraging data with CPP-conjugated oligonucleotides have been obtained both in vitro and in vivo in animal models of diseases such as Duchenne muscular dystrophy. Whether CPP-cargo conjugates enter cells by direct translocation across the plasma membrane or by endocytosis remains controversial. In many instances, however, endosomal escape appears as a major limitation of this new delivery strategy.

Oligonucleotides-Based Therapeutics.

This Special Issue of Biomedicines aims to outline nucleic-acid-based strategies that have emerged as tools to regulate specific gene expression and, more recently, as a new class of medicines [...].

Synthesis and splice-redirecting activity of branched, arginine-rich peptide dendrimer conjugates of peptide nucleic acid oligonucleotides.

Arginine-rich cell-penetrating peptides have found excellent utility in cell and in vivo models for enhancement of delivery of attached charge-neutral PNA or PMO oligonucleotides. We report the synthesis of dendrimeric peptides containing 2- or 4-branched arms each having one or more R-Ahx-R motifs and their disulfide conjugation to a PNA705 splice-redirecting oligonucleotide. Conjugates were assayed in a HeLa pLuc705 cell assay for luciferase up-regulation and splicing redirection. Whereas 8-Arg branched peptide-PNA conjugates showed poor activity compared to a linear (R-Ahx-R)(4)-PNA conjugate, 2-branched and some 4-branched 12 and 16 Arg peptide-PNA conjugates showed activity similar to that of the corresponding linear peptide-PNA conjugates. Many of the 12- and 16-Arg conjugates retained significant activity in the presence of serum. Evidence showed that biological activity in HeLa pLuc705 cells of the PNA conjugates of branched and linear (R-Ahx-R) peptides is associated with an energy-dependent uptake pathway, predominantly clathrin-dependent, but also with some caveolae dependence.

Improved cell-penetrating peptide-PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle.

Steric blocking peptide nucleic acid (PNA) oligonucleotides have been used increasingly for redirecting RNA splicing particularly in therapeutic applications such as Duchenne muscular dystrophy (DMD). Covalent attachment of a cell-penetrating peptide helps to improve cell delivery of PNA. We have used a HeLa pLuc705 cell splicing redirection assay to develop a series of PNA internalization peptides (Pip) conjugated to an 18-mer PNA705 model oligonucleotide with higher activity compared to a PNA705 conjugate with a leading cell-penetrating peptide being developed for therapeutic use, (R-Ahx-R)(4). We show that Pip-PNA705 conjugates are internalized in HeLa cells by an energy-dependent mechanism and that the predominant pathway of cell uptake of biologically active conjugate seems to be via clathrin-dependent endocytosis. In a mouse model of DMD, serum-stabilized Pip2a or Pip2b peptides conjugated to a 20-mer PNA (PNADMD) targeting the exon 23 mutation in the dystrophin gene showed strong exon-skipping activity in differentiated mdx mouse myotubes in culture in the absence of an added transfection agent at concentrations where naked PNADMD was inactive. Injection of Pip2a-PNADMD or Pip2b-PNADMD into the tibealis anterior muscles of mdx mice resulted in approximately 3-fold higher numbers of dystrophin-positive fibres compared to naked PNADMD or (R-Ahx-R)(4)-PNADMD.

Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure-activity studies.

Redirecting the splicing machinery through the hybridization of high affinity, RNase H- incompetent oligonucleotide analogs such as phosphoramidate morpholino oligonucleotides (PMO) might lead to important clinical applications. Chemical conjugation of PMO to arginine-rich cell penetrating peptides (CPP) such as (R-Ahx-R)(4) (with Ahx standing for 6-aminohexanoic acid) leads to sequence-specific splicing correction in the absence of endosomolytic agents in cell culture at variance with most conventional CPPs. Importantly, (R-Ahx-R)(4)-PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy. Unfortunately, active doses in some applications might be close to cytotoxic ones thus presenting challenge for systemic administration of the conjugates in those clinical settings. Structure-activity relationship studies have thus been undertaken to unravel CPP structural features important for the efficient nuclear delivery of the conjugated PMO and limiting steps in their internalization pathway. Affinity for heparin (taken as a model heparan sulfate), hydrophobicity, cellular uptake, intracellular distribution and splicing correction have been monitored. Spacing between the charges, hydrophobicity of the linker between the Arg-groups and Arg-stereochemistry influence splicing correction efficiency. A significant correlation between splicing correction efficiency, affinity for heparin and ability to destabilize model synthetic vesicles has been observed but no correlation with cellular uptake has been found. Efforts will have to focus on endosomal escape since it appears to remain the limiting factor for the delivery of these splice-redirecting ON analogs.

Anti-apoptotic peptide for long term cardioprotection in a mouse model of myocardial ischemia-reperfusion injury.

Reperfusion therapy during myocardial infarction (MI) leads to side effects called ischemia-reperfusion (IR) injury for which no treatment exists. While most studies have targeted the intrinsic apoptotic pathway to prevent IR injury with no successful clinical translation, we evidenced recently the potent cardioprotective effect of the anti-apoptotic Tat-DAXXp (TD) peptide targeting the FAS-dependent extrinsic pathway. The aim of the present study was to evaluate TD long term cardioprotective effects against IR injury in a MI mouse model. TD peptide (1 mg/kg) was administered in mice subjected to MI (TD; n = 21), 5 min prior to reperfusion, and were clinically followed-up during 6 months after surgery. Plasma cTnI concentration evaluated 24 h post-MI was 70%-decreased in TD (n = 16) versus Ctrl (n = 20) mice (p***). Strain echocardiography highlighted a 24%-increase (p****) in the ejection fraction mean value in TD-treated (n = 12) versus Ctrl mice (n = 17) during the 6 month-period. Improved cardiac performance was associated to a 54%-decrease (p**) in left ventricular fibrosis at 6 months in TD (n = 16) versus Ctrl (n = 20). In conclusion, targeting the extrinsic pathway with TD peptide at the onset of reperfusion provided long-term cardioprotection in a mouse model of myocardial IR injury by improving post-MI cardiac performance and preventing cardiac remodeling.

A novel therapeutic peptide targeting myocardial reperfusion injury.

AIMS: Regulated cell death is a main contributor of myocardial ischaemia-reperfusion (IR) injury during acute myocardial infarction. In this context, targeting apoptosis could be a potent therapeutical strategy. In a previous study, we showed that DAXX (death-associated protein) was essential for transducing the FAS-dependent apoptotic signal during IR injury. The present study aims at evaluating the cardioprotective effects of a synthetic peptide inhibiting FAS:DAXX interaction. METHODS AND RESULTS: An interfering peptide was engineered and then coupled to the Tat cell penetrating peptide (Tat-DAXXp). Its internalization and anti-apoptotic properties were demonstrated in primary cardiomyocytes. Importantly, an intravenous bolus injection of Tat-DAXXp (1 mg/kg) 5 min before reperfusion in a murine myocardial IR model decreased infarct size by 48% after 24 h of reperfusion. In addition, Tat-DAXXp was still efficient after a 30-min delayed administration, and was completely degraded and eliminated within 24 h thereby reducing risks of potential side effects. Importantly, Tat-DAXXp reduced mouse early post-infarction mortality by 67%. Mechanistically, cardioprotection was supported by both anti-apoptotic and pro-survival effects, and an improvement of myocardial functional recovery as evidenced in ex vivo experiments. CONCLUSIONS: Our study demonstrates that a single dose of Tat-DAXXp injected intravenously at the onset of reperfusion leads to a strong cardioprotection in vivo by inhibiting IR injury validating Tat-DAXXp as a promising candidate for therapeutic application.

A peptide-based dendrimer that enhances the splice-redirecting activity of PNA conjugates in cells.

The full therapeutic potential of oligonucleotide (ON)-based agents has been hampered by cellular delivery challenges. Cell-penetrating peptides (CPP) represent promising delivery vectors for nucleic acids, and their potential has recently been evaluated using a functional splicing redirection assay, which capitalizes on the nuclear delivery of splice-correcting steric-block ON analogues such as peptide nucleic acids (PNA). Despite encouraging in vitro and in vivo data with arginine-rich CPP-steric block conjugates, mechanistic studies have shown that entrapment within the endosome/lysosome compartment after endocytosis remains a limiting factor. Previous work from our group has shown that CPP oligomerization greatly improves cellular delivery and increases transfection of plasmid DNA. We now report the chemical synthesis and the evaluation of multivalent CPP-PNA constructs incorporating monomeric (p53(mono)) and dendrimer-like tetrameric (p53(tet)) forms of the p53 tetramerization domain containing peptide, a 10 arginine CPP domain (R10), and a splice redirecting PNA (PNA705). These CPP-PNA conjugates were termed R10p53(tet)-PNA705 and R10p53(mono)-PNA705, referring to their oligomerization state. The present study demonstrates that the splicing redirection efficiency of PNA705 is much greater in the context of the tetrameric R10p53(tet)-PNA705 construct than for the monomeric and occurs at nanomolar concentrations, demonstrating that multivalency is an important factor in delivering PNA into cells.

Peptide-based delivery of steric-block PNA oligonucleotides.

Several strategies based on synthetic oligonucleotides (ON) have been proposed to control gene expression. As for most biomolecules, however, delivery has remained a major roadblock for in vivo applications. Conjugation of steric-block neutral DNA mimics such as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligonucleotides (PMO) to cell penetrating peptides (CPP) has recently been proposed as a new delivery strategy. It is particularly suitable to interfere sequence-specifically with pre-mRNA splicing thus offering various applications in fundamental research and in therapeutics. The chemical synthesis of these CPP conjugates as well as methodologies to monitor their cellular uptake and their efficiency in a reliable and easy to implement assay of splicing correction will be described.

Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide.

Sequence-specific interference with the nuclear pre-mRNA splicing machinery has received increased attention as an analytical tool and for development of therapeutics. It requires sequence-specific and high affinity binding of RNaseH-incompetent DNA mimics to pre-mRNA. Peptide nucleic acids (PNA) or phosphoramidate morpholino oligonucleotides (PMO) are particularly suited as steric block oligonucleotides in this respect. However, splicing correction by PNA or PMO conjugated to cell penetrating peptides (CPP), such as Tat or Penetratin, has required high concentrations (5-10 microM) of such conjugates, unless an endosomolytic agent was added to increase escape from endocytic vesicles. We have focused on the modification of existing CPPs to search for peptides able to deliver more efficiently splice correcting PNA or PMO to the nucleus in the absence of endosomolytic agents. We describe here R6-Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) as the most active of all CPPs tested so far in a splicing correction assay in which masking of a cryptic splice site allows expression of a luciferase reporter gene. Efficient and sequence-specific correction occurs at 1 muM concentration of the R6Pen-PNA705 conjugate as monitored by luciferase luminescence and by RT-PCR. Some aspects of the R6Pen-PNA705 structure-function relationship have also been evaluated.

Cell penetrating peptide conjugates of steric block oligonucleotides.

Charge neutral steric block oligonucleotide analogues, such as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO), have promising biological and pharmacological properties for antisense applications, such as for example in mRNA splicing redirection. However, cellular uptake of free oligomers is poor and the utility of conjugates of PNA or PMO to cell penetrating peptides (CPP), such as Tat or Penetratin, is limited by endosomal sequestration. Two new families of arginine-rich CPPs named (R-Ahx-R)(4) AhxB and R(6)Pen allow efficient nuclear delivery of splice correcting PNA and PMO at micromolar concentrations in the absence of endosomolytic agents. The in vivo efficacy of (R-Ahx-R)(4) AhxB PMO conjugates has been demonstrated in mouse models of Duchenne muscular dystrophy and in various viral infections.

Comparison of basic peptides- and lipid-based strategies for the delivery of splice correcting oligonucleotides.

Expression of alternatively spliced mRNA variants at specific stages of development or in specific cells and tissues contributes to the functional diversity of the human genome. Aberrations in alternative splicing were found as a cause or a contributing factor to the development, progression, or maintenance of numerous diseases. The use of antisense oligonucleotides (ON) to modify aberrant expression patterns of alternatively spliced mRNAs is a novel means of potentially controlling such diseases. Oligonucleotides can be designed to repair genetic mutations, to modify genomic sequences in order to compensate for gene deletions, or to modify RNA processing in order to improve the effects of the underlying gene mutation. Steric block ON approach have proven to be effective in experimental model for various diseases. Here, we describe our experience in investigating two strategies for ON delivery: ON conjugation with basic peptides and lipid-based particulate system (lipoplex). Basic peptides or Cell Penetrating Peptides (CPP) such as the TAT-derived peptide appear to circumvent many problems associated with ON and drug delivery. This strategy may represent the next paradigm in our ability to modulate cell function and offers a unique avenue for the treatment of disease. Lipoplexes result from the intimate interaction of ON with cationic lipids leading to ON carrying particles able to be taken up by cells and to release ON in the cytoplasm. We have used as an experimental model the correction of a splicing alteration of the mutated beta-globin intron causing thalassemia. Data on cell penetration and efficacy of correction of specific steric block ON delivered either by basic peptides or lipoplex are described. A comparison of the properties of both delivery systems is made respective to the use of this new class of therapeutic molecules.

Lipoplex and peptide-based strategies for the delivery of steric-block oligonucleotides.

Synthetic oligonucleotides offer interesting prospects for the control of gene expression but clinical applications have been severely limited by their poor bioavailability. Cationic lipids have been widely used for the delivery of charged oligonucleotide (ON) analogues but most of the commercial formulations are toxic and poorly stable in the presence of serum proteins. We have developed a DOGS/DOPE liposome formulation named DLS (for delivery liposomal system), that allows for the efficient nuclear delivery of negatively charged antisense ON analogues as monitored by fluorescence microscopy and by their ability to correct deficient pre-mRNA splicing, even in serum-supplemented cell culture. Uncharged DNA mimics such as peptide nucleic acids (PNA), or phosphorodiamidate morpholino (PMO) ON are particularly interesting for their high metabolic stability and affinity for complementary RNA targets but they cannot be delivered with cationic lipids. Cell penetrating peptides (CPP), such as Tat or penetratin, have been used widely as conjugates for the delivery of various biomolecules and might be appropriate for neutral ON analogues. However, entrapment within endocytic vesicles severely limits the efficiency of PNA delivery by CPPs in the absence of endosomolytic drugs, such as chloroquine. The conjugation of new arginine-rich CPPs to PNA allows efficient nuclear delivery in the absence of chloroquine as monitored in a splicing correction assay. Both strategies have their advantages but DLS-mediated delivery remains more efficient than CPP delivery for the nuclear targeting of splice correcting ON analogues in vitro.

Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells.

The trans-activation response (TAR) RNA stem-loop that occurs at the 5' end of HIV RNA transcripts is an important antiviral target and is the site of interaction of the HIV-1 Tat protein together with host cellular factors. Oligonucleotides and their analogues targeted to TAR are potential antiviral candidates. We have investigated a range of cell penetrating peptide (CPP) conjugates of a 16mer peptide nucleic acid (PNA) analogue targeted to the apical stem-loop of TAR and show that disulfide-linked PNA conjugates of two types of CPP (Transportan or a novel chimeric peptide R6-Penetratin) exhibit dose-dependent inhibition of Tat-dependent trans-activation in a HeLa cell assay when incubated for 24 h. Activity is reached within 6 h if the lysosomotropic reagent chloroquine is co-administered. Fluorescein-labelled stably-linked conjugates of Tat, Transportan or Transportan TP10 with PNA were inactive when delivered alone, but attained trans-activation inhibition in the presence of chloroquine. Confocal microscopy showed that such fluorescently labelled CPP-PNA conjugates were sequestered in endosomal or membrane-bound compartments of HeLa cells, which varied in appearance depending on the CPP type. Co-administration of chloroquine was seen in some cases to release fluorescence from such compartments into the nucleus, but with different patterns depending on the CPP. The results show that CPP-PNA conjugates of different types can inhibit Tat-dependent trans-activation in HeLa cells and have potential for development as antiviral agents. Endosomal or membrane release is a major factor limiting nuclear delivery and trans-activation inhibition.

Local RNA target structure influences siRNA efficacy: a systematic global analysis.

The efficiency with which small interfering RNAs (siRNAs) down-regulate specific gene expression in living cells is variable and a number of sequence-governed, biochemical parameters of the siRNA duplex have been proposed for the design of an efficient siRNA. Some of these parameters have been clearly identified to influence the assembly of the RNA-induced silencing complex (RISC), or to favour the sequence preferences of the RISC endonuclease. For other parameters, it is difficult to ascertain whether the influence is a determinant of the siRNA per se, or a determinant of the target RNA, especially its local structural characteristics. In order to gain an insight into the effects of local target structure on the biological activity of siRNA, we have used large sets of siRNAs directed against local targets of the mRNAs of ICAM-1 and survivin. Target structures were classified as accessible or inaccessible using an original, iterative computational approach and by experimental RNase H mapping. The effectiveness of siRNA was characterized by measuring the IC50 values in cell culture and the maximal extent of target suppression. Mean IC50 values were tenfold lower for accessible local target sites, with respect to inaccessible ones. Mean maximal target suppression was improved. These data illustrate that local target structure does, indeed, influence the activity of siRNA. We suggest that local target screening can significantly improve the hit rate in the design of biologically active siRNAs.

Successful silencing of plasminogen activator inhibitor-1 in human vascular endothelial cells using small interfering RNA.

Clinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator, may be involved in the pathophysiology of atherosclerosis and cardiovascular disease. We investigated the feasibility, efficacy and functional effects of PAI-1 gene silencing in human vascular endothelial cells using small interfering RNA. Double-stranded 21 bp-RNA molecules targeted at sequences within the human PAI-1 gene were constructed. Successful siRNA transfection of HUVEC was confirmed using fluorescence microscopy and flow cytometry. One of five candidate siRNA sequences reduced PAI-1 mRNA and protein in a concentration- and time-dependent manner. Suppression of PAI-1 mRNA was detected up to 72 hours after transfection. Moreover, siRNA treatment reduced the activity of PAI-1 released from HUVEC, and prevented the oxLDL- or LPS-induced upregulation of PAI-1 secretion. Importantly, siRNA treatment did not affect the expression of other endothelial-cell markers. Moreover, downregulation of PAI-1 significantly enhanced the ability of endothelial cells to adhere to vitronectin, and this effect could be reversed upon addition of recombinant PAI-1. SiRNA-mediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis.

Vectorization of morpholino oligomers by the (R-Ahx-R)4 peptide allows efficient splicing correction in the absence of endosomolytic agents.

The efficient and non-toxic nuclear delivery of steric-block oligonucleotides (ON) is a prerequisite for therapeutic strategies involving splice correction or exon skipping. Cationic cell penetrating peptides (CPPs) have given rise to much interest for the intracellular delivery of biomolecules, but their efficiency in promoting cytoplasmic or nuclear delivery of oligonucleotides has been hampered by endocytic sequestration and subsequent degradation of most internalized material in endocytic compartments. In the present study, we compared the splice correction activity of three different CPPs conjugated to PMO(705), a steric-block ON targeted against the mutated splicing site of human beta-globin pre-mRNA in the HeLa pLuc705 splice correction model. In contrast to Tat48-60 (Tat) and oligoarginine (R(9)F(2)) PMO(705) conjugates, the 6-aminohexanoic-spaced oligoarginine (R-Ahx-R)(4)-PMO(705) conjugate was able to promote an efficient splice correction in the absence of endosomolytic agents. Our mechanistic investigations about its uptake mechanisms lead to the conclusion that these three vectors are internalized using the same endocytic route involving proteoglycans, but that the (R-Ahx-R)(4)-PMO(705) conjugate has the unique ability to escape from lysosomial fate and to access to the nuclear compartment. This vector, which has displays an extremely low cytotoxicity, the ability to function without chloroquine adjunction and in the presence of serum proteins. It thus offers a promising lead for the development of vectors able to enhance the delivery of therapeutic steric-block ON in clinically relevant models.

Cationic phosphoramidate alpha-oligonucleotides efficiently target single-stranded DNA and RNA and inhibit hepatitis C virus IRES-mediated translation.

A potential means to improve the efficacy of steric-blocking antisense oligonucleotides (ON) is to increase their affinity for a target RNA. The grafting of cationic amino groups to the backbone of the ON is one way to achieve this, as it reduces the electrostatic repulsion between the ON and its target. We have examined the duplex stabilising effects of introducing cationic phosphoramidate internucleoside linkages into ON with a non-natural alpha-anomeric configuration. Cationic alpha-ON bound with high affinity to single-stranded DNA and RNA targets. Duplex stabilisation was proportional to the number of cationic modifications, with fully cationic ON having particularly high thermal stability. The average stabilisation was greatly increased at low ionic strength. The duplex formed between cationic alpha-ON and their RNA targets were not substrates for RNase H. The penalty in T(m) inflicted by a single mismatch, however, was high; suggesting that they are well suited as sequence-specific, steric-blocking, antisense agents. Using a well-described target sequence in the internal ribosome entry site of the human hepatitis C virus, we have confirmed this potential in a cell-free translation assay as well as in a whole cell assay. Interestingly, no vectorisation was necessary for the cationic alpha-ON in cell culture.