Steric-blocking Oligonucleotides Research Articles

Human disease-causing mutations result in loss of leiomodin 2 through nonsense-mediated mRNA decay.

The leiomodin (Lmod) family of actin-binding proteins play a critical role in muscle function, highlighted by the fact that mutations in all three family members (LMOD1-3) result in human myopathies. Mutations in the cardiac predominant isoform, LMOD2 lead to severe neonatal dilated cardiomyopathy. Most of the disease-causing mutations in the LMOD gene family are nonsense, or frameshift, mutations predicted to result in expression of truncated proteins. However, in nearly all cases of disease, little to no LMOD protein is expressed. We show here that nonsense-mediated mRNA decay, a cellular mechanism which eliminates mRNAs with premature termination codons, underlies loss of mutant protein from two independent LMOD2 disease-causing mutations. Furthermore, we generated steric-blocking oligonucleotides that obstruct deposition of the exon junction complex, preventing nonsense-mediated mRNA decay of mutant LMOD2 transcripts, thereby restoring mutant protein expression. Our investigation lays the initial groundwork for potential therapeutic intervention in LMOD-linked myopathies.

LncRNA MIAT Promotes Spinal Cord Injury Recovery in Rats by Regulating RBFOX2-Mediated Alternative Splicing of MCL-1.

LncRNA myocardial infarction-associated transcript (MIAT) alleviates acute spinal cord injury (ASCI)-induced neuronal cell apoptosis, but the specific mechanism of it involved in regulating SCI progression needs further exploration. Here, a SCI rat model was established, followed by administration with adenovirus-mediated MIAT overexpression vector (Ad-MIAT) alone or together with Ad-RBFOX2 (RNA binding fox-1 homolog 2). The data indicated that MIAT overexpression promoted motor function recovery, improved morphology of injured tissues, and restrained neuron loss and cell apoptosis in SCI rats. Then, PC-12 cells were treated with H2O2 to induce cell injury. And highly expressed MIAT suppressed H2O2-caused decrease in cell viability and increase in cell apoptosis. MIAT stabilized RBFOX2 protein expression by binding to RBFOX2, thereby promoting RBFOX2-induced upregulation of anti-apoptotic MCL-1L (myeloid cell leukemia sequence 1) and reduction of pro-apoptotic MCL-1S. And silencing RBFOX2 in vitro blocked the inhibitory effect of MIAT on cell apoptosis. Moreover, MCL-1-specific steric-blocking oligonucleotides (SBOs) were used to transfer the MCL-1 pre-mRNA splicing pattern from MCL-1L to MCL-1S. SBOs reversed the protection effect of RBFOX2 overexpression on H2O2-induced cell injury. Furthermore, overexpression of MCL-1L instead of MCL-1S facilitated autophagy activation in H2O2-stimulated cells. Interestingly, co-overexpression of MIAT and RBFOX2 had a better promoting effect on SCI recovery. In conclusion, MIAT mitigated SCI by promoting RBFOX2-mediated alternative splicing of MCL-1. Our findings might provide a promising therapeutic target for SCI.

Transcriptome-Wide Off-Target Effects of Steric-Blocking Oligonucleotides.

Steric-blocking oligonucleotides (SBOs) are short, single-stranded nucleic acids designed to modulate gene expression by binding to RNA transcripts and blocking access from cellular machinery such as splicing factors. SBOs have the potential to bind to near-complementary sites in the transcriptome, causing off-target effects. In this study, we used RNA-seq to evaluate the off-target differential splicing events of 81 SBOs and differential expression events of 46 SBOs. Our results suggest that differential splicing events are predominantly hybridization driven, whereas differential expression events are more common and driven by other mechanisms (including spurious experimental variation). We further evaluated the performance of in silico screens for off-target splicing events, and found an edit distance cutoff of three to result in a sensitivity of 14% and false discovery rate (FDR) of 99%. A machine learning model incorporating splicing predictions substantially improved the ability to prioritize low edit distance hits, increasing sensitivity from 4% to 26% at a fixed FDR of 90%. Despite these large improvements in performance, this approach does not detect the majority of events at an FDR <99%. Our results suggest that in silico methods are currently of limited use for predicting the off-target effects of SBOs, and experimental screening by RNA-seq should be the preferred approach.

Modification of Mcl-1 alternative splicing induces apoptosis and suppresses tumor proliferation in gastric cancer.

Splicing dysregulation, which leads to apoptosis resistance, has been recognized as a major hallmark for tumorigenesis and cancer progression. Targeting alternative splicing by either increasing pro-apoptotic proteins or inhibiting anti-apoptotic proteins in tumor cells may be an effective approach for gastric cancer (GC) therapy. However, the role of modulation of alternative splicing in GC remains poorly understood. In this study, to the best of our knowledge, the unbalanced expression of the myeloid cell leukemia-1 (Mcl-1) splicing variants, Mcl-1L and Mcl-1S, was identified in GC patients for the first time. Increasing anti-apoptotic Mcl-1L and decreasing pro-apoptotic Mcl-1S expression levels were correlated with tumor proliferation and poor survival. In vitro data showed that a shift in splicing from Mcl-1L to Mcl-1S induced by treatment with Mcl-1-specific steric-blocking oligonucleotides (SBOs) efficiently decreased Mcl-1L expression, increased Mcl-1S expression, and accelerated tumor cell apoptosis in a dose-dependent manner. Additionally, mouse xenotransplant models confirmed that modification of Mcl-1 alternative splicing increased tumor cell death and suppressed tumor proliferation. This study demonstrated that the modification of Mcl-1 splicing might stimulate the pro-apoptotic factor and inhibit the anti-apoptotic protein to induce significant apoptosis. Thus, this finding provided a strategy for cancer therapy, according to which SBOs could be used to change the Mcl-1 splicing pattern, thereby inducing apoptosis.

Cell-Penetrating Peptide Conjugates of Steric Blocking Oligonucleotides as Therapeutics for Neuromuscular Diseases from a Historical Perspective to Current Prospects of Treatment.

The review starts with a historical perspective of the achievements of the Gait group in synthesis of oligonucleotides (ONs) and their peptide conjugates toward the award of the 2017 Oligonucleotide Therapeutic Society Lifetime Achievement Award. This acts as a prelude to the rewarding collaborative studies in the Gait and Wood research groups aimed toward the enhanced delivery of charge neutral ON drugs and the development of a series of Arg-rich cell-penetrating peptides called Pip (peptide nucleic acid/phosphorodiamidate morpholino oligonucleotide [PNA/PMO] internalization peptides) as conjugates of such ONs. In this review we concentrate on these developments toward the treatment of the neuromuscular diseases Duchenne muscular dystrophy and spinal muscular atrophy toward a platform technology for the enhancement of cellular and in vivo delivery suitable for widespread use as neuromuscular and neurodegenerative ON drugs.

Oligonucleotide Therapies: The Past and the Present.

In this review we address the development of oligonucleotide (ON) medicines from a historical perspective by listing the landmark discoveries in this field. The various biological processes that have been targeted and the corresponding ON interventions found in the literature are discussed together with brief updates on some of the more recent developments. Most ON therapies act through antisense mechanisms and are directed against various RNA species, as exemplified by gapmers, steric block ONs, antagomirs, small interfering RNAs (siRNAs), micro-RNA mimics, and splice switching ONs. However, ONs binding to Toll-like receptors and those forming aptamers have completely different modes of action. Similar to other novel medicines, the path to success has been lined with numerous failures, where different therapeutic ONs did not stand the test of time. Since the first ON drug was approved for clinical use in 1998, the therapeutic landscape has changed considerably, but many challenges remain until the expectations for this new form of medicine are met. However, there is room for cautious optimism.

RNA therapeutics: beyond RNA interference and antisense oligonucleotides.

Here, we discuss three RNA-based therapeutic technologies exploiting various oligonucleotides that bind to RNA by base pairing in a sequence-specific manner yet have different mechanisms of action and effects. RNA interference and antisense oligonucleotides downregulate gene expression by inducing enzyme-dependent degradation of targeted mRNA. Steric-blocking oligonucleotides block the access of cellular machinery to pre-mRNA and mRNA without degrading the RNA. Through this mechanism, steric-blocking oligonucleotides can redirect alternative splicing, repair defective RNA, restore protein production or downregulate gene expression. Moreover, they can be extensively chemically modified to acquire more drug-like properties. The ability of RNA-blocking oligonucleotides to restore gene function makes them best suited for the treatment of genetic disorders. Positive results from clinical trials for the treatment of Duchenne muscular dystrophy show that this technology is close to achieving its clinical potential.

Peptide-mediated Cell and In Vivo Delivery of Antisense Oligonucleotides and siRNA

Antisense oligonucleotides and short interfering RNAs (siRNAs) are nucleic acids-targeting reagents for gene expression modulation that are being developed as drugs for many applications. A number of useful synthetic nucleic acids analogues have been introduced recently to greatly improve their properties for use as therapeutics. However, their full effectiveness in cells and in vivo has often only been realized through development of suitable nonviral delivery systems. Among these, a range of natural and synthetic peptides have been found useful for enhancing cellular uptake and/or cell targeting of oligonucleotide analogues and siRNA. Such peptides are synthetically conjugated, used as noncovalent complexes, or used in combination with polymer, liposomal or exosome formulation techniques. This review begins by describing the modes of action of antisense reagents and siRNA and goes on to focus on recent advances in their peptide-mediated cell and in vivo delivery and how peptide use has influenced drug development. The review discusses the challenges associated with understanding the physiological and toxicological aspects of peptide-mediated delivery. Developments towards clinical use are also highlighted, with particular emphasis on peptide conjugates of oligonucleotide analogues used for treatment of neuromuscular diseases.

A non-covalent strategy combining cationic lipids and CPPs to enhance the delivery of splice correcting oligonucleotides

Modulation of pre-mRNA splicing by steric-block oligonucleotides constitutes a promising strategy for the treatment of many diseases, but requires efficient delivery to cell nuclei. In the present study, we evaluated the efficacy of a non-covalent strategy that combines a cell penetrating peptide with a lipoplex-based formulation to mediate the delivery of splice-switching oligonucleotides. The splice correcting ability of these new formulations was assessed using splice-switching oligonucleotides targeted towards the mutated splicing site of human β-globin pre-mRNA in the HeLa pLuc/705 splice correction model. Importantly, the optimal splice correcting activity was exhibited by the formulation containing both lipid and peptide components, the order of component addition in these formulations being crucial for their efficacy. Our results demonstrate that the inclusion of cationic liposomes in the formulation provides the ability to improve release from endocytic vesicles, a barrier that severely limits the efficiency of oligonucleotide delivery by cell penetrating peptides. On the other hand, cell penetrating peptides potentiate the cellular uptake and delivery of the oligonucleotides by the lipoplexes. Moreover, when combining cell penetrating peptides with the lipoplex formulations, a significant reduction in the amount of required cationic lipid could be achieved, while maintaining or even enhancing biological activity.

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.

Noncovalent Peptide-Mediated Delivery of Chemically Modified Steric Block Oligonucleotides Promotes Splice Correction: Quantitative Analysis of Uptake and Biological Effect

Despite numerous encouraging reports in the literature, the efficiency of cell penetrating peptides (CPPs) in promoting cellular delivery of bioactive cargos is still limited. To extend our current understanding of the underlying limitations of such approaches, we performed quantitative uptake studies of different chemically modified (2'-O-methyl, LNA and PNA) steric block oligonucleotides, targeted against a mutated splice site inserted in a firefly luciferase reporter gene construct, applying the peptide carrier MPGalpha as a model system. The peptide formed stable noncovalent complexes with phosphorothioate oligonucleotide (PTO) and locked nucleic acid (LNA) modified oligonucleotides, whereas the neutral peptide nucleic acid (PNA) had to be hybridized to an unmodified DNA to allow for complex formation. Detailed quantitative uptake studies revealed comparable numbers of intracellular PTO and LNA oligonucleotides after peptide-mediated delivery. Surprisingly, the PTO derivative showed the strongest upregulation of reporter gene activity of about 100-fold followed by the PNA (40-fold) and LNA (10-fold). Electroporation and microinjection studies proved that delivery itself was not the limiting factor for the low activity of the LNA derivative. Maximal achievable reporter gene activity could be observed only after addition of chloroquine (CQ), indicative of an endosomal pathway involved. This is in line with nuclear microinjection experiments, which show that the minimal number of steric block molecules needed to trigger the observed reporter upregulation is about two orders of magnitude lower than determined after peptide or cationic lipid delivery.

Peptide-Mediated Cellular Delivery of Oligonucleotide-Based Therapeutics In Vitro: Quantitative Evaluation of Overall Efficacy Employing Easy to Handle Reporter Systems

Cellular uptake of therapeutic oligonucleotides and subsequent intracellular trafficking to their target sites represents the major technical hurdle for the biological effectiveness of these potential drugs. Accordingly, laboratories worldwide focus on the development of suitable delivery systems. Among the different available non-viral systems like cationic polymers, cationic liposomes and polymeric nanoparticles, cell-penetrating peptides (CPPs) represent an attractive concept to bypass the problem of poor membrane permeability of these charged macromolecules. While uptake per se in most cases does not represent the main obstacle of nucleic acid delivery in vitro, it becomes increasingly apparent that intracellular trafficking is the bottleneck. As a consequence, in order to optimize a given delivery system, a side-by-side analysis of nucleic acid cargo internalized and the corresponding biological effect is required to determine the overall efficacy. In this review, we will concentrate on peptide-mediated delivery of siRNAs and steric block oligonucleotides and discuss different methods for quantitative assessment of the amount of cargo taken up and how to correlate those numbers with biological effects by applying easy to handle reporter systems. To illustrate current limitations of non-viral nucleic acid delivery systems, we present own data as an example and discuss options of how to enhance trafficking of molecules entrapped in cellular compartments.

Arginine‐rich cell penetrating peptides: Design, structure–activity, and applications to alter pre‐mRNA splicing by steric‐block oligonucleotides

Rerouting the splicing machinery with steric-block oligonucleotides (ON) might lead to new therapeutic strategies in the treatment of diseases such as beta-thalassemia, Duchenne muscular dystrophy, or cancers. Interfering with splicing requires the sequence-specific and stable hybridization of RNase H-incompetent ON as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO). Unfortunately, these uncharged DNA mimics are poorly taken up by most cell types and conventional delivery strategies that rely on electrostatic interaction do not apply. Likewise, conjugation to cell penetrating peptides (CPPs) as Tat, Arg9, Lys8, or Pen leads to poor splicing correction efficiency at low concentration essentially because PNA- and PMO-CPP conjugates remain entrapped within endocytotic vesicles. Recently, we have designed an arginine-rich peptide (R-Ahx-R)4 (with Ahx for aminohexanoic acid) and an arginine-tailed Penetratin derivative which allow sequence-specific and efficient splicing correction at low concentration in the absence of endosomolytic agents. Both CPPs are undergoing structure-activity relationship studies for further optimization as steric-block ON delivery vectors.

Cell-penetrating-peptide-based delivery of oligonucleotides: an overview

Cationic CPPs (cell-penetrating peptides) have been used largely for intracellular delivery of low-molecular-mass drugs, biomolecules and particles. Most cationic CPPs bind to cell-associated glycosaminoglycans and are internalized by endocytosis, although the detailed mechanisms involved remain controversial. Sequestration and degradation in endocytic vesicles severely limits the efficiency of cytoplasmic and/or nuclear delivery of CPP-conjugated material. Re-routing the splicing machinery by using steric-block ON (oligonucleotide) analogues, such as PNAs (peptide nucleic acids) or PMOs (phosphorodiamidate morpholino oligomers), has consequently been inefficient when ONs are conjugated with standard CPPs such as Tat (transactivator of transcription), R(9) (nona-arginine), K(8) (octalysine) or penetratin in the absence of endosomolytic agents. New arginine-rich CPPs such as (R-Ahx-R)(4) (6-aminohexanoic acid-spaced oligo-arginine) or R(6) (hexa-arginine)-penetratin conjugated to PMO or PNA resulted in efficient splicing correction at non-cytotoxic doses in the absence of chloroquine. SAR (structure-activity relationship) analyses are underway to optimize these peptide delivery vectors and to understand their mechanisms of cellular internalization.

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 μM) 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 μM 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.

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.

Peptide-based delivery of nucleic acids: design, mechanism of uptake and applications to splice-correcting oligonucleotides

CPPs (cell-penetrating peptides) have given rise to much interest for the delivery of biomolecules such as peptides, proteins or ONs (oligonucleotides). CPPs and their conjugates were initially thought to translocate through the cell membrane by a non-endocytotic mechanism which has recently been re-evaluated. Basic-amino-acid-rich CPPs first interact with cell-surface proteoglycans before being internalized by endocytosis. Sequestration and degradation in endocytotic vesicles severely limits the cytoplasmic and nuclear delivery of the conjugated biomolecules. Accordingly, splicing correction by CPP-conjugated steric-block ON analogues is inefficient in the absence of endosomolytic agents. New arginine-rich CPPs allowing efficient splicing correction by conjugated PNAs (peptide nucleic acids) or PMO (phosphorodiamidate morpholino oligomer) steric blockers in the absence of endosomolytic agents have recently been defined in our group and are currently being characterized. They offer promising leads for the development of efficient cellular delivery vectors for therapeutic steric-block ON analogues.

Anti‐HIV Activity of Steric Block Oligonucleotides

The unabated increase in spread of HIV infection worldwide has redoubled efforts to discover novel antiviral and virucidal agents that might be starting points for clinical development. Oligonucleotides and their analogs targeted to form complementary duplexes with highly conserved regions of the HIV RNA have shown significant antiviral activity, but to date clinical studies have been dominated by RNase H-inducing oligonucleotide analog phosphorothioates (GEM 91 and 92) that have specificity and efficacy limitations. However, they have proven the principle that oligonucleotides can be safe anti-HIV drugs. Newer oligonucleotide analogs are now available, which act as strong steric block agents of HIV RNA function. We describe our ongoing studies targeting the HIV-1 trans-activation responsive region (TAR) and the viral packaging signal (psi) with steric block oligonucleotides of varying chemistry and demonstrate their great potential for steric blocking of viral protein interactions in vitro and in cells and describe the first antiviral studies. Peptide nucleic acids (PNA) disulfide linked to cell-penetrating peptides (CPP) have been found to have particular promise for the lipid-free direct delivery into cultured cells and are excellent candidates for their development as antiviral and virucidal agents.

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 β-globin pre-mRNA in the HeLa pLuc705 splice correction model. In contrast to Tat48–60 (Tat) and oligoarginine (R 9F 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.

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.