NEW DELIVERY SYSTEMS FOR ANTISENSE PEPTIDE NUCLEIC ACIDS (original) (raw)
Related papers
Comparison of novel delivery systems for antisense peptide nucleic acids
Journal of Controlled Release, 2005
Peptide nucleic acids (PNAs) provide a powerful tool to study the mechanism of transcription and translation, an innovative strategy to regulate target gene expression. They have been successfully used in antisense technology, for their ability to specifically bind to messenger RNA (mRNA) targets and to inhibit translation of the target genes. However, unlike most of the DNA and RNA oligonucleotides, PNAs are poorly penetrated through the cell membrane, partially due to their uncharged property. To enhance the efficiency in PNA delivery, many strategies have been explored.
Bioconjugate Chemistry, 2006
Cell-penetrating peptides (CPPs) are characterized by their ability to be internalized in mammalian cells. To investigate the relative potency of CPPs as carriers of medicinally relevant cargo, a positive read-out assay based on the ability of a peptide nucleic acid (PNA) oligomer to promote correct expression of a recombinant luciferase gene was employed. Seven different CPPs were included in the study: Transportan, oligo-arginine (R 7-9 ), pTat, Penetratin, KFF, SynB3, and NLS. The CPP-PNA conjugates were synthesized by different conjugation chemistries: continuous synthesis, maleimide coupling, and ester or disulfide linkage. Under serum-free conditions PNA-SS-Transportan-amide (ortho)-PNA was found to be the most potent conjugate, resulting in maximum luciferase signal at a concentration of 1-2 µM. (D-Arg) 9 -PNA showed optimal efficacy at 5 µM but gave rise to only one-third of the luciferase signal obtained with the Transportan conjugate. The pTat-and KFF-PNA conjugates showed significantly lower efficacy. The penetratin-, SynB3-. and NLS-PNA conjugates showed only minimal or no activity. Serum was found to have a drastic negative impact on CPP-driven cellular uptake. PNA-SS-Transportan-acid (ortho) and (D-Arg) 9 -PNA were least sensitive to the presence of serum. Both the chemical nature and, in the case of Transportan, the position of the peptide PNA coupling were found to have a major impact on the transport capacity of the peptides. However, no simple relationship between linker type and antisense activity of the conjugates could be deduced from the data.
Enhanced antisense effect of modified PNAs delivered through functional PMMA microspheres
International Journal of Pharmaceutics, 2006
Peptide nucleic acids (PNA) are very promising antisense agents, but their in vivo application is often hampered by their low bioavailability, mainly due to their limited uptake through cellular and nuclear membranes. However, PNA chemical synthesis easily allows modification with functional structures able to improve the intrinsically low permeability and great interest is arising in finding specific and efficient delivery protocols. Polymeric core-shell microspheres with anionic functional groups on the surface were tested for their ability to reversibly bind lysine modified PNA sequences, whose antisense activity against COX-2 mRNA was already demonstrated in murine macrophages.
Artificial DNA: PNA & XNA, 2012
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 PNApolyethyleneimine 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.
Nanoparticle for delivery of antisense γPNA oligomers targeting CCR5
Artificial DNA: PNA & XNA, 2013
human peripheral blood mononuclear cells and (2) difficulty in intracellular delivery. In attempts to resolve these limitations, strides have been made by incorporation of numerous cationic residues, 11 inclusion of polar groups in the backbone 12 and nucleobases, 13 and conjugation of high molecular weight polyethylene glycol (PEG) to one of the termini. 14 Though these chemical modifications have led to improvements in solubility and cellular uptake properties, it is often achieved at the expense of binding affinity and sequence specificity. These limitations can be addressed by inducing chirality at the gamma position of regular PNA: one class of chiral PNA is known as gamma PNA (γPNA). Biophysical characterization and NMR structural studies have shown that installation of a chiral center at the gamma position pre-organizes the PNA oligomer and also increases the binding affinity to the cDNA or RNA sequences. 15-20 Recently, by performing a series of thermodynamic studies, we have revealed that inclusion of diethylene glycol at the gamma position (also known as miniPEG-based γPNA) increases the solubility properties of PNA and enhances its binding with cDNA and RNA strands. 17 In addition, we have also shown that mini-PEG-based gamma PNA (MP γPNA) has the potential to invade duplex DNA in a sequence-unrestricted manner. 21 However, more effective intracellular delivery methods are required for broader impact of new generations of gamma PNA in gene therapy-based applications. Recently, we have developed new methods for delivery of PNAs for gene silencing and gene editing. McNeer et al. demonstrated the use of poly(lactide-co-glycolide) (PLGA) nanoparticles for ©2013 Landes Bioscience. Do not distribute.
PLoS ONE, 2014
Peptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine. We show that chloroquine not only induced escape from endosomes but also enhanced the cellular uptake of the conjugate. Mechanistic studies revealed that (R/W)9-PNA conjugates were internalized via pinocytosis. Replacement of arginines with lysines reduced the uptake of the conjugate by six-fold, resulting in the abolition of intracellular target inhibition. Our results show that the arginines play a crucial role in the conjugate uptake and antisense activity. To determine whether specificity of the interactions of arginines with cell surface proteoglycans result in the internalization, we used flow cytometry to examine uptake of arginine-and lysine-rich conjugates in wild-type CHO-K1 and proteoglycan-deficient A745 cells. The uptake of both conjugates was decreased by four fold in CHO-745 cells; therefore proteoglycans promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Our results show that arginine-rich cellpenetrating peptides, especially (R/W)9, are a promising tool for PNA internalization.
Cellular delivery of peptide nucleic acids (PNAs)
Methods in molecular biology (Clifton, N.J.), 2014
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.
An Efficient Biodelivery System for Antisense Polyamide Nucleic Acid (PNA)
Oligonucleotides, 2008
With the aim of developing a general and straightforward procedure for the intracellular delivery of naked peptide nucleic acids (PNAs), we designed an intracellularly biodegradable triphenylphosphonium (TPP) cation based transporter system. In this system, TPP is linked, via a biolabile disul de bridge, to an activated mercaptoethoxycarbonyl moiety, allowing its direct coupling to the N-terminal extremity of a free PNA through a carbamate bond. We found that such TPP-PNA-carbamate conjugates were highly stable in a cell culture medium containing fetal calf serum. In a glutathione-containing medium mimicking the cytosol, the conjugates were rapidly degraded into an unstable intermediate, which spontaneously decomposed, releasing the free PNA. Using a uorescence-labeled PNA-TPP conjugate, we demonstrated that conjugates were taken up by cells. Ef cient cellular uptake and release of the PNA into the cytosol was further con rmed by the anti-HIV activity measured for the TPP-conjugate of a 16-mer PNA targeting the TAR region of the HIV-1 genome. This conjugate exhibited an IC 50 value of 1 µM, while the free 16-mer PNA did not inhibit replication of HIV in the same cellular test.
Nucleic Acids Research, 2008
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 bisphosphonate 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.