Cellular Antisense Activity of PNA-Oligo(bicycloguanidinium) Conjugates Forming Self-Assembled Nanoaggregates.

A series of peptide nucleic acid-oligo(bicycloguanidinium) (PNA-BGn ) conjugates were synthesized and characterized in terms of cellular antisense activity by using the pLuc750HeLa cell splice correction assay. PNA-BG4 conjugates exhibited low micromolar antisense activity, and their cellular activity required the presence of a hydrophobic silyl terminal protecting group on the oligo(BG) ligand and a minimum of four guanidinium units. Surprisingly, a nonlinear dose-response with an activity threshold around 3-4 µM, indicative of large cooperativity, was observed. Supported by light scattering and electron microscopy analyses, we propose that the activity, and thus cellular delivery, of these lipo-PNA-BG4 conjugates is dependent on self-assembled nanoaggregates. Finally, cellular activity was enhanced by the presence of serum. Therefore we conclude that the lipo-BG-PNA conjugates exhibit an unexpected mechanism for cell delivery and are of interest for further in vivo studies.

Nanomolar cellular antisense activity of peptide nucleic acid (PNA) cholic acid ("umbrella") and cholesterol conjugates delivered by cationic lipids.

Limited cellular uptake and low bioavailability of peptide nucleic acids (PNAs) have restricted widespread use of PNAs as antisense/antigene agents for cells in culture and not least for in vivo applications. We now report the synthesis and cellular antisense activity in cultured HeLa pLuc705 cells of cholesterol and cholic acid ("umbrella") derivatives of splice correction antisense PNA oligomers. While the conjugates alone were practically inactive up to 1 µM, their activity was dramatically improved when delivered by a cationic lipid transfection agent (LipofectAMINE2000). In particular, PNAs, conjugated to cholesterol through an ester hemisuccinate linker or to cholic acid, exhibited low nanomolar activity (EC(50) ∼ 25 nM). Excellent sequence specificity was retained, as mismatch PNA conjugates did not show any significant antisense activity. Furthermore, we show that increasing the transfection volume improved transfection efficiency, suggesting that accumulation (condensation) of the PNA/lipid complex on the cellular surface is part of the uptake mechanism. These results provide a novel, simple method for very efficient cellular delivery of PNA oligomers, especially using PNA-cholic acid conjugates which, in contrast to PNA-cholesterol conjugates, exhibit sufficient water solubility. The results also question the generality of using cholic acid "umbrella" derivatives as a delivery modality for antisense oligomers.

Cellular delivery and antisense effects of peptide nucleic acid conjugated to polyethyleneimine via disulfide linkers.

Peptide nucleic acid (PNA) is potentially an attractive antisense and antigene agent for which more efficient cellular delivery systems are still warranted. The cationic polymer polyethylenimine (PEI) is commonly used for cellular transfection of DNA and RNA complexes, but is not readily applicable for PNA due to the (inherent) charge neutrality of PNA. However, PEI could function as an efficient scaffold for PNA via chemical conjugation. Accordingly, we modified PEI with the amine-reactive heterobifunctional linker agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (with and without a PEG moiety) and further reacted this with a cysteine PNA. The level of modification was determined spectrophotometrically with high accuracy, and the PNA transfection efficiency of the conjugates was evaluated in an antisense luciferase splice-correction assay using HeLa pLuc705 cells. We find that PEI is an efficient vector for PNA delivery yielding significantly higher (up to 10-fold) antisense activity than an analogous PNA-octaarginine conjugate, even in the presence of chloroquine, which only slightly enhances the PEI-PNA activity. The PEI-PEG conjugates are preferred due to lower acute cellular toxicity. Finally, the method can be easily modified to allow for co-conjugation of other small molecules in a high-throughput screening assay that does not require a purification step.

Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid) conjugates targeting intron-exon junctions.

BACKGROUND: Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human cancer gene in JAR cells. METHODS: We screened 10 different 15 mer PNAs targeting intron2 at both the 5' - and the 3'-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512) targeting the 3'-splice site of intron3 with a complementarity of 4 bases to intron3 and 11 bases to exon4 for its splicing modulation effect. This PNA2512 was further tested for the effects on the mdm2 protein level as well as for inhibition of cell growth in combination with the DNA damaging agent camptothecin (CPT). RESULTS: We show that several of these PNAs effectively inhibit the splicing thereby producing a larger mRNA still containing intron2, while skipping of exon3 was not observed by any of these PNAs. The most effective PNA (PNA2406) targeting the 3'-splice site of intron2 had a complementarity of 4 bases to intron2 and 11 bases to exon3. PNA (2512) targeting the 3'-splice site of intron3 induced both splicing inhibition (intron3 skipping) and skipping of exon4. Furthermore, treatment of JAR cells with this PNA resulted in a reduction in the level of MDM2 protein and a concomitant increase in the level of tumor suppressor p53. In addition, a combination of this PNA with CPT inhibited cell growth more than CPT alone. CONCLUSION: We have identified several PNAs targeting the 5'- or 3'-splice sites in intron2 or the 3'-splice site of intron3 of mdm2 pre-mRNA which can inhibit splicing. Antisense targeting of splice junctions of mdm2 pre-mRNA may be a powerful method to evaluate the cellular function of MDM2 splice variants as well as a promising approach for discovery of mdm2 targeted anticancer drugs.

Subnanomolar antisense activity of phosphonate-peptide nucleic acid (PNA) conjugates delivered by cationic lipids to HeLa cells.

In the search of facile and efficient methods for cellular delivery of peptide nucleic acids (PNA), we have synthesized PNAs conjugated to oligophosphonates via phosphonate glutamine and bis-phosphonate lysine amino acid derivatives thereby introducing up to twelve phosphonate moieties into a PNA oligomer. This modification of the PNA does not interfere with the nucleic acid target binding affinity based on thermal stability of the PNA/RNA duplexes. When delivered to cultured HeLa pLuc705 cells by Lipofectamine, the PNAs showed dose-dependent nuclear antisense activity in the nanomolar range as inferred from induced luciferase activity as a consequence of pre-mRNA splicing correction by the antisense-PNA. Antisense activity depended on the number of phosphonate moieties and the most potent hexa-bis-phosphonate-PNA showed at least 20-fold higher activity than that of an optimized PNA/DNA hetero-duplex. These results indicate that conjugation of phosphonate moieties to the PNA can dramatically improve cellular delivery mediated by cationic lipids without affecting on the binding affinity and sequence discrimination ability, exhibiting EC(50) values down to one nanomolar. Thus the intracellular efficacy of PNA oligomers rival that of siRNA and the results therefore emphasize that provided sufficient in vivo bioavailability of PNA can be achieved these molecules may be developed into potent gene therapeutic drugs.

In Vitro Cellular Delivery of Peptide Nucleic Acid (PNA).

Cellular delivery methods are a prerequisite for cellular studies with PNA. This chapter describes PNA cellular delivery using cell-penetrating peptide (CPP)-PNA conjugates and transfection of PNA-ligand conjugates mediated by cationic lipids. Furthermore, two endosomolytic procedures employing chloroquine treatment or photochemical internalization (PCI) for significantly improving PNA delivery efficacy are described.

Enzyme-Triggered Release of the Antisense Octaarginine-PNA Conjugate from Phospholipase A2 Sensitive Liposomes.

Phospholipase sensitive liposomes (PSLs) have attracted great attention in targeted anticancer drug delivery due to cargo release triggered by tumor-secreted phospholipase A2 (sPLA2). Such liposomes could also serve as a vehicle for tissue-specific delivery of antisense therapeutics to (solid) tumors. While extensive studies on developing PSL formulations for small molecules exist, hardly any data are available on delivering larger molecules such as antisense agents. The present study demonstrates PSL encapsulation and phospholipase A2 triggered the release of a splice correcting, antisense octaarginine-peptide nucleic acid (octaarginine-PNA) conjugate. The results show that, although PNA can be efficiently encapsulated in PSL and also released using sPLA2 in serum-free conditions, the release is inhibited in the presence of serum. This is ascribed to the adsorption of serum proteins, including serum albumin and apolipoprotein C-III, to the surface of PSL (corona formation) and consequent prevention of sPLA2-mediated PNA release.

Cooperative Cellular Uptake and Activity of Octaarginine Antisense Peptide Nucleic acid (PNA) Conjugates.

Cellular uptake and antisense activity of d-octaarginine conjugated peptide nucleic acids (PNAs) is shown to exhibit pronounced cooperativity in serum-containing medium, in particular by being enhanced by analogous mis-match PNA-cell-penetrating peptide (PNA-CPP) conjugates without inherent antisense activity. This cooperativity does not show cell or PNA sequence dependency, suggesting that it is a common effect in cationic CPP conjugated PNA delivery. Interestingly, our results also indicate that Deca-r8-PNA and r8-PNA could assist each other and even other non-CPP PNAs as an uptake enhancer agent. However, the peptide itself (without being attached to the PNA) failed to enhance uptake and antisense activity. These results are compatible with an endosomal uptake mechanism in which the endocytosis event is induced by multiple CPP-PNA binding to the cell surface requiring a certain CPP density, possibly in terms of nanoparticle number and/or size, to be triggered. In particular the finding that the number of endosomal events is dependent on the total CPP-PNA concentration supports such a model. It is not possible from the present results to conclude whether endosomal escape is also cooperatively induced by CPP-PNA.

Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain.

Conjugation to cationic cell penetrating peptides (such as Tat, Penetratin, or oligo arginines) efficiently improves the cellular uptake of large hydrophilic molecules such as oligonucleotides and peptide nucleic acids, but the cellular uptake is predominantly via an unproductive endosomal pathway and therefore mechanisms that promote endosomal escape (or avoid the endosomal route) are required for improving bioavailability. A variety of auxiliary agents (chloroquine, calcium ions, or lipophilic photosensitizers) has this effect, but improved, unaided delivery would be highly advantageous in particular for future in vivo applications. We find that simply conjugating a lipid domain (fatty acid) to the cationic peptide (a CatLip conjugate) increases the biological effect of the corresponding PNA (CatLip) conjugates in a luciferase cellular antisense assay up to 2 orders of magnitude. The effect increases with increasing length of the fatty acid (C8-C16) but in parallel also results in increased cellular toxicity, with decanoic acid being optimal. Furthermore, the relative enhancement is significantly higher for Tat peptide compared to oligoarginine. Confocal microscopy and chloroquine enhancement indicates that the lipophilic domain increases the endosomal uptake as well as promoting significantly endosomal escape. These results provide a novel route for improving the (cellular) bioavailability of larger hydrophilic molecules.

Effective photo-enhancement of cellular activity of fluorophore-octaarginine antisense PNA conjugates correlates with singlet oxygen formation, endosomal escape and chromophore lipophilicity.

Photochemical internalization (PCI) is a cellular drug delivery method based on the generation of light-induced reactive oxygen species (ROS) causing damage to the endosomal membrane and thereby resulting in drug release to the cytoplasm. In our study a series of antisense fluorophore octaarginine peptide nucleic acid (PNA) conjugates were investigated in terms of PCI assisted cellular activity. It is found that tetramethylrhodamine and Alexa Fluor 555 conjugated octaarginine PNA upon irradiation exhibit more than ten-fold increase in antisense activity in the HeLa pLuc705 luciferase splice correction assay. An analogous fluorescein conjugate did not show any significant enhancement due to photobleaching, and neither did an Alexa Fluor 488 conjugate. Using fluorescence microscopy a correlation between endosomal escape and antisense activity was demonstrated, and in parallel a correlation to localized formation of ROS assigned primarily to singlet oxygen was also observed. The results show that tetramethylrhodamine (and to lesser extent Alexa Fluor 555) conjugated octaarginine PNAs are as effectively delivered to the cytosol compartment by PCI as by chloroquine assisted delivery and also indicate that efficient photodynamic endosomal escape is strongly dependent on the quantum yield for photochemical singlet oxygen formation, photostability as well as the lipophilicity of the chromophore.

Photochemically enhanced cellular delivery of cell penetrating peptide-PNA conjugates.

Recent studies have shown that endosomal release is a major rate-limiting step for cellular delivery via a variety of cationic cell penetrating peptides. Thus, methods and/or protocols for effective release of endosomally entrapped drugs are highly warranted. Photochemical internalization (PCI) has previously been proposed for this purpose. Here, we demonstrate an enhancement of up to two orders of magnitude of the antisense effects (cytosolic/nuclear) of peptide nucleic acid-peptide conjugates (Tat, Arg7, KLA) in HeLa cells by a PCI approach using the photosensitizer AlPc2a. These results emphasize the importance of endosomal release for cellular activity of this type of drug delivery and also raise hope that methods like PCI which have applications for in vivo use may also enhance the bioavailability and in vivo efficacy of these types of conjugates.

Evaluation of transfection protocols for unmodified and modified peptide nucleic acid (PNA) oligomers.

We have compared the efficacy of different transfection protocols reported for peptide nucleic acid (PNA) oligomers. A precise evaluation of uptake efficacy was achieved by using a positive readout assay based on the ability of a PNA oligomer to correct aberrant splicing of a recombinant luciferase gene. The study comprised transfection of PNA conjugated to acridine, adamantyl, decanoic acid, and porphyrine (acr-PNA, ada-PNA, deca-PNA, and por-RNA, respectively) and unmodified PNA partially hybridized to a DNA oligomer (PNA/DNA cotransfection). Furthermore, the effect of conjugation to a nuclear localization signal (NLS) was evaluated as part of the PNA/DNA cotransfection protocol. Transfection of the tested PNAs was systematically optimized. PNA/DNA cotransfection was found to produce the highest luciferase activity, but only after careful selection of the DNA oligonucleotide. Both a cationic lipid, Lipofectamine, and a nonliposomal cationic polymer, polyethylenimine (PEI, ExGen 500), were efficient transfection reagents for the PNA/DNA complex. However, Lipofectamine, in contrast to PEI, showed severe side effects, such as cytotoxicity. acr-PNA, ada-PNA, and por-PNA were transfectable with efficacies between 5 and 10 times lower than that seen with PNA/DNA cotransfection. Conjugation of PNA to NLS had no effect on PNA/DNA cotransfection efficacy. An important lesson from the study was the finding that because of uncontrollable biologic variations, even optimal transfection conditions differed to a certain extend from experiment to experiment in an unpredictable way.

Calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic tat and oligoarginine peptides.

Cell-penetrating peptides have been widely used to improve cellular delivery of a variety of proteins and antisense agents. However, recent studies indicate that such cationic peptides are predominantly entering cells via an endosomal pathway. We now show that the nuclear antisense effect in HeLa cells of a variety of peptide nucleic acid (PNA) peptide conjugates is significantly enhanced by addition of 6 mM Ca(2+) (as well as by the lysosomotrophic agent chloroquine). In particular, the antisense activities of Tat(48-60) and heptaarginine-conjugated PNAs were increased 44-fold and 8.5-fold, respectively. Evidence is presented that the mechanism involves endosomal release. The present results show that Ca(2+) can be used as an effective enhancer for in vitro cellular delivery of cationic peptide-conjugated PNA oligomers, and also emphasize the significance of the endosomal escape route for such peptides.

Down-regulation of MDM2 and activation of p53 in human cancer cells by antisense 9-aminoacridine-PNA (peptide nucleic acid) conjugates.

A series of peptide nucleic acid (PNA) oligomers targeting the mdm2 oncogene mRNA has been tested for the ability to inhibit the growth of JAR cells. The effect of these PNAs on the cells was also reflected in reduced levels of the MDM2 protein and increased levels of the p53 tumor suppressor protein, which is negatively regulated by MDM2. Initially, PNA oligomers were delivered as DNA complexes with lipofectamine, but it was discovered that PNA conjugated to the DNA intercalator 9-aminoacridine (Acr) (Acr-PNA) could be effectively delivered to JAR cells (as well as to HeLa pLuc705 cells) even in the absence of a DNA carrier. Using such lipofectamine-delivered Acr-PNA conjugates, one PNA targeting a cryptic AUG initiation site was identified that at a concentration of 2 microM caused a reduction of MDM2 levels to approximately 20% (but no reduction in mdm2 mRNA levels) and a 3-fold increase in p53 levels, whereas a 2-base mismatch control had no such effects. Furthermore, transcriptional activation by p53 was also increased (6-fold), and cell viability was reduced to 80%. Finally, this PNA acted cooperatively with camptothecin treatment both with regard to p53 activity induction as well as cell viability. Using this novel cell delivery system, we have identified a target on the mdm2 mRNA that appears sensitive to antisense inhibition by PNA and therefore could be used as a lead for further development of mdm2-targeted antisense (PNA and other) gene therapeutic anticancer drugs.

Electroporation Enhanced Effect of Dystrophin Splice Switching PNA Oligomers in Normal and Dystrophic Muscle.

Peptide nucleic acid (PNA) is a synthetic DNA mimic that has shown potential for discovery of novel splice switching antisense drugs. However, in vivo cellular delivery has been a limiting factor for development, and only few successful studies have been reported. As a possible modality for improvement of in vivo cellular availability, we have investigated the effect of electrotransfer upon intramuscular (i.m.) PNA administration in vivo. Antisense PNA targeting exon 23 of the murine dystrophin gene was administered by i.m. injection to the tibialis anterior (TA) muscle of normal NMRI and dystrophic mdx mice with or without electroporation. At low, single PNA doses (1.5, 3, or 10 µg/TA), electroporation augmented the antisense exon skipping induced by an unmodified PNA by twofold to fourfold in healthy mouse muscle with optimized electric parameters, measured after 7 days. The PNA splice switching was detected at the RNA level up to 4 weeks after a single-dose treatment. In dystrophic muscles of the MDX mouse, electroporation increased the number of dystrophin-positive fibers about 2.5-fold at 2 weeks after a single PNA administration compared to injection only. In conclusion, we find that electroporation can enhance PNA antisense effects in muscle tissue.

Quantitation of formate by solid-phase microextraction and gas chromatography--mass spectrometry utilizing a [13C]formate internal standard.

A new method for the analysis of formic acid was developed using gas chromatography-electron impact ionization mass spectrometry in the selected ion monitoring mode and solid-phase microextraction. Using this method with [13C]formic acid as an internal standard, the peak area ratio of [12C]formic acid/[13C]formic acid was not affected by differing methanol or sulfuric acid concentrations during the esterification and fiber adsorbing step. In comparison, the peak area ratio of formic acid/acetonitrile as detected by conventional GC with flame ionization detection was greatly affected by methanol or sulfuric acid concentrations. The formic acid calibration curve of our method showed excellent linearity over the range 5 to 200 microM. The within- and between-run assay relative standard deviations for the formic acid concentration were all less than 1.70%.

Methylamine treatment changes the allocation of carbohydrate to roots in rice plants.

Methylamine (MA), an ammonia analogue, has been used to investigate ammonia uptake. This compound competes with ammonia to be taken up and also inhibits the nitrate assimilation pathway. However, the effect of MA on plant growth is unknown. In this paper, we describe the responses of the rice plant to MA. The growth of MA-treated plants was inhibited in the aerial parts and stimulated in the roots. MA-treatment also induced a decrease of starch and hexose in shoots whereas hexose, sucrose and starch contents are increased in MA-treated roots. These results indicate that MA can change the mass allocation of biomass to the roots. The properties of MA suggest that a plant alters its growth via a change in the distribution of carbohydrate in resposes to the nitrogen status.

Cellular delivery of peptide nucleic acids (PNAs).

Cellular delivery methods are a prerequisite for cellular studies with PNA. This chapter describes PNA cellular delivery using cell-penetrating peptide (CPP)-PNA conjugates and transfection of PNA-ligand conjugates mediated by cationic lipids. Furthermore, two endosomolytic procedures employing chloroquine treatment or photochemical internalization (PCI) for significantly improving PNA delivery efficacy are described.

Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods.

Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA-octaarginine conjugate upon varying the cell culture transfection volume (and cell density) at fixed PNA concentration. The results show that for all delivery modalities the cellular antisense activity increases (less than proportionally) with increasing volume (in some cases accompanied with increased toxicity), and that this effect is more pronounced at higher cell densities. These results emphasize that transfection efficacy using cationic carriers is critically dependent on parameters such as transfection volume and cell density, and that these must be taken into account when comparing different delivery regimes.

Enhanced cellular delivery of cell-penetrating peptide-peptide nucleic acid conjugates by photochemical internalization.

Cell-penetrating peptides (CPPs) have been widely used for a cellular delivery of biologically relevant cargoes including antisense peptide nucleic acids (PNAs). Although chemical conjugation of PNA to a variety of CPPs significantly improves the cellular uptake of the PNAs, bioavailability (antisense activity) is still limited by endocytotic entrapment. We have shown that this low bioavailability can be greatly improved by combining CPP-PNA conjugate administration with a photochemical internalization technique using photosensitizers such as aluminum phthalocyanine (AlPcS(2a)) or tetraphenylporphyrin tetrasulfonic acid (TPPS). Cellular uptake of the PNA conjugates were evaluated by using a sensitive cellular method with HeLa pLuc705 cells based on the splicing correction of luciferase gene by targeting antisense oligonucleotides to a cryptic splice site of the mutated luciferase gene. The cellular efficacy of CPP conjugates were evaluated by measuring luciferase activity as a result of splicing correction and was also confirmed by RT-PCR analysis of luciferase pre-mRNA.