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In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine β-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies.

MicroRNAs are a family of small noncoding RNAs regulating gene expression by sequence-selective mRNA targeting, leading to a translational repression or mRNA degradation. The oncomiR miR-221 is highly expressed in human gliomas, as confirmed in this study in samples of low and high grade gliomas, as well in the cell lines U251, U373 and T98G. In order to alter the biological functions of miR-221, a peptide nucleic acid targeting miR-221 (R8-PNA-a221) was produced, bearing a oligoarginine peptide (R8) to facilitate uptake by glioma cells. The effects of R8-PNA-a221 were analyzed in U251, U373 and T98G glioma cells and found to strongly inhibit miR-221. In addition, the effects of R8-PNA-a221 on p27(Kip1) (a target of miR-221) were analyzed in U251 and T98G cells by RT-qPCR and by Western blotting. No change of p27(Kip1) mRNA content occurs in U251 cells in the presence of PNA-a221 (lacking the R8 peptide), whereas significant increase of p27(Kip1) mRNA was observed with the R8-PNA-a221. These data were confirmed by Western blot assay. A clear increment of p27(Kip1) protein expression in the samples treated with R8-PNA-a221 was detected. In addition, R8-PNA-a221 was found able to increase TIMP3 expression (another target of miR-221) in T98G cells. These results suggest that PNAs against oncomiRNA miR-221 might be proposed for experimental treatment of human gliomas.

Molecules able to interfere in miRNA genesis and function are potent tools to unravel maturation and processing pathways. Antisense oligonucleotides or analogs are actually employed for the inhibition of miRNA function. Here we illustrate how Peptide Nucleic Acids oligomers targeting pre-miRNA are exploited to inhibit miRNA maturation.

The involvement of microRNAs in human pathologies is a firmly established fact. Accordingly, the pharmacological modulation of their activity appears to be a very appealing issue in the development of new types of drugs (miRNA therapeutics). One of the most interesting issues is the possible development of miRNA therapeutics for development of anti-cancer molecules. In this respect appealing molecules are based on peptide nucleic acids (PNAs), displaying a pseudo-peptide backbone composed of N-(2-aminoethyl)glycine units and found to be excellent candidates for antisense and antigene therapies. The major limit in the use of PNAs for alteration of gene expression is the low uptake by eukaryotic cells. The aim of this chapter is to describe methods for determining the activity of PNAs designed to target oncomiRNAs, using as model system miR-221 and its target p27(Kip1) mRNA. The effects of PNAs targeting miR-221 are here presented discussing data obtained using as model system the human breast cancer cell line MDA-MB-231, in which miR-221 is up-regulated and p27(Kip1) down-regulated.

The activity of a peptide nucleic acid (PNA) targeting cancer-associated microRNA-221 is described. PNAs against miR-221 were designed in order to bind very efficiently to the target RNA strand and to undergo efficient uptake in the cells. A polyarginine-PNA conjugate targeted against miR-221 (Rpep-PNA-a221) showed both very high affinity for RNA and efficient cellular uptake without the addition of transfection reagents. Unmodified PNA with the same sequence displayed RNA binding, but cellular uptake was very poor. Consistently, only Rpep-PNA-a221 strongly inhibited miR-221. Targeting miR-221 by PNA resulted in i) lowering of the hybridization levels of miR-221 measured by RT-qPCR, ii) upregulation of p27Kip1 gene expression, measured by RT-qPCR and western blot analysis. The major conclusion of this study is that efficient delivery of anti‑miR PNA through a suitable peptide carrier (Rpep‑PNA-a221) leads to inhibition of miR-221 activity, altering the expression of miR-221-regulated functions in breast cancer cells.

A series of 18-mer peptide nucleic acids (PNAs) targeted against micro-RNA miR-210 was synthesised and tested in a cellular system. Unmodified PNAs, R(8) -conjugated PNAs and modified PNAs containing eight arginine residues on the backbone, either as C2-modified (R) or C5-modified (S) monomers, all with the same sequence, were compared. Two different models were used for the modified PNAs: one with alternated chiral and achiral monomers and one with a stretch of chiral monomers at the N terminus. The melting temperatures of these derivatives were found to be extremely high and 5 M urea was used to assess differences between the different structures. FACS analysis and qRT-PCR on K562 chronic myelogenous leukaemic cells indicated that arginine-conjugated and backbone-modified PNAs display good cellular uptake, with best performances for the C2-modified series. Resistance to enzymatic degradation was found to be higher for the backbone-modified PNAs, thus enhancing the advantage of using these derivatives rather than conjugated PNAs in the cells in serum, and this effect is magnified in the presence of peptidases such as trypsin. Inhibition of miR-210 activity led to changes in the erythroid differentiation pathway, which were more evident in mithramycin-treated cells. Interestingly, the anti-miR activities differed with use of different PNAs, thus suggesting a role of the substituents not only in the cellular uptake, but also in the mechanism of miR recognition and inactivation. This is the first report relating to the use of backbone-modified PNAs as anti-miR agents. The results clearly indicate that backbone-modified PNAs are good candidates for the development of very efficient drugs based on anti-miR activity, due to their enhanced bioavailabilities, and that overall anti-miR performance is a combination of cellular uptake and RNA binding., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

PNAs conjugated to carrier peptides have been employed for the targeting of miRNA precursor, with the aim to develop molecules able to interfere in the pre-miRNA processing. The capability of the molecules to bind pre-miRNA has been tested in vitro by fluorescence assayes on Thiazole Orange labeled molecules and in vivo, in K562 cells, evaluating the amount of miRNA produced after treatment of cells with two amounts of PNAs.

Herein we describe the activity of a peptide nucleic acid (PNA) that targets microRNA-210 (miR-210), which is associated with hypoxia and is modulated during erythroid differentiation. PNAs directed against miR-210 were designed to bind with high affinity to the target RNA strand and to undergo efficient uptake in target cells. A polyarginine-PNA conjugate directed against miR-210 (Rpep-PNA-a210) showed both very high affinity for RNA and efficient uptake into target cells without the need for transfection reagents. An unmodified PNA of the same sequence displayed the ability to bind RNA, but cellular uptake was very poor. Consistent with this, only Rpep-PNA-a210 strongly inhibited miR-210 activity, as evaluated by assays on undifferentiated K562 cells and on cells treated with mithramycin, which was found to induce erythroid differentiation and miR-210 overexpression. Targeting miR-210 by Rpep-PNA-a210 resulted in: 1) a decrease in miR-210 levels as measured by RT-PCR, 2) up-regulation of raptor mRNA, 3) a decrease in γ-globin mRNA, and 4) decreased expression of differentiated functions (i.e., proportion of benzidine-positive cells, content of embryo-fetal hemoglobins). The efficient delivery of anti-miR PNAs through a suitable peptide carrier (Rpep-PNA-a210) leads to the inhibition of miR-210 activity, altering the expression of miR-210-regulated erythroid functions., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Peptide nucleic acids (PNAs) are DNA/RNA mimics extensively used for pharmacological regulation of gene expression in a variety of cellular and molecular systems, and they have been described as excellent candidates for antisense and antigene therapies. At present, very few data are available on the use of PNAs as molecules targeting miRNAs. miRNAs are a family of small nc RNAs that regulate gene expression by sequence-selective targeting of mRNAs, leading to a translational repression or mRNA degradation to the control of highly regulated biological functions, such as differentiation, cell cycle and apoptosis. The aim of this article is to present the state-of-the-art concerning the possible use of PNAs to target miRNAs and modify their biological metabolism within the cells. The results present in the literature allow to propose PNA-based molecules as very promising reagents to modulate the biological activity of miRNAs. In consideration of the involvement of miRNAs in human pathologies, PNA-mediated targeting of miRNAs has been proposed as a potential novel therapeutic approach.

The ability of PNA to interact with DNA double stranded has been recently investigated. In a decoy approach these interactions are of great importance as may lead to inhibition of interactions of DNA sequences to specific transcription factors and may be employed as a strategy for the inhibition of gene transcription alternative to the antisense strategy (targeting transcription factors mRNAs) and the transcription factor decoy approach (targeting transcription factors). We explored the ability of PNA and PNAs with modified monomers to bind to DNA and to interfere in the formation of DNA/transcription factor complex. We report a procedure for the synthesis of Fmoc-gamma-hydroxymetyl PNA, the synthesis and CD analysis of PNA oligomers containing the modified monomer in different positions and EMSA assays to test the: (a) binding to double stranded DNA and (b) inhibition of DNA-protein interactions., (2010 Elsevier Inc. All rights reserved.)

Peptide nucleic acids (PNAs) are oligonucleotide mimics in which the sugar-phosphate backbone has been replaced by a pseudo-peptide backbone. Among PNA-based molecules, PNA-DNA conjugates characterized by tracts of DNA bound to N and/or C terminus of PNA are very soluble in aqueous media, are able to recognize exclusively single strands of DNA and RNA in antiparallel fashion, activate RNAse H, bind to transcription factors and are more stable than DNA to nucleases degradation. Very little information is available on chimeras constituted of alternating monomers of PNA and DNA. In this article, we describe a simple fully automated strategy for the synthesis of 6-mer and 10-mer alternate PNA-DNA chimeras consisting of polythymine oligomers, stability assays in fetal calf serum, UV and CD studies of the single strand alternate chimeras and of alternate chimera/DNA and alternate chimera/RNA duplexes. Evidences supporting the formation of duplex hybrids were found. Furthermore, the ability of forming Hoogsteen base pairing with duplex DNA was investigated. Finally, we tested the ability of the PNA-DNA alternates in (a) interfering with reverse transcription of eukaryotic mRNA and (b) inhibiting DNA-protein interactions., ((c) 2007 Wiley Periodicals, Inc.)

The major aim of this paper was to determine whether cationic microspheres (CM), consisting of the permeable polymer Eudragit RS 100 plus the cationic surfactant dioctadecyl-dimethyl-ammonium bromide (DDAB(18)), could bind to double-stranded peptide nucleic acid PNA-DNA-PNA (PDP) chimeras exhibiting decoy activity against NF-kappaB transcription factors. Microspheres were produced by the 'solvent evaporation method' and centrifugation at 500, 1,000 and 3,000 rpm to obtain different-sized microparticles. Microsphere morphology, size and size distribution were determined by optical and electron microscopy observations. In order to determine their binding activity, double-stranded DNA-based and PDP-based decoy molecules were incubated with different amounts of microparticles in the presence of 100 ng of either (32)P-labeled DNA-DNA or DNA-PDP hybrid molecules or cold PDP-PDP hybrids. The complexes were analyzed by agarose gel electrophoresis. The resistance of (32)P-labeled DNA-DNA and DNA-PDP molecules in the presence of serum or cellular extracts was evaluated after binding to CM by gel electrophoresis analysis. DDAB(18) Eudragit RS 100 microspheres are able to bind to DNA-PDP and PDP-PDP hybrids, to deliver these molecules to target cells and to protect DNA-PDP molecules from enzymatic degradation in simulated biological fluids. In addition, when assayed in ex vivo conditions, DDAB(18) Eudragit RS 100 microspheres exhibited low toxicity. The results presented in this paper demonstrate that CM can be considered suitable formulations for pharmacogenomic therapy employing double-stranded PDP chimeras.

Peptide nucleic acids (PNAs) are DNA-mimicking molecules in which the sugar-phosphate backbone is replaced by a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units. We determined whether double-stranded molecules based on PNAs and PNA-DNA-PNA (PDP) chimeras could be capable of stable interactions with nuclear proteins belonging to the Sp1 transcription factor family and, therefore, could act as decoy reagents able to inhibit molecular interactions between Sp1 and DNA. Since the structure of PNA/PNA hybrids is very different from that of the DNA/DNA double helix, they could theoretically alter the molecular structure of the double-stranded PNA-DNA-PNA chimeras, perturbing interactions with specific transcription factors. We found that PNA-based hybrids do not inhibit Sp1/DNA interactions. In contrast, hybrid molecules based on PNA-DNA-PNA chimeras are very effective decoy molecules, encouraging further experiments focused on the possible use of these molecules for the development of potential agents for a decoy approach in gene therapy. In this respect, the finding that PDP-based decoy molecules are more resistant than DNA/DNA hybrids to enzymatic degradation appears to be of great interest. Furthermore, their resistance can even be improved after complexation with cationic liposomes to which PDP/PDP chimeras are able to bind by virtue of their internal DNA structure.

Double-stranded molecules based on peptide nucleic acids (PNAs)-DNA chimeras carrying binding sites for known transcription factors could be of great interest in decoy pharmacotherapy of neoplastic diseases. For instance, decoy molecules recognizing Sp1 and NF-kappaB transcription factors were found to inhibit tumor cell growth and invasion activity. In this respect, we have recently found that double-stranded PNA-DNA chimeras carrying NF-kappaB binding sites inhibit the binding of NF-kappaB p52 and p50 transcription factors to target DNA molecules. In this article we determined the resistance of double-stranded decoy molecules based on PNA-DNA chimeras to exonucleases (both 3'-->5' and 5'-->3' exonucleases), endonucleases, and 5'-phosphatases. In addition, we performed experiments aimed at determining the resistance of these molecules in cellular extracts and serum. Finally, we used liposomes as protective agents in experimental conditions in which the decoy molecules employed were found to be unstable (high concentrations of enzymes, cellular extracts, or serum). The results obtained demonstrated that decoy molecules based on PNA-DNA chimeras are more resistant than DNA-based decoys to exo- and endonucleases, serum, and cellular extracts. In addition, the resistance of DNA/PNA hybrids in the presence of high concentrations of serum and cellular extracts was increased after complexation to cationic liposomes, due to the fact that double-stranded PNA-DNA-PNA chimeras bind to these delivery systems. The results obtained in the present study support the proposal of molecules based on PNA-DNA chimeras for an efficient decoy treatment of tumor cells both in vitro and in vivo.

Peptide nucleic acids (PNAs) have been recently proposed as useful molecules in pharmacogenetic therapy, especially due to the fact that they show a very high stability with respect to DNA and RNA. However, PNAs are not efficient decoy molecules, are characterized by negligible cell internalization and low solubility and are not suitable to be delivered by liposomes. With respect to the biological activity of PNA-based molecules, PDP deserve great consideration, due to the fact that they exhibit high levels of solubility, and are expected to be resistant to proteinases and exonucleases. In this manuscript we determined whether double-stranded molecules based on PNA-DNA chimeras containing NF-kappaB binding sites, exhibit decoy activity against NF-kappaB transcription factors. In addition, we determined whether they can be complexed by cationic liposomes. The results obtained demonstrated that hybrids based on PNA-DNA chimeras are powerful decoy molecules against NF-kappaB p52 transcription factor. In addition, we found that cationic liposomes can be proposed for in vitro delivery to target cells of these decoy molecules. The results presented in this paper are thus of practical importance, since the simplicity and the versatility of the cationic liposome technology have made cationic liposomes useful nonviral gene delivery systems for human gene therapy.

The inhibition of gene transcription mediated by peptide nucleic acids (PNAs) has been described as mainly due to a process that involves strand invasion of target DNA containing homopurine stretches with generation of PNA/DNA/PNA triplexes. Scarce information is available on DNA strand invasion of mixed purine-pyrimidine stretches and theirs possible use as molecules able to interact with transcription factors. With this aim, we compared the effects of DNA/DNA, DNA/PNA or PNA/PNA hybrid molecules mimicking the NF-kappaB binding sites of HIV-1 on LTR-driven in vitro transcription. The results allowed us to conclude that PNA/PNA molecules and DNA/PNA hybrids are capable of inhibiting transcription, like the DNA/DNA. We investigated the mechanisms by which PNA/PNA and DNA/PNA hybrids affect the transcription. The results suggest that DNA/PNA and PNA/PNA molecules inhibit transcription in distinct manner; the inhibitory effects are due to strand invasion in the case of PNA/PNA hybrids and decoy effects on transcription factors belonging the NF-kappaB/Rel family in the case of DNA/PNA hybrids. It is noteworthy that DNA/PNA hybrids are more resistant to nucleases than DNA/DNA hybrids.

The decoy approach against nuclear factor κB (NF-κB) is a useful tool to alter NF-κB dependent gene expression using synthetic oligonucleotides (ODNs) carrying NF-κB specific cis-elements. Unfortunately, ODNs are not stable and need to be be extensively modified to be used in vivo or ex vivo. We have previously evaluated the possible use of peptide nucleic acids (PNAs) as decoy molecules. The backbone of PNAs is composed of N-(2-aminoethyl)glycine units, rendering these molecules resistant to both nucleases and proteases. We found that the binding of NF-κB transcription factors to PNAs was either very low (binding to PNA–PNA hybrids) or exhibited low stability (binding to PNA–DNA hybrids). The main consideration of the present paper was to determine whether PNA–DNA chimeras mimicking NF-κB binding sites are capable of stable interactions with proteins belonging to the NF-κB family. Molecular modeling was employed for the design of PNA–DNA chimeras; prediction of molecular interactions between chimeras and NF-κB nuclear proteins were investigated by molecular dynamics simulations, and interactions between PNA–DNA chimeras and NF-κB proteins were studied by gel shifts. We found significant differences between the structure of duplex NF-κB PNA–DNA chimera and duplex NF-κB DNA–DNA. However, it was found that these differences do not prevent the duplex PNA–DNA chimera from binding to NF-κB transcription factors, being able to suppress the molecular interactions between HIV-1 LTR and p50, p52 and nuclear factors from B-lymphoid cells. Therefore, these results demonstrate that the designed NF-κB DNA–PNA chimeras could be used for a decoy approach in gene therapy. [ABSTRACT FROM AUTHOR]

Abstract: The identification of all epigenetic modifications (i.e. DNA methylation, histone modifications and expression of noncoding RNAs such as microRNAs) involved in gene regulation is one of the major steps forward for understanding human biology in both normal and pathological conditions and for development of novel drugs. In this context, microRNAs play a pivotal role. This review article focuses on the involvement of microRNAs in the regulation of gene expression, on the possible role of microRNAs in the onset and development of human pathologies, and on the pharmacological alteration of the biological activity of microRNAs. RNA and DNA analogs, which can selectively target microRNAs using Watson–Crick base pairing schemes, provide a rational and efficient way to modulate gene expression. These compounds, termed antago-miR or anti-miR have been described in many examples in the recent literature and have proved to be able to perform regulatory as well as therapeutic functions. Among these, a still not fully exploited class is that of peptide nucleic acids (PNAs), promising tools for the inhibition of miRNA activity, with important applications in gene therapy and in drug development. PNAs targeting miR-122, miR-155 and miR-210 have already been developed and their biological effects studied both in vitro and in vivo. [Copyright &y& Elsevier]