Induction of splice correction by cell-penetrating peptide nucleic acids

BACKGROUND: Directing splicing using oligonucleotides constitutes a promising therapeutic tool for a variety of diseases such as beta-thalassemia, cystic fibrosis, and certain cancers. The rationale is to block aberrant splice sites, thus directing the splicing of the pre-mRNA towards the desired protein product. One of the difficulties in this setup is the poor bioavailability of oligonucleotides, as the most frequently used transfection agents are unsuitable for in vivo use. Here we present splice-correcting peptide nucleic acids (PNAs), tethered to a variety of cell-penetrating peptides (CPPs), evaluating their mechanism of uptake and ability to correct aberrant splicing. METHODS: HeLa cells stably expressing luciferase containing an aberrant splice site were used. A previously described PNA sequence, capable of correcting the aberrant splicing, was conjugated to the CPPs, Tat, penetratin and transportan, via a disulfide bridge. The ability of the CPP-PNA conjugates to correct splicing was measured, and membrane disturbance and cell viability were evaluated using LDH leakage and WST-1 assays. Lysosomotropic agents, inhibition of endocytosis at 4 degrees C and confocal microscopy were used to investigate the importance of endocytosis in the uptake of the cell-penetrating PNAs. RESULTS: All the three CPPs were able to promote PNA translocation across the plasma membrane and induce splice correction. Transportan (TP) was the most potent vector and significantly restored splicing in a concentration-dependent manner. Interestingly, TP also rendered a concentration-dependent splice correction in serum, in contrast to Tat and penetratin. Addition of the lysosomotrophic agent chloroquine increases the splice correction efficacy of the CPP-PNA conjugates up to 4-fold, which together with experiments at 4 degrees C and the visual information from confocal microscopy, indicate that the mechanism of uptake responsible for internalization of CPP-PNA conjugates is mainly endocytic. Finally, co-localization studies with dextran further indicate that conjugates, at least in the case of TP, internalize via endocytosis and in particular macropinocytosis. CONCLUSIONS: These data demonstrate that CPPs can be used for the delivery of splice-correcting PNAs, with potential to be used as a therapeutic approach for regulating splicing in a variety of diseases. Transportan presents itself as the overall most suitable vector in this study, generating the most efficient conjugates for splice correction.     

Solid-Phase synthesis of peptide nucleic acids

Peptide nucleic acids (PNA) were synthesized by a modified Merrifield method using several improvements. Activation by O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in combination with in situ neutralization of the resin allowed efficient coupling of all four Boc-protected PNA monomers within 30 min. HPLC analysis of the crude product obtained from a fully automated synthesis of the model PNA oligomer H-CGGACTAAGTCCATTGC-Gly-NH₂, indicated an average yield per synthetic cycle of 97.1%. N¹-benzyloxycarbonyl-N6³-methylimidazole triflate substantially outperformed acetic anhydride as a capping reagent. The resin-bound PNAs were successfully cleaved by the ‘low–high’ trifluoromethanesulphonic acid procedure.     

Imaging gene expression in the brain with peptide nucleic acid (PNA) antisense radiopharmaceuticals and drug targeting technology

Summary Antisense oligomers are potential pharmaceutical and radiopharmaceutical agents that can be used to modulate and image gene expression. Progress within vivo gene targeting using antisense-based therapeutics has been slower than expected during the last decade, owing to poor trans-cellular delivery of antisense agents. This chapter suggests that if antisense pharmacology is merged with drug targeting technology, then membrane barriers can be circumvented and antisense agents can be delivered to tissuesin vivo. Without the application of drug targeting, the likelihood of success for an antisense drug development program is low, particularly for the brain which is protected by the blood-brain barrier (BBB). Among the different classes of antisense agents, peptide nucleic acids (PNA) present advantages forin vivo applications over conventional and modified oligodeoxynucleotides (ODN), including phosphorothioates (PS)-ODN. Some advantages of PNAs include their electrically neutral backbone, low toxicity to neural cells, resistance to nucleases and peptidases, and lack of binding to plasma proteins. PNAs are poorly transported through cellular membranes, however, including the BBB and the brain cell membrane (BCM). Because the mRNA target for the antisense agent lies within the cytosol of the target cell, the BBB and the BCM must be circumventedin vivo, which is possible with the use of chimeric peptide drug targeting technology. Chimeric peptides are formed by conjugation of a non-transportable drug, such as a PNA, to a drug delivery vector. The vector undergoes receptor-mediated transcytosis (RMT) through the BBB and receptor-mediated endocytosis through the BCMin vivo. When labeled with a radioisotope (e.g.,¹²⁵I or¹¹¹In), the antisense chimeric peptide provides imaging of gene expression in the brainin vivo in a sequence-specific manner. Further development of antisense radiopharmaceutical agents may allow forin vivo imaging of genes in pathological states, and may provide tools for the analysis of novel genes with functional genomics.     

Imaging gene expression in the brain with peptide nucleic acid (PNA) antisense radiopharmaceuticals and drug targeting technology

Antisense oligomers are potential pharmaceutical and radiopharmaceutical agents that can be used to modulate and image gene expression. Progress with in vivogene targeting using antisense-based therapeutics has been slower than expected during the last decade, owing to poor trans-cellular delivery of antisense agents. This chapter suggests that if antisense pharmacology is merged with drug targeting technology, then membrane barriers can be circumvented and antisense agents can be delivered to tissues in vivo. Without the application of drug targeting, the likelihood of success for an antisense drug development program is low, particularly for the brain which is protected by the blood-brain barrier (BBB). Among the different classes of antisense agents, peptide nucleic acids (PNA) present advantages for in vivoapplications over conventional and modified oligodeoxynucleotides (ODN), including phosphorothioates (PS)-ODN. Some advantages of PNAs include their electrically neutral backbone, low toxicity to neural cells, resistance to nucleases and peptidases, and lack of binding to plasma proteins. PNAs are poorly transported through cellular membranes, however, including the BBB and the brain cell membrane (BCM). Because the mRNA target for the antisense agent lies within the cytosol of the target cell, the BBB and the BCM must be circumvented in vivo, which ispossible with the use of chimeric peptide drug targeting technology. Chimeric peptides are formed by conjugation of a non-transportable drug, such as a PNA, to a drug delivery vector. The vector undergoes receptor-mediated transcytosis (RMT) through the BBB and receptor-mediated endocytosis through the BCM in vivo. When labeled with a radioisotope (e.g., ¹²⁵I or ¹¹¹In), the antisense chimeric peptide provides imaging of gene expressionin the brain in vivoin a sequence-specific manner. Further development of antisense radiopharmaceutical agents may allow for in vivoimaging of genes in pathological states, and may provide tools for the analysis of novel genes with functional genomics.     

Light-directed synthesis of peptide nucleic acids (PNAs) chips

We report herein the light-directed synthesis of peptide nucleic acids (PNAs) microarray using PNA monomers protected by photolabile protecting groups and a maskless technique that uses a digital micromirror array system to form virtual masks. An ultraviolet image from the virtual mask was cast onto the active surface of a glass substrate, which was mounted in a flow cell reaction chamber connected to a peptide synthesizer. Light exposure was followed by automatic chemical coupling cycles and these steps were repeated with different virtual masks to grow the desired PNA probes in a selected pattern. In a preliminary experiment, an array of PNA probes with dimensions of 4.11 mm × 4.11 mm was generated on each slide. Each synthesis region in the final array measured 210 μm × 210 μm for a total of 256 sites. The center-to-center space was 260 μm. It was observed from the hybridization pattern of the fluorescently labeled oligonucleotide targets that the fluorescence intensities of the matched, and mismatched sequences showed substantial difference, demonstrating specificity in the identification of complementary sequences. This opens the way to exploit processes from the microelectronics industry for the fabrication of PNA microarrays with high densities.     

Inhibition of Mycobacterium smegmatis gene expression and growth using antisense peptide nucleic acids

Antisense agents that inhibit genes at the mRNA level are attractive tools for genome-wide studies and drug target validation. The approach may be particularly well suited to studies of bacteria that are difficult to manipulate with standard genetic tools. Antisense peptide nucleic acids (PNA) with attached carrier peptides can inhibit gene expression in Escherichia coli and Staphylococcus aureus. Here we asked whether peptide-PNAs could mediate antisense effects in Mycobacterium smegmatis. We first targeted the gfp reporter gene and observed dose- and sequence-dependent inhibition at low micromolar concentrations. Sequence alterations within both the PNA and target mRNA sequences eliminated inhibition, strongly supporting an antisense mechanism of inhibition. Also, antisense PNAs with various attached peptides showed improved anti-gfp effects. Two peptide-PNAs targeted to the essential gene inhA were growth inhibitory and caused cell morphology changes that resemble that of InhA-depleted cells. Therefore, antisense peptide-PNAs can efficiently and specifically inhibit both reporter and endogenous essential genes in mycobacteria.     

Inhibition of bacterial translation and growth by peptide nucleic acids targeted to domain II of 23S rRNA.

The objective of this work was to study the inhibitory effects of antisense peptide nucleic acids (PNAs) targeted to domain II of 23S rRNA on bacterial translation and growth. In this paper, we report that PNA(G1138) or peptide-PNA(G1138) targeted to domain II of 23S rRNA can inhibit both translation in vitro (in a cell-free translation system) and bacterial growth in vivo. The inhibitory concentration (IC50) and the minimum inhibiting concentration (MIC) are 0.15 and 10 microM, respectively. The inhibition effect of PNA(G1138) in vitro is somewhat lower than that of tetracycline (IC50 = 0.12 microM), but the MIC of peptide-PNA(G1138) against Escherichia coli is significantly higher than that of tetracycline (MIC = 4 microM). Further studies based on similar colony-forming unit (CFU) assays showed that peptide-PNA(G1138) at 10 microM is bactericidal, but the bactericidal effect is less effective than that of tetracycline. Nevertheless, the results demonstrated that the peptide-PNA(G1138) treatment is bactericidal in a dose- and sequence-dependent manner and that the G1138 site of 23S rRNA is a possible sequence target for designing novel PNA-based antibiotics.     

Thiolated pyrrolidinyl peptide nucleic acids for the detection of DNA hybridization using surface plasmon resonance

Thiolated pyrrolidinyl peptide nucleic acids (HS-PNAs) bearing d-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones with different lengths and types of thiol modifiers were synthesized and then characterized by MALDI–TOF mass spectrometry. These HS-PNAs were immobilized on gold-coated glass by self-assembled monolayer (SAM) formation via S atom linkage for the detection of DNA hybridization using surface plasmon resonance (SPR). The amount and the stability of the immobilized HS-PNAs, as well as the effects of spacer and blocking thiol on DNA hybridization efficiency, were determined. SPR results indicated that the hybridization efficiency was enhanced when the distance between the PNA portion and the thiol terminal was increased and/or when blocking thiol was used following the HS-PNA immobilization. The immobilized HS-PNA could discriminate between fully complementary DNA from one or two base mismatched DNA with a relatively high degree of mismatch discrimination (>45%) in PBS buffer at 25 °C. The lowest DNA concentration at which reliable discrimination between fully complementary and single mismatched DNA could still occur was at about 0.2 μM, which is equivalent to 10 pmol of DNA. This research demonstrates that using these novel thiolated PNAs in combination with the SPR technique offers a direct, rapid and non-label based method that could potentially be applied for the analysis of genomic or PCR-amplified DNA in the future.     

Preparation of radioiodinated peptide nucleic acids with high specific activity

Peptide nucleic acids (PNAs) have stronger affinity and greater specificity than do oligonucleotides for binding to DNA and RNA and, as such, have potential utility as probes in molecular biology applications. In this study, a novel approach for labeling the PNA with radioiodine that avoided solubility issues and poor labeling encountered when trying to radioiodinate PNAs directly in solution was developed. For this approach, a purpose-designed prosthetic group that incorporated both a radioiodinatable tyrosine and a triphenylphosphonium (TPP) moiety was synthesized. The latter is an organic cation that combines the properties of good solubility in both aqueous and organic solvents with a strong retention by reverse phase HPLC. Following radioiodination of the TPP-based prosthetic group in phosphate buffer, the prosthetic group was purified and coupled to the terminal amine of 15-mer PNA on the solid phase resin. After cleavage and deprotection of the PNA from the resin, the presence of the TPP group resulted in a clean separation of radioiodinated PNA from unlabeled PNA, yielding a high-specific activity probe in a single HPLC run. As an example of a potential molecular biology application of the resultant (125)I-labeled PNA probe, it was used to detect mRNA for the Lcn2 gene in Northern blotting.     

Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Genomic sequencing makes it possible to identify all the genes of an organism, now including Homo sapiens. Yet measurement of the expression of each gene of interest still presents a dauntingprospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately,no reliable method has been available to measure levels of specificmRNAs in vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless,we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cellsare taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAsin human cancer xenografts in a mouse model. The oncogenes cyclinD1, ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experimentsprovide a proof-of-principle for noninvasive detection of oncogeneexpression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.     

Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Summary Genomic sequencing makes it possible to identify all the genes of an organism, now including Homo sapiens. Yet measurement of the expression of each gene of interest still presents a daunting prospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately, no reliable method has been available to measure levels of specific mRNAs in vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless, we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cells are taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAs in human cancer xenografts in a mouse model. The oncogenes cyclin D1, ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experiments provide a proof-of-principle for noninvasive detection of oncogene expression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.     

Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Summary Genomic sequencing makes it possible to identify all the genes of an organism, now includingHomo sapiens. Yet measurement of the expression of each gene of interest still presents a daunting prospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately, no reliable method has been available to measure levels of specific mRNAsin vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless, we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cells are taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAs in human cancer xenografts in a mouse model. The oncogenes cyclin D1,ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experiments provide a proof-of-principle for noninvasive detection of oncogene expression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.     

The peptide nucleic acids (PNAs): a new generation of probes for genetic and cytogenetic analyses

Peptide nucleic acids (PNAs) are synthetic homologs of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible pseudo-peptide polymer to which the nucleobases are linked. This structure gives PNAs the capacity to hybridize with high affinity and specificity to complementary sequences of DNA and RNA, and also confers remarkable resistance to DNAses and proteinases. The unique physico-chemical characteristics of PNAs have led to the development of a wide range of biological assays. Several exciting new applications of PNA technology have been published recently in genetics and cytogenetics. Also, PNA-based hybridization technology is developing rapidly within the field of in situ fluorescence hybridization, pointing out the great potential of PNA probes for chromosomal investigations.     

A rapid coupling protocol for the synthesis of peptide nucleic acids

With the current interest in anti-sense and anti-gene technologies, an efficient, fast and less toxic synthesis protocol would be advantageous for the oligomerisation of Peptide Nucleic Acids (PNA). Most of the methods currently in use for the t-Boc synthesis of PNA's use TFA/m-cresol, pyridine, piperidine and capping reagents. In this work, a rapid synthesis protocol has been adapted from an earlier published peptide synthesis method allowing a reduction in cycle time from around 30 min down to 16 min. By utilising quantitative deprotection with 100% TFA, a coupling time of 10 min and a four-fold excess of monomer, this synthesis protocol has been used to synthesise a number of PNA's incorporating all four nucleotides of varying sequence, up to 17 residues in length.     

A rapid coupling protocol for the synthesis of peptide nucleic acids

Summary With the current interest in anti-sense and anti-gene technologies, an efficient, fast and less toxic synthesis protocol would be advantageous for the oligomerisation of Peptide Nucleic Acids (PNA). Most of the methods currently in use for thet-Boc synthesis of PNA's use TFA/m-cresol, pyridine, piperidine and capping reagents. In this work, a rapid synthesis protocol has been adapted from an earlier published peptide synthesis method allowing a reduction in cycle time from around 30 min down to 16 min. By utilising quantitative deprotection with 100% TFA, a coupling time of 10 min and a four-fold excess of monomer, this synthesis protocol has been used to synthesise a number of PNA's incorporating all four nucleotides of varying sequence, up to 17 residues in length.     

Medicinal chemistry of plasmid DNA with peptide nucleic acids: A new strategy for gene therapy

Summary In this chapter, we describe an approach using a peptide nucleic acid (PNA) clamp to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. This strategy enables investigators to functionalize their gene of interest by direct coupling of ligands (fluorophores, peptide, proteins, sugars or oligonucleotides) to plasmid DNA. This approach provides versatile tools to study the mechanisms of gene delivery and to circumvent some of the main obstacles of synthetic gene delivery systems, such as specific targeting and efficient delivery. The proof-of-principal of PNA-dependent gene chemistry (PDGC) was demonstrated with a fluorescently labeled PNA that allowed generation of a highly fluorescent preparation of plasmid DNA that was functionally and conformationally intact. Fluorescent-PNA/DNA was used to identify critical parameters involved in naked DNA and non-viral gene delivery technology. The greatest potential of PDGC lies in the ability to attach specific ligands (e.g., peptides, proteins) to the plasmid DNA in order to overcome cellular barriers of non-viral gene delivery systems. In this regard, specific examples of ligands coupled to DNA are described and their effect on increasing the efficacy of gene therapy is presented     

Inhibition of gene expression by peptide nucleic acids in cultured cells

We have tested in cultured cells the capacity of antisense and antigene PNAs to inhibit, in a sequence specific manner, the expression of oncogenes in leukaemia and pancreatic carcinoma cells. The results observed appeared promising and suggest that PNA may play in the future an important role in targeting disease-related genes.