Efficient and selective kidney targeting by chemically modified carbohydrate conjugates

Peptide nucleic acids (PNAs) are a potentially powerful approach for the recognition of cellular mRNA and the inhibition of gene expression. Despite their promise, the rules for using antisense PNAs have remained obscure, and antisense PNAs have been used sparingly in research. Here we investigate the ability of PNAs to be effective antisense agents inside mammalian cells, to inhibit expression of human caveolin-1 (hCav-1), and to discriminate between its alpha and beta isoforms. Many human genes are expressed as isoforms. Isoforms may play different roles within a cell or within different tissues, and defining these roles is a challenge for functional genomics and drug discovery. PNAs targeted to the translation start codons for the alpha and beta isoforms inhibit expression of hCav-1. Inhibition is dependent on PNA length. The potency and duration of inhibition by PNAs are similar to inhibition of gene expression by short interferring RNA (siRNA). Expression of the alpha isoform can be blocked selectively by a PNA. Cell proliferation is halted by inhibition of expression of both hCav-1 isoforms, but not by inhibition of the alpha hCav-1 isoform alone. Efficient antisense inhibition and selective modulation of isoform expression suggest that PNAs are versatile tools for controlling gene expression and dissecting the roles of closely related protein variants. Potent inhibition by PNAs may supply a "knock down" technology that can complement and "cross-check" siRNA and other approaches to antisense gene inhibition that rely on oligomers with phosphate or phosphorothioate backbone linkages.

Targeted Gene Modification of Hematopoietic Progenitor Cells in Mice Following Systemic Administration of a PNA-peptide Conjugate

Antibiotics have long been used for anti-infective control of bacterial infections, growth promotion in husbandry, and prophylactic protection against plant pathogens. However, their inappropriate use results in the emergence and spread of multiple drug resistance (MDR) especially among various bacterial populations, which limits further administration of conventional antibiotics. Therefore, the demand for novel anti-infective approaches against MDR diseases becomes increasing in recent years. The peptide nucleic acid (PNA)-based technology has been proposed as one of novel anti-infective and/or therapeutic strategies. By definition, PNA is an artificially synthesized nucleic acid mimic structurally similar to DNA or RNA in nature and linked one another via an unnatural pseudo-peptide backbone, rendering to its stability in diverse host conditions. It can bind DNA or RNA strands complimentarily with high affinity and sequence specificity, which induces the target-specific gene silencing by inhibiting transcription and/or translation. Based on these unique properties, PNA has been widely applied for molecular diagnosis as well as considered as a potential anti-infective agent. In this review, we discuss the general features of PNAs and their application to various bacterial pathogens as new anti-infective or antimicrobial agents.

Re-expression of the death-signalling p75 neurotrophin receptor (p75NTR) is associated with injury and neurodegeneration in the adult nervous system. The induction of p75NTR expression in mature degenerating spinal motor neurons of humans and transgenic mice with amyotrophic lateral sclerosis (ALS) suggests a role of p75NTR in the progression of motor neuron disease (MND). In this study, we designed, synthesized and evaluated novel antisense peptide nucleic acid (PNA) constructs targeting p75NTR as a potential gene knockdown therapeutic strategy for ALS. An 11-mer antisense PNA directed at the initiation codon, but not downstream gene sequences, dose-dependently inhibited p75NTR expression and death-signalling by nerve growth factor (NGF) in Schwann cell cultures. Antisense phosphorothioate oligonucleotide (PS-ODN) sequences used for comparison failed to confer such inhibitory activity. Systemic intraperitoneal administration of this antisense PNA to mutant superoxide dismutase 1 (SOD1G93A) transgenic mice significantly delayed locomotor impairment and mortality compared with mice injected with nonsense or scrambled PNA sequences. Reductions in p75NTR expression and subsequent caspase-3 activation in spinal cords were consistent with increased survival in antisense PNA-treated mice. The uptake of fluorescent-labelled antisense PNA in the nervous system of transgenic mice was also confirmed. This study suggests that p75NTR may be a promising antisense target in the treatment of ALS.

Design and application of a peptide nucleic acid sequence targeting the p75 neurotrophin receptor.

Vitamin B12 also called cobalamin (Cbl) is an important enzymatic cofactor taken up by mammalian and also by many bacterial cells. Peptide nucleic acid (PNA) is a synthetic DNA analogue that has the ability to bind in a complementary manner to natural nucleic acids. Provided that PNA is efficiently delivered to cells, it could act as a steric blocker of functional DNA or RNA and regulate gene expression at the level of transcription or translation. Recently, Cbl has been examined as a transporter of various molecules to cells. Also, PNA, if covalently linked with Cbl, can be delivered to bacterial cells, but it is crucial to verify that Cbl does not change the desired PNA biological properties. We have analyzed the structure and conformational dynamics of conjugates of Cbl with a PNA monomer and oligomer. We synthesized a cyanocobalamin derivative with a PNA monomer C connected via the triazole linker and determined its NMR spectra. Using microsecond-long molecular dynamics simulations, we examined the internal dynamics of cyanocobalamin-C, its conjugate with a 14-mer PNA, and free PNA. The results suggest that all compounds acquire rather compact structures but the PNA oligomer conformations vary. For the Cbl-C conjugate the cross-peaks from the ROESY spectrum corroborated with the clusters from molecular dynamics trajectories. Within PNA the dominant interaction is stacking but the stacking bases are not necessarily neighboring in the PNA sequence. More bases stack in free PNA than in PNA of the conjugate, but stacking is less stable in free PNA. PNA in the conjugate is slightly more exposed to solvent. Overall, cyanocobalamin attached to a PNA oligomer increases the flexibility of PNA in a way that could be beneficial for its hybridization with natural nucleic acid oligomers.

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.

Peptide nucleic acid (PNA) is a peptide-like DNA mimic that was introduced almost ten years ago. It was immediately predicted that PNA would have a bright future in gene therapeutic drug development, but progress in this direction has been rather modest thus far. This is predominantly due to inefficient uptake of PNA by most living cells. However, within the past couple of years a variety of methods have been devised to address this problem and the stage should now be set for more rapid progress. Several studies have demonstrated antisense effects ex vivo in cells in culture and two reports on direct injection of PNA into the brain of rats are also interesting. Only a few studies have addressed the possible exploitation of the antisense principle for development of antibacterial drugs. However, the first in vitro results using antiribosomal RNA PNAs and antisense PNAs targeted to the β-lactamase gene on Escherichia coli cultures were quite promising. Most recently, these preliminary studies have been extended to demonstrate in vivo efficacy of antibacterial PNAs in an E. coli peritonitis/sepsis mouse model. Therefore, PNA drug development again is rapidly picking up pace.

Nucleic acid biosensors have a growing number of applications in genetics and biomedicine. This contribution is a critical review of the current state of the art concerning the use of nucleic acid analogues, in particular peptide nucleic acids (PNA) and locked nucleic acids (LNA), for the development of high-performance affinity biosensors. Both PNA and LNA have outstanding affinity for natural nucleic acids, and the destabilizing effect of base mismatches in PNA- or LNA-containing heterodimers is much higher than in double-stranded DNA or RNA. Therefore, PNA- and LNA-based biosensors have unprecedented sensitivity and specificity, with special applicability in DNA genotyping. Herein, the most relevant PNA- and LNA-based biosensors are presented, and their advantages and their current limitations are discussed. Some of the reviewed technology, while promising, still needs to bridge the gap between experimental status and the harder reality of biotechnological or biomedical applications.

An antisense peptide nucleic acid against Pseudomonas aeruginosa inhibiting bacterial-induced inflammatory responses in the cystic fibrosis IB3-1 cellular model system

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) causes threatening infection-related mortality worldwide. Currently, spread of multi-drug resistance (MDR) MRSA limits therapeutic options and requires new approaches to “druggable” target discovery, as well as development of novel MRSA-active antibiotics. RNA polymerase primary σ70 (encoded by gene rpoD) is a highly conserved prokaryotic factor essential for transcription initiation in exponentially growing cells of diverse S. aureus, implying potential for antisense inhibition.Methodology/Principal FindingsBy synthesizing a serial of cell penetrating peptide conjugated peptide nucleic acids (PPNAs) based on software predicted parameters and further design optimization, we identified a target sequence (234 to 243 nt) within rpoD mRNA conserved region 3.0 being more sensitive to antisense inhibition. A (KFF)3K peptide conjugated 10-mer complementary PNA (PPNA2332) was developed for potent micromolar-range growth inhibitory effects against four pathogenic S. aureus strains with different resistance phenotypes, including clinical vancomycin-intermediate resistance S. aureus and MDR-MRSA isolates. PPNA2332 showed bacteriocidal antisense effect at 3.2 fold of MIC value against MRSA/VISA Mu50, and its sequence specificity was demonstrated in that PPNA with scrambled PNA sequence (Scr PPNA2332) exhibited no growth inhibitory effect at higher concentrations. Also, PPNA2332 specifically interferes with rpoD mRNA, inhibiting translation of its protein product σ70 in a concentration-dependent manner. Full decay of mRNA and suppressed expression of σ70 were observed for 40 µM or 12.5 µM PPNA2332 treatment, respectively, but not for 40 µM Scr PPNA2332 treatment in pure culture of MRSA/VISA Mu50 strain. PPNA2332 (≥1 µM) essentially cleared lethal MRSA/VISA Mu50 infection in epithelial cell cultures, and eliminated viable bacterial cells in a time- and concentration- dependent manner, without showing any apparent toxicity at 10 µM.ConclusionsThe present result suggested that RNAP primary σ70 is a very promising candidate target for developing novel antisense antibiotic to treat severe MRSA infections.

In recent years, there has been considerable interest in developing gene‐targeting drugs. Synthetic molecules that bind effectively with high sequence specificity to a desired target in a particular “destined” gene sequence are of major interest in medicine and molecular biotechnological research. They show hope for the development of gene therapeutic agents, high‐precision diagnostic devices for genetic analysis and convenient tools for nucleic acid manipulations. Peptide nucleic acid (PNA) is a nucleic acid analog in which the sugar–phosphate backbone of natural nucleic acid has been replaced by a synthetic peptide backbone usually formed from N‐(2‐aminoethyl)glycine units resulting in an achiral and uncharged mimic. It is chemically stable and, in contrast to nucleic acids and peptides, is naturally resistant to hydrolytic (enzymatic) cleavage and thereby not expected to be degraded by cell extracts or inside a living cell. It is capable of sequence‐specific recognition of DNA and RNA obeying a Watson–Crick hydrogen bonding scheme and the hybrid complexes exhibit extraordinary thermal stability and unique ionic strength effects. It also recognizes duplex homopurine sequences of DNA to which it binds by strand invasion, forming a stable PNA·DNA–PNA triplex with a looped‐out DNA strand. This potential synthetic mimic of DNA, since its discovery in 1991, has attracted great attention within medicinal chemistry and molecular biology and also various other fields such as organic and physical chemistry because of its interesting chemical and physical properties. It has been regarded to have great potential for both diagnostic and pharmaceutical applications. In vitro studies have shown successful attempts to use PNA as an inhibitor for both transcription and translation of specific genes. This holds promise for using PNA for antigene and antisense therapy. However, the most exciting use of PNA, as an antisense or antigene drug, requires an efficient and safe method to deliver PNA into living cells, a problem that still remains to be solved.

Single-stranded peptide nucleic acid (PNA) probes interact strongly with several nanomaterials, and the interaction was diminished in the presence of complementary nucleic acid targets which forms the basis of many nucleic acid sensing platforms. As opposed to the negatively charged DNA probes, the charges on the PNA probes may be fine-tuned by incorporating amino acids with charged side chains. The contribution of electrostatic effects to the interaction between PNA probes and nanomaterials has been largely overlooked. This work reveals that electrostatic effects substantially enhanced the quenching of dye-labeled conformationally constrained pyrrolidinyl PNA probes by several nanomaterials including graphene oxide (GO), reduced graphene oxide, gold nanoparticles (AuNPs), and silver nanoparticles. The fluorescence quenching and the color change from red to purple in the case of AuNPs because of aggregation were inhibited in the presence of complementary nucleic acid targets. Thus, fluorescence and colorimetric assays for DNA and RNA that can distinguish even single-base-mismatched nucleic acids with improved sensitivity over conventional DNA probes were established. Both the GO- and AuNP-based sensing platforms have been successfully applied for the detection of real DNA and RNA samples in vitro and in living cells. This study emphasizes the active roles of electrostatic effects in the PNA-nanomaterial interactions, which paves the way toward improving the performance of PNA-nanomaterial based assays of nucleic acids.

This chapter will compare and contrast the properties and applications of two leading antisense molecules, Peptide Nucleic Acids (PNAs)1,2 and Morpholinos3,4. Where appropriate, I discuss the compelling advantages which these two advanced ‘blocker’ types provide relative to ‘modifier’ types of antisense molecules. Some of the properties which are compared include chemical synthesis strategies, chemical stabilities, backbone flexibilities, aqueous solubilities, target selection criteria, target binding affinities, and sequence specificities. Since the mid-1980s, phosphorothioate-linked DNA oligos (S-DNAs) have dominated the antisense field. For many antisense applications, however, advanced non-ionic oligos provide a far better combination of properties, including stability in biological systems, high efficacy and specificity, lack of toxicity, and freedom from non-antisense effects. Prior to the discovery of PNAs by Nielsen et al., I was the first person to conceptualize and to synthesize morpholinos, recognizing their advantages for antisense chemistry. Morpholinos and PNAs share a number of key properties, such as non-ionic backbones whose structures differ radically from that of nucleic acids, resistance to enzymatic degradation, and high (Morpholinos) or very high (PNAs) affinity for complementary RNA sequences. In the context of diagnostics, a particularly valuable property of both structural types is that they strongly pair to complementary genetic sequences under conditions which disrupt secondary structures of nucleic acids. Another particular advantage of morpholinos, and part of the original impetus to develop them, is the fact that they are relatively cheap to produce; the subunits of Morpholinos can be assembled into antisense oligos via simple and efficient coupling to the morpholine nitrogen, without the expensive catalysts and post-coupling oxidation steps required in the production of most nucleic acid analogs. In spite of their many similarities, Morpholinos and PNAs also exhibit significant differences which translate to differing advantages in particular applications. Two key differences which bear on their preferred applications are: 1) PNAs have higher affinities for RNA than do Morpholinos, though both structural types form duplexes with RNA which are more stable than DNA/RNA duplexes, and much more stable than S-DNA/RNA duplexes; 2) Morpholinos are highly soluble in aqueous solutions, generally 5 to 30 mM for 25-mers, depending on sequence, whereas PNAs are typically several hundred-fold less soluble. As a consequence of these differing properties, it appears that PNAs are better suited for high-affinity applications such as targeting short sequences (e.g., the exposed segment of telomerase RNA) and for discriminating between single base differences, as in SNPs (single nucleotide polymorphisms). Conversely, Morpholinos excel in applications which require high aqueous solubility and exquisite discrimination between a targeted mRNA and tens of thousands of non-target mRNAs, such as in vivo applications with developing embryos and other complex systems.

We read with great interest the article titled “MicroRNA-145 Antagonism Reverses TGF-b Inhibition of F508del CFTR Correction in Airway Epithelia” by Lutful Kabir and colleagues (1). In this paper, the authors demonstrate that miR-145 mediates TGF-b (transforming growth factor-b) inhibition of synthesis and function of CFTR (cystic fibrosis transmembrane conductance regulator) in cystic fibrosis (CF) airway epithelia. Interestingly, they found that antagonists of miR-145 were able to interrupt TGF-b signaling and restore F508del CFTR modulation. Therefore, they suggested that miR-145 targeting may provide a novel therapeutic opportunity to enhance the benefit of F508del CFTR correction in CF epithelia. In agreement with this, we have elsewhere published data supporting the use of miR-145 targeting in CF, based on an antisense peptide nucleic acid (PNA) to target miR-145-5p (PNAa145) and enhance expression of the CFTR gene, which we analyzed at mRNA (qRT-PCR) and protein (Western blotting) levels (2). In support of the conclusion by Lutful Kabir and colleagues, our data suggest the use of suitably delivered antisense molecules targeting miR-145-5p to enhance the expression of CFTR (2). With respect to a possible microRNA (miRNA)-based therapeutic option, one limitation is of course the presence of more than 300 CFTR gene disease-causing mutations (www.genet.sickkids.on.ca/cftr/) (3). Besides the CFTR mutation leading to the deletion of the phenylalanine in position 508 (F508del CFTR), accounting for 50–90% CF chromosomes, most CFcausing mutations are missense (42%), nonsense (10%), frameshift (15%), splicing (13%), in-frame deletion/insertion (2%), and promoter (0.5%) mutations, which are now operationally categorized in six classes of molecular defects of the CFTR protein (3).