Nanoparticles Deliver Triplex-forming PNAs for Site-specific Genomic Recombination in CD34+ Human Hematopoietic Progenitors

Triplex-forming peptide nucleic acids (PNAs) are powerful gene therapy agents that can enhance recombination of short donor DNAs with genomic DNA, leading to targeted and specific correction of disease-causing genetic mutations. Therapeutic use of PNAs is severely limited, however, by challenges in intracellular delivery, particularly in clinically relevant targets such as hematopoietic stem and progenitor cells. Here, we demonstrate efficient and nontoxic PNA-mediated recombination in human CD34⁺ cells using poly(lactic-co-glycolic acid) (PLGA) nanoparticles for intracellular oligonucleotide delivery. Treatment of progenitor cells with nanoparticles loaded with PNAs and DNAs targeting the β-globin locus led to levels of site-specific modification in the range of 0.5–1% in a single treatment, without detectable loss in cell viability, resulting in a 60-fold increase in modified and viable cells as compared to nucleofection. As well, the differentiation capacity of the progenitor cells treated with nanoparticles did not change relative to untreated progenitor cells, indicating that nanoparticles are safe and minimally disruptive delivery vectors for PNAs and DNAs to mediate gene modification in human primary cells. This is the first demonstration of the use of biodegradable nanoparticles to deliver genome-editing agents to human primary cells, and provides a strong rationale for systemic delivery of complex nucleic acid mixtures designed for gene correction.     

Enhanced gene transfection efficiency in CD13-positive vascular endothelial cells with targeted poly(lactic acid)-poly(ethylene glycol) nanoparticles through caveolae-mediated endocytosis

The efficient delivery of therapeutic gene into cells of interest is a critical challenge to broad application of non-viral vectors. The approach of introducing ligands that lead gene vectors to target caveolae-mediated endocytosis on nanoparticle surface might serve as a promising strategy for the effective gene transfection. Recently, in an attempt to enhance the possibility of caveolae-mediated endocytosis, we fabricated a peptide-targeted gene vector for highly efficient receptor-mediated intracellular delivery. Cyclic Asn-Gly-Arg (cNGR) peptide was used to target gene loaded poly(lactic acid)-poly(ethylene glycol) nanoparticles (PLA-PEG NPs) to HUVEC over-expressing CD13. Using 6-lauroxyhexyl lysinate (LHLN) as cationic surfactant, cNGR modified PLA-PEG NPs (cNGR-PEG-PLA NPs) were capable of complexing and compacting DNA into homogeneous small-sized complexes (< 200 nm) with positive charge (~ 10 mV). Fortunately, the results of in vitro cellular uptake tests and mechanism studies were consistent with our original hypothesis. The cNGR peptide presented on nanoparticles' surface could specifically mediate the fast and efficient internalization of cNGR-PEG-PLA NPs into HUVEC. Moreover, free cNGR inhibited their intracellular uptake into HUVEC revealing the mechanism of receptor-mediated endocytosis. Furthermore, the inspiring results of the mechanism studies and transfection assays demonstrated that caveolae-mediated endocytosis was indeed mainly involved in the internalization of cNGR-PEG-PLA NPs into HUVEC and led to significant gene transfection efficiency in contrast with cNGR non-modified PLA-PEG NPs. Given such encouraging and favorable properties including biocompatibility, high transfer efficiency, low cytotoxicity, and fast uptake by nondestructive endocytic pathways, cNGR-PEG-PLA NPs could be a promising carrier for the intracellular delivery of therapeutic agents.     

Increased receptor-mediated gene delivery to the liver by protamine-enhanced-asialofetuin-lipoplexes

A novel lipidic vector composed of DOTAP/Chol liposomes, asialofetuin (AF), protamine sulfate and DNA has been developed. The resulting protamine-AF-lipoplexes improved significantly the levels of gene expression in cultured cells and in the liver upon i.v. administration. Lipoplexes containing the optimal amount of AF (1 microg/microg DNA) showed a 16-fold higher transfection activity in HepG2 cells than non-targeted (plain) complexes. The uptake by cells having asialoglycoprotein receptors (ASGPr) on their plasma membrane was decreased by the addition of free AF, indicating that AF-lipoplexes were taken up specifically by cells via ASGPr-mediated endocytosis. Results from transfections performed in cells defective in ASGPr, ie HeLa cells, confirmed this mechanism. By addition of the condensing peptide, protamine sulfate, smaller complexes were obtained, which enhanced even more the uptake of AF-complexes in HepG2 cells and in the liver. The optimal amount of protamine was 0.4 microg/mcirog DNA, and gene expression was about 5-fold over that obtained with AF-lipoplexes in the absence of the peptide, and 75-fold higher than that with plain conventional lipoplexes. Protamine-AF-lipoplexes increased by a factor of 12 luciferase gene expression in the liver of mice administered systemically via the tail vein, compared to plain complexes. In summary, our findings extend the scope of previous studies where AF-lipoplexes were used to introduce DNA into hepatocytes. The combination of targeting and protamine condensation obviated the need for partial hepatectomy, commonly required to obtain efficient gene delivery in this organ. Since protamine sulfate has been proven to be non-toxic in humans, the novel liver-specific vector described here may be useful for the delivery of clinically important genes to this organ.     

Polymer delivery systems for site-specific genome editing

Triplex-forming peptide nucleic acids (PNAs) can be used to coordinate the recombination of short 50-60 bp “donor DNA” fragments into genomic DNA, resulting in site-specific correction of genetic mutations or the introduction of advantageous genetic modifications. Site-specific gene editing in hematopoietic stem and progenitor cells (HSPCs) could result in the treatment or cure of inherited disorders of the blood such as β-thalassemia or sickle cell anemia. Gene editing in HSPCs and differentiated T cells could also help combat HIV infection by modifying the HIV co-receptor CCR5, which is necessary for R5-tropic HIV entry. However, translation of genome modification technologies to clinical practice is limited by challenges in intracellular delivery, especially in difficult-to-transfect hematolymphoid cells. Here, we review the use of engineered biodegradable polymer nanoparticles for site-specific genome editing in human hematopoietic cells, which represent a promising approach for ex vivo and in vivo gene therapy.     

Liposomes as carriers for DNA-PNA hybrids.

Peptide nucleic acids (PNAs) are DNA mimics composed of N-(2-aminoethyl)glycine units. This structure gives to PNAs (a) resistance to DNases and proteinases, (b) capacity to hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, and (c) capacity to form highly stable (PNA)(2)-RNA triplexes with RNA targets. Furthermore, DNA-PNA hybrid molecules are capable to reversibly interact with DNA-binding proteins, being therefore of interest for studies on regulation of gene expression by the decoy approach. The major conclusion of this paper is that cationic liposomes are able to efficiently complexate DNA-PNA hybrid molecules and mediate their binding to target cells. Our results are of some interest, since, unlike commonly used nucleic acids analogs, PNA oligomers are not taken up spontaneously into the cells. In addition, they are not suitable for an efficient delivery with commonly used liposomal formulations. Transfection of PNA-DNA hybrid molecules to in vitro cultured cells could be of great interest to determine the applications of these new reagents to experimental alteration of gene expression.     

Peptide nucleic acid delivery to human mitochondria

Peptide nucleic acids (PNAs) are synthetic polynucleobase molecules, which bind to DNA and RNA with high affinity and specificity. Although PNAs have enormous potential as anti-sense agents, the success of PNA-mediated gene therapy will require efficient cellular uptake and sub-cellular trafficking. At present these mechanisms are poorly understood. To address this, we have studied the uptake of biotinylated PNAs into cultured cell lines using fluorescence confocal microscopy. In human myoblasts, initial punctate staining was followed by the release of PNAs into the cytosol and subsequent localisation and concentration in the nucleus. To determine whether PNAs could also be used as therapeutic agents for mtDNA disease, we attempted to localise PNAs to the mitochondrial matrix. When attached to the presequence peptide of the nuclear-encoded human cytochrome c oxidase (COX) subunit VIII, the biotinylated PNA was successfully imported into isolated organelles in vitro. Furthermore, delivery of the biotinylated peptide-PNA to mitochondria in intact cells was confirmed by confocal microscopy. These studies demonstrate that biotinylated PNAs can be directed across cell membranes and to a specific sub-cellular compartment within human cells - highlighting the importance of these novel molecules for human gene therapy.     

Glutathione-responsive nano-vehicles as a promising platform for targeted intracellular drug and gene delivery

The past couple of years have witnessed a tremendous progress in the development of glutathione-responsive nano-vehicles for targeted intracellular drug and gene delivery, as driven by the facts that (i) many therapeutics (e.g. anti-cancer drugs, photosensitizers, and anti-oxidants) and biotherapeutics (e.g. peptide and protein drugs, and siRNA) exert therapeutical effects only inside cells like the cytosol and cell nucleus, and (ii) several intracellular compartments such as cytosol, mitochondria, and cell nucleus contain a high concentration of glutathione (GSH) tripeptides (about 2-10 mM), which is 100 to 1000 times higher than that in the extracellular fluids and circulation (about 2-20 μM). Glutathione has been recognized as an ideal and ubiquitous internal stimulus for rapid destabilization of nano-carriers inside cells to accomplish efficient intracellular drug release. In this paper, we will review recent results on GSH-responsive nano-vehicles in particular micelles, nanoparticles, capsules, polymersomes, nanogels, dendritic and macromolecular drug conjugates, and nano-sized nucleic acid complexes for controlled delivery of anti-cancer drugs (e.g. doxorubicin and paclitaxel), photosensitizers, anti-oxidants, peptides, protein drugs, and nucleic acids (e.g. DNA, siRNA, and antisense oligodeoxynucleotide). The unique disulfide chemistry has enabled novel and versatile designs of multifunctional delivery systems addressing both intracellular and extracellular barriers. We are convinced that GSH-responsive nano-carrier systems have enormous potential in targeted cancer therapy.     

Recent advances in the development of peptide nucleic acid as a gene-targeted drug

Peptide nucleic acid (PNA) is a non-ionic mimic of DNA that binds to complementary DNA and RNA sequences with high affinity and selectivity. Targeting of single-stranded RNA leads to antisense effects, whereas PNAs directed toward double-stranded DNA exhibit antigene properties. Recent advances in cell uptake and in antisense and antigene effects in biological systems are summarised in this review. In addition to traditional targets, namely genomic DNA and messenger RNA, applications for PNA as a bacteriocidal antibiotic, for regulating splice site selection and as a telomerase inhibitor are described.     

Anti-JL1 antibody-conjugated poly (L-lysine) for targeted gene delivery to leukemia T cells.

We have designed the gene delivery carrier targeted to Molt 4 cells, human leukemia T cells, using monoclonal antibody against leukemia-specific JL1 antigen, anti-JL1 antibody, as a targeting moiety. Anti-JL1 antibody has been proven to bind to JL1 antigen and subsequently be internalized into Molt 4 cells, demonstrating that anti-JL1 antibody has the potential as a targeting ligand for leukemia-specific gene transfer. Anti-JL1 antibody was modified with the heterobifunctional crosslinker, PDPH, at carbohydrate sites and conjugated to thiolated poly-L-lysine (PLL) via disulfide bridges. The composition and antigen binding affinity of antibody-PLL conjugates were analyzed by the amino acid analysis and the flow cytometry, respectively. Antibody-PLL conjugates neutralized pSV-beta-galactosidase plasmid DNA at 5:1 weight ratio and condensed into about 200--300-nm complexes. DNA/antibody-PLL complexes were effectively internalized into Molt 4 cells after 4 h incubation at 37 degrees C and showed significantly higher in vitro transfection efficiency than DNA/PLL complexes and DNA/Lipofectin formulation due to the targeting effect of receptor-mediated endocytosis induced by anti-JL1 antibody.     

Unconventional internalization mechanisms underlying functional delivery of antisense oligonucleotides via cationic lipoplexes and polyplexes

There is mounting interest in developing antisense and siRNA oligonucleotides into therapeutic entities; however, this potential has been limited by poor access of oligonucleotides to their pharmacological targets within cells. Transfection reagents, such as cationic lipids and polymers, are commonly utilized to improve functional delivery of nucleic acids including oligonucleotides. Cellular entry of large plasmid DNA molecules with the assistance of these polycationic carriers is mediated by some form of endocytosis; however, the mechanism for delivery of small oligonucleotide molecules has not been well established. In this study, splice-shifting oligonucleotides have been formulated into cationic lipoplexes and polyplexes, and their internalization mechanisms have been examined by using pharmacological and genetic inhibitors of endocytosis. The results showed that intercellular distribution of the oligonucleotides to the nucleus governs their pharmacological response. A mechanistic study revealed that oligonucleotides delivered by lipoplexes enter the cells partially by membrane fusion and this mechanism accounts for the functional induction of the target gene. In contrast, polyplexes are internalized by unconventional endocytosis pathways that do not require dynamin or caveolin. These studies may help rationally design novel delivery systems with superior transfection efficiency but lower toxicity.     

Combination of a new generation of PNAs with a peptide-based carrier enables efficient targeting of cell cycle progression

The design of potent systems for the delivery of charged and noncharged molecules that target genes of interest remains a challenge. We describe a novel technology that combines a new generation of peptide nucleic acids (PNAs), or HypNA-pPNAs, with a new noncovalent peptide-based delivery system, Pep-2, which promotes efficient delivery of PNAs into several cell lines. We have validated the potential of this technology by showing that Pep2-mediated delivery of an antisense HypNA-pPNA chimera directed specifically against cyclin B1 induces rapid and robust downregulation of its protein levels and efficiently blocks cell cycle progression of several cell lines, as well as proliferation of cells derived from a breast cancer. Pep-2-based delivery system was shown to be 100-fold more efficient in delivering HypNA-pPNAs than classical cationic lipid-based methods. Whereas Pep-2 is essential for improving the bioavailability of PNAs and HypNA-pPNAs, the latter contribute significantly to the efficiency and specificity of the biological response. We have found that Pep-2/HypNA-pPNA strategy promotes potent antisense effects, which are approximately 25-fold greater than with classical antisense oligonucleotide directed specifically against the same cyclin B1 target. Taken together, these data demonstrate that peptide-mediated delivery of HypNA-pPNAs constitutes a very promising technology for therapeutic applications.     

Branched cationic peptides for gene delivery: role of type and number of cationic residues in formation and in vitro activity of DNA polyplexes

To examine the suitability of synthetic peptides as DNA-binding and -compacting agents for receptor-mediated gene delivery, we have synthesized and characterized a series of branched oligocationic peptides that differ in the number and type (lysine, arginine, ornithine) of cationic amino acids in the DNA-binding moiety. The peptides were designed as branched molecules to provide a coupling site via a spacer for the attachment of effectors at a flexible distance from the DNA-binding moiety. This design provides torsional flexibility in the peptide backbone of the DNA-binding moiety to maximize cation-DNA phosphate interactions and also minimizes the potential for interference by the effector with DNA binding. The branched peptides bind DNA with affinities that increase with the number of cationic groups. The peptides compact DNA into microparticulate structures as judged by an ethidium bromide displacement assay, dynamic light scattering, and electron microscopy. In general, differences in DNA binding and compaction owing to variation in the cationic side chain were modest, with the rank order being arginyl > lysyl approximately ornithyl. Incorporation of tryptophans into the DNA-binding moiety had no major effect on apparent binding affinity but clearly reduced the DNA-compacting potency of the peptides. Compared with polylysine, the peptides and their DNA complexes are weak activators of the complement system. Complement activation by an octaarginyl peptide was stronger than that induced by an octalysyl peptide. The microparticulate peptide-DNA complexes are suitable for receptor-mediated gene delivery as evidenced by transferrinfection of K562 cells in the presence of chloroquine. The results obtained in gene delivery in vitro suggest that a minimum chain length of six to eight cationic amino acids is required to compact DNA into structures active in receptor-mediated gene delivery.     

Synergistic Effects of Anti-CmeA and Anti-CmeB Peptide Nucleic Acids on Sensitizing Campylobacter jejuni to Antibiotics

The CmeABC efflux pump in Campylobacter jejuni confers resistance to structurally divergent antimicrobials, and inhibition of CmeABC represents a promising strategy to control antibiotic-resistant Campylobacter. Antisense peptide nucleic acids (PNAs) targeting the three components of CmeABC were evaluated for inhibition of CmeABC expression. The result revealed a synergistic effect of the PNAs targeting CmeA and CmeB on sensitizing C. jejuni to antibiotics. This finding further demonstrates the feasibility of using PNAs to potentiate antibiotics against antibiotic-resistant Campylobacter.     

Gold Nanoparticles for Nucleic Acid Delivery

Gold nanoparticles provide an attractive and applicable scaffold for delivery of nucleic acids. In this review, we focus on the use of covalent and noncovalent gold nanoparticle conjugates for applications in gene delivery and RNA-interference technologies. We also discuss challenges in nucleic acid delivery, including endosomal entrapment/escape and active delivery/presentation of nucleic acids in the cell.     

Inhibition of gene expression and growth by antisense peptide nucleic acids in a multiresistant beta-lactamase-producing Klebsiella pneumoniae strain

Klebsiella pneumoniae causes common and severe hospital- and community-acquired infections with a high incidence of multidrug resistance. The emergence and spread of beta-lactamase-producing K. pneumoniae strains highlight the need to develop new therapeutic strategies. In this study, we developed antisense peptide nucleic acids (PNAs) conjugated to the (KFF)(3)K peptide and investigated whether they could mediate gene-specific antisense effects in K. pneumoniae. No outer membrane permeabilization was observed with antisense PNAs when used alone. Antisense peptide-PNAs targeted at two essential genes, gyrA and ompA, were found to be growth inhibitory at concentrations of 20 muM and 40 muM, respectively. Mismatched antisense peptide-PNAs with sequence variations of the gyrA and ompA genes when used as controls were not growth inhibitory. Bactericidal effects exerted by peptide-anti-gyrA PNA and peptide-anti-ompA PNA on cells were observed within 6 h of treatment. The antisense peptide-PNAs specifically inhibited expression of DNA gyrase subunit A and OmpA from the respective targeted genes in a dose-dependent manner. Both antisense peptide-PNAs cured IMR90 cell cultures that were infected with K. pneumoniae (10(4) CFU) in a dose-dependent manner without any noticeable toxicity to the human cells.     

Selective recognition of human telomeric G-quadruplex with designed peptide via hydrogen bonding followed by base stacking interactions

We described a novel synthetic peptide in which a glutamine residue binds through hydrogen bonding to a guanine-base and a trytophan residue intercalates with K⁺ resulting in stabilization of a human telomeric G-quadruplex with high selectivity over its complementary c-rich strand and a double-stranded DNA and its complementary C-rich strand. This peptide offers great potential for cancer treatment by inhibiting the telomere extension by telomerase.

A new synthetic peptide is presented. A Glu residue binds through H-bonding to a guanine-base and a Trp residue intercalates with K⁺ resulting in stabilization of a human telomeric G-quadruplex with high selectivity over a complementary c-rich strand and double-stranded DNA.     

Cytosolic delivery of a p16-peptide oligoarginine conjugate for inhibiting proliferation of MCF7 cells.

One of the major limitations in protein and peptide therapeutics is the requirement of delivery to the cytosol or nucleus of cells. It has recently been shown that a small peptide derived from the p16 protein is able to inhibit cell cycle progression when delivered to the cytosol after conjugation to cell penetrating peptides, however the correlation between delivery efficiency and biological activity has not been made. Additionally, whether or not the biological activity attained was due to membrane transduction has not been established. In this paper, the total internalization, and internalization via endocytosis and transduction of 125I-p16, 125I-p16-C(R)9, and 125I-p16-C(K)9 were determined in MCF7 cultured cell monolayers. The results showed that while p16 and p16-oligopeptide conjugates have similar total internalization, 125I-p16-C(R)9 is predominantly internalized via membrane transduction, while p16 and p16-oligolysine are primarily endocytosed. Therefore, the amount of 125I-p16-C(R)9 delivered to the cytosol is significantly higher than both 125I-p16-C(K)9 and 125I-p16. These results show that biological activity is correlated with membrane transduction efficiency, and not total internalization. Additionally, the biological activity and delivery to the cytosol were not sensitive to endocytic inhibitors, verifying that the biological effect is due to membrane transduction, and not endocytosis.     

Targeting Essential Genes in Salmonella enterica Serovar Typhimurium with Antisense Peptide Nucleic Acid

We investigated the capability of antisense peptide nucleic acids (PNAs) conjugated to the (KFF)₃K cell-penetrating peptide to target possible essential genes (ligA, rpoA, rpoD, engA, tsf, and kdtA) in Salmonella enterica serovar Typhimurium and inhibit bacterial growth in vitro and in cell culture. All targeted PNA-based gene inhibition has shown great potency in gene expression inhibition in a sequence-specific and dose-dependent manner at micromolar concentrations. Among tested PNAs, the anti-rpoA and -rpoD PNAs showed the greatest potency.     

Ligand substitution of receptor targeted DNA complexes affects gene transfer into hepatoma cells

We have targeted the serpin enzyme complex receptor for gene transfer in human hepatoma cell lines using peptides < 30 amino acids in length which contain the five amino acid recognition sequence for this receptor, coupled to poly K of average chain length 100 K, using the heterobifunctional coupling reagent sulfo-LC SPDP. The number of sulfo-LC SPDP modified poly-L-lysine residues, as well as the degree of peptide substitution was assessed by nuclear magnetic resonance spectroscopy. Conjugates were prepared in which 3.5%, 7.8% or 26% of the lysine residues contained the sulfo-LC SPDP moiety. Each of these conjugates was then coupled with ligand peptides so that one in 370, one in 1039, or one in 5882 lysines were substituted with receptor ligand. Electron microscopy and atomic force microscopy were used to assess complex structure and size. HuH7 human hepatoma cells were transfected with complexes of these conjugates with the plasmid pGL3 and luciferase expression measured 2 to 16 days after treatment. All the protein conjugates in which 26% of the K residues were modified with sulfo-LC SPDP were poor gene transfer reagents. Complexes containing less substituted poly K, averaged 17 +/- 0.5 nm in diameter and gave peak transgene expression of 3-4 x 10(6) ILU/mg which persisted (> 7 x 10(5) ILU) at 16 days. Of these, more substituted polymers condensed DNA into complexes averaging 20 +/- 0.7 nm in diameter and gave five-fold less luciferase than complexes containing less substituted conjugates. As few as eight to 11 ligands per complex are optimal for DNA delivery via the SEC receptor. The extent of substitution of receptor-mediated gene transfer complexes affects the size of the complexes, as well as the intensity and duration of transgene expression. These observations may permit tailoring of complex construction for the usage required.