Manipulating the pH response of 2,3-diaminopropionic acid rich peptides to mediate highly effective gene silencing with low-toxicity

Cationic amphipathic pH responsive peptides possess high in vitro and in vivo nucleic acid delivery capabilities and function by forming a non-covalent complex with cargo, protecting it from nucleases, facilitating uptake via endocytosis and responding to endosomal acidification by being released from the complex and inserting into and disordering endosomal membranes. We have designed and synthesised peptides to show how Coulombic interactions between ionizable 2,3-diaminopropionic acid (Dap) side chains can be manipulated to tune the functional pH response of the peptides to afford optimal nucleic acid transfer and have modified the hydrogen bonding capabilities of the Dap side chains in order to reduce cytotoxicity. When compared with benchmark delivery compounds, the peptides are shown to have low toxicity and are highly effective at mediating gene silencing in adherent MCF-7 and A549 cell lines, primary human umbilical vein endothelial cells and both differentiated macrophage-like and suspension monocyte-like THP-1 cells.     

Influence of cationic lipid composition on gene silencing properties of lipid nanoparticle formulations of siRNA in antigen-presenting cells

Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases.     

Enhanced folate receptor mediated gene therapy using a novel pH-sensitive lipid formulation.

Liposomal gene therapy vectors that penetrate cells by endocytosis must escape an endosomal compartment in order to enter the target cell's nucleus. Because such endosomal compartments are generally acidic in nature, pH-sensitive liposomes have been designed that are stable at extracellular pH ( approximately pH 7.4) but fusogenic at endosomal pH values ( approximately pH 5). We report here the use of a novel folate-targeted, pH-sensitive, anionic liposomal vector that mediates the efficient delivery of DNA into folate receptor-bearing cells and discharges the DNA into the cytoplasm. N-Citraconyl-dioleoylphosphatidylethanolamine (C-DOPE), a derivative of dioleylphosphatidylethanolamine (DOPE) that hydrolyzes rapidly at pH 5 to yield DOPE, was synthesized and incorporated with DOPE and folate-polyethyleneglycol-DOPE into liposomes. The resulting liposomes were stable at neutral pH but fusogenic at pH 5. Folate-labeled gene transfer vectors were prepared by compacting plasmid DNA with polylysine at a 1:0.75 (w/w) ratio and complexing the condensed cationic plasmid with the above anionic liposomes. Association of the polylysine-DNA with the liposomes was confirmed by sucrose gradient centrifugation, where migration of the folate-labeled vectors was midway between that of the free liposomes and condensed polylysine-DNA. Transfection of cultured cancer cells with the pH-sensitive liposomal vectors was found to be significantly more efficient than transfection with DOPE-cholesterol hemisuccinate-based vectors, the more commonly used pH-dependent, liposomal transfection formulation. Optimization studies revealed that inclusion of only 3% C-DOPE and 0.1% folate-derivatized DOPE yielded the highest transfection activity. Nearly quantitative competition with free folic acid as well as direct correlation of transfection efficiency with folate receptor density for several different cell lines further documented that vector uptake was mediated by folate receptor endocytosis. Taken together, these data argue that C-DOPE warrants further consideration as a pH sensitive component of lipid-based gene delivery formulations.     

Ultrasound and Microbubble-Targeted Delivery of Small Interfering RNA Into Primary Endothelial Cells Is More Effective Than Delivery of Plasmid DNA

Ultrasound and microbubble-targeted delivery (UMTD) is a promising non-viral technique for genetic-based therapy. We found that UMTD of small interfering RNA (siRNA) is more effective than delivery of plasmid DNA (pDNA). UMTD (1 MHz, 0.22 MPa) of fluorescently labeled siRNA resulted in 97.9 ± 1.5% transfected cells, with siRNA localized homogenously in the cytoplasm directly after ultrasound exposure. UMTD of fluorescently labeled pDNA resulted in only 43.0 ± 4.2% transfected cells, with localization mainly in vesicular structures, co-localizing with endocytosis markers clathrin and caveolin. Delivery of siRNA against GAPDH (glyceraldehyde-3-phosphate dehydrogenase) effectively decreased protein levels to 24.3 ± 7.9% of non-treated controls (p < 0.01). In contrast, 24 h after delivery of pDNA encoding GAPDH, no increase in protein levels was detected. Transfection efficiency, verified with red fluorescently labeled pDNA encoding enhanced green fluorescent protein, revealed that of the transfected cells, only 2.0 ± 0.7% expressed the transgene. In conclusion, the difference in localization between siRNA and pDNA after UMTD is an important determinant of the effectiveness of these genetic-based technologies.     

Evaluation of transportan 10 in PEI mediated plasmid delivery assay.

Cell-penetrating peptides (CPPs) are novel high-capacity delivery vectors for different bioactive cargoes. We have evaluated the CPP transportan 10 (TP10) as a delivery vector in different in vitro plasmid delivery assays. Tested methods include: TP10 crosslinked to a plasmid via a peptide nucleic acid (PNA) oligomer, TP10 conjugation with polyethyleneimine (PEI), and addition of unconjugated TP10 to standard PEI transfection assay. We found that without additional DNA condensing agents, TP10 has poor transfection abilities. However, the presence of TP10 increases the transfection efficiency several folds compared to PEI alone. At as low concentrations as 0.6 nM, TP10-PNA constructs were found to enhance plasmid delivery up to 3.7-fold in Neuro-2a cells. Interestingly, the transfection efficiency was most significant at low PEI concentrations, allowing reduced PEI concentration without loss of gene delivery. No increase in cytotoxicity due to TP10 was observed and the uptake mechanism was determined to be endocytosis, as previously reported for PEI mediated transfection. In conclusion, TP10 can enhance PEI mediated transfection at relatively low concentrations and may help to develop future gene delivery systems with reduced toxicity.     

The internalization route resulting in successful gene expression depends on both cell line and polyethylenimine polyplex type.

Understanding cellular uptake and intracellular processing of nonviral gene delivery systems is a key aspect in developing more efficient vectors. In this study, the impact of clathrin- and caveolae/lipid-raft-dependent endocytosis on cell entry and overall transfection efficiency of polyethylenimine (PEI) polyplexes was evaluated. Most remarkably, the internalization pathway mediating successful transfection depended on both cell type and polyplex type applied. Colocalization studies with transferrin and cholera toxin B revealed that at least two specific endocytosis pathways--the clathrin-dependent and the lipid-raft-dependent--mediated cellular uptake of PEI polyplexes. With the help of specific uptake inhibitors (chlorpromazine and filipin III), cell-line-dependent variations regarding the route of successful transfection were observed (HUH-7, COS-7, HeLa). In COS-7 cells, the clathrin-dependent pathway was the main contributor to the transfection process. In HUH-7 cells, gene transfer by linear PEI polyplexes succeeded mainly via the clathrin-dependent route, whereas transfection by branched PEI polyplexes was mediated by both pathways. In HeLa cells, both pathways were able to mediate successful gene delivery. However, the lipid-raft-dependent pathway was more relevant. The study also revealed that the concentration window between specific inhibitory function and nonspecific toxicity of the uptake inhibitors was very narrow.     

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.     

A novel environment-sensitive biodegradable polydisulfide with protonatable pendants for nucleic acid delivery

Clinical application of nucleic acid-based therapies is limited by the lack of safe and efficient delivery systems. The purpose of this study is to design and evaluate novel biodegradable polymeric carriers sensitive to environmental changes for efficient delivery of nucleic acids, including plasmid DNA and siRNA. A novel polydisulfide with protonatable pendants was synthesized by the oxidative polymerization of a dithiol monomer, which was readily prepared by solid phase chemistry. The polydisulfide exhibited good buffering capacity and low cytotoxicity. It formed stable complexes with both plasmid DNA and siRNA. The particle sizes of the complexes decreased with the increase of the N/P ratios in the range of 100 to 750 nm. The complexes were stable in the presence of salt and heparin under normal physiological conditions, but dissociated to release nucleic acids in a reductive environment similar to cytoplasm. The polydisulfide demonstrated N/P ratio dependent transfection efficiency for plasmid DNA and gene silencing efficiency for siRNA. The presence of an endosomal disrupting agent, chloroquine, did not affect the DNA transfection efficiency of the polydisulfide. The transfection or gene silencing efficiency of the polydisulfide/DNA or siRNA complexes was comparable to or slightly lower than that of corresponding PEI complexes. Moreover, the polydisulfide showed better serum-friendly feature than PEI when delivering either DNA or siRNA in the presence of 10% FBS. This novel polydisulfide is a promising lead for further design and development of safe and efficient delivery systems for nucleic acids.     

Successful target cell transduction of capsid-engineered rAAV vectors requires clathrin-dependent endocytosis

Cell surface targeting of recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to modify AAV's natural tropism. As modification of the capsid surface is likely to affect the mechanism of vector internalization and consequently the vector's intracellular fate, we investigated early steps in cell transduction of rAAV capsid insertion mutants. Mutants displaying peptides with neutral overall charge at position 587 transduced cells independently of AAV2's primary receptor heparan sulfate proteoglycan (HSPG), whereas mutants carrying positively charged insertions were capable of HSPG binding with affinities correlating with their net positive charge. Whereas rAAV2 is internalized via an HSPG- and clathrin-dependent pathway, HSPG-binding mutants used a clathrin- and caveolin-independent mechanism. Surprisingly, although this pathway was as efficient in mediating vector entry as the one used by rAAV2, successful cell transduction was hampered at a post-entry step, presumably caused by inefficient endosomal escape. In contrast, HSPG-independent, clathrin-dependent internalization used by non-HSPG-binding mutants correlated with efficient nuclear delivery of vector genomes and robust transgene expression. These findings indicate that cell surface targeting strategies should direct uptake of rAAV targeting vectors to clathrin-mediated endocytosis, the naturally evolved entry route of AAV, to promote successful intracellular processing and re-targeting of rAAV's tropism.     

Thermoresponsive polymers as gene delivery vectors: cell viability, DNA transport and transfection studies.

A range of gene delivery vectors containing the thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAm) was evaluated for effects on cell viability, intracellular trafficking and transgene expression in C2C12 mouse muscle cells. Polymers were complexed with plasmid DNA at pH 7.4 and the ability of the resulting particles to transfect cells was assessed via confocal microscopy and protein expression studies in tissue culture. Cell viability assays indicated that these polymers were toxic at high concentrations when not complexed to DNA or at certain polymer:DNA ratios. Poly(ethyleneimine) co-polymers with side-chain grafted PNIPAm were shown to be less toxic than poly(ethyleneimine) alone or PNIPAm-co-(N,N'-dimethylaminoethylmethacrylate) linear co-polymers and the effects were concentration dependent. Confocal micrographs of labeled polymers and DNA indicated rapid cellular entry for all the complexes but expression of Green Fluorescent Protein was achieved only when the branched PEI-PNIPAm co-polymers were used as vectors. The results indicate that design of appropriate co-polymer components and overall polymer architecture can be used to mediate, and perhaps ultimately control, DNA transport and transgene expression.     

Progress and prospects: naked DNA gene transfer and therapy

Increases in efficiency have made naked DNA gene transfer a viable method for gene therapy. Intravascular delivery results in effective gene delivery to liver and muscle, and provides in vivo transfection methods for basic and applied gene therapy and antisense strategies with oligonucleotides and small interfering RNA (siRNA). Delivery via the tail vein in rodents provides an especially simple and effective means for in vivo gene transfer. Electroporation methods significantly enhance direct injection of naked DNA for genetic immunization. The availability of plasmid DNA expression vectors that enable sustained high level expression, allows for the development of gene therapies based on the delivery of naked plasmid DNA.     

Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation

The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors.     

Gene therapy from the perspective of systems biology

Gene therapy research has expanded from its original concept of replacing absent or defective DNA with functional DNA to include the manipulation (increase or decrease) of gene expression by the delivery of modified genes, siRNA or other genetic material via multiple vectors, including naked plasmid DNA, viruses and even cells. Specific tissues or cell types are targeted in order to decrease the risks of systemic or side effects. As with the development of any drug, there is an amount of empiricism in the choice of gene target, route of administration, dosing and, in particular, the scaling-up from preclinical models to clinical trials. High-throughput experimental and computational systems biology studies that account for the complexities of host-disease-therapy interactions hold significant promise in assisting in the development and optimization of gene therapies, including personalized therapies and the identification of biomarkers to evaluate the success of such strategies. This review describes some of the obstacles and successes in gene therapy, using the specific example of growth factor gene delivery to promote angiogenesis and blood vessel remodeling in ischemic diseases; anti-angiogenic gene therapy in cancer is also discussed. In addition, the opportunities for systems biology and in silico modeling to improve on current outcomes are highlighted.     

Development and characterisation of chitosan nanoparticles for siRNA delivery.

Gene silencing mediated by double-stranded small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for diseases with genetic defects. The use of siRNA, however, is hampered by its rapid degradation and poor cellular uptake into cells in vitro or in vivo. Therefore, we have explored chitosan as a siRNA vector due to its advantages such as low toxicity, biodegradability and biocompatibility. Chitosan nanoparticles were prepared by two methods of ionic cross-linking, simple complexation and ionic gelation using sodium tripolyphosphate (TPP). Both methods produced nanosize particles, less than 500 nm depending on type, molecular weight as well as concentration of chitosan. In the case of ionic gelation, two further factors, namely chitosan to TPP weight ratio and pH, affected the particle size. In vitro studies in two types of cells lines, CHO K1 and HEK 293, have revealed that preparation method of siRNA association to the chitosan plays an important role on the silencing effect. Chitosan-TPP nanoparticles with entrapped siRNA are shown to be better vectors as siRNA delivery vehicles compared to chitosan-siRNA complexes possibly due to their high binding capacity and loading efficiency. Therefore, chitosan-TPP nanoparticles show much potential as viable vector candidates for safer and cost-effective siRNA delivery.     

Cell delivery, intracellular trafficking and expression of an integrin-mediated gene transfer vector in tracheal epithelial cells

The mechanism of cell entry and intracellular fate of a gene transfer vector composed of a receptor-targeting, DNA-condensing peptide, RGD-oligolysine, a luciferase encoding plasmid DNA (pDNA) and a cationic liposome was examined. We demonstrate by confocal microscopy, electron microscopy and subcellular fractionation that the major mechanism of entry of the vector is endocytic. The vector complex rapidly (5 min) internalizes into early endosomes, then late endosomes and lysosomes. Entry involves, at least in part, clathrin-coated pit-mediated endocytosis since different conditions or drugs known to influence this pathway modify both uptake of pDNA and its expression. The observed increase in expression with addition of a lip some correlated with an increase in the rate of transfer of the pDNA to lysosomes, a decrease in intracellular recycling and exocytosis of the pDNA and an increase in the amount of pDNA in the nuclear fraction. Trafficking within the cell involved endosome fusion and the acid environment of the endosomes-lysosomes was beneficial for expression. After 30 min both the peptide and pDNA localized to the nucleus and the amount of intact pDNA in the nuclear fraction was highest with liposome and peptide. A better understanding of the cellular mechanisms by which vectors transfer to and traffic in cells should help design improved vectors.     

Therapeutic plasmid DNA versus siRNA delivery: common and different tasks for synthetic carriers

Gene therapy offers great opportunities for the treatment of severe diseases including cancer. In recent years the design of synthetic carriers for nucleic acid delivery has become a research field of increasing interest. Studies on the delivery of plasmid DNA (pDNA) have brought up a variety of gene delivery vehicles. The more recently emerged gene silencing strategy by the intracellular delivery of small interfering RNA (siRNA) takes benefit from existing expertise in pDNA transfer. Despite common properties however, delivery of siRNA also faces distinct challenges due to apparent differences in size, stability of the formed nucleic acid complexes, the location and mechanism of action. This review emphasizes the common aspects and main differences between pDNA and siRNA delivery, taking into consideration a wide spectrum of polymer-based, lipidic and peptide carriers. Challenges and opportunities which result from these differences as well as the recent progress made in the optimization of carrier design are presented.     

A Cell-penetrating Helical Polymer For siRNA Delivery to Mammalian Cells

Cell-penetrating peptides (CPPs) are routinely used for intracellular delivery of a variety of cargo, including drugs, genes, and short interfering RNA (siRNA). Most CPPs are active only upon exposure to acidic environments inside of late endosomes, thereby facilitating the endosomal escape of internalized vectors. Here, we describe the generation of a synthetic polypeptide—PVBLG_n-8—that is able to adopt a helical structure independent of pH. Like other CPPs, the helical structure of PVBLG_n-8 allows the polypeptide to destabilize membranes. However, since the helix is stable at all physiologically relevant pH values between pH 2 and pH 7.4, the membrane permeation properties of PVBLG_n-8 are irreversible. Given its pH-insensitive activity, our results suggest that PVBLG_n-8 is able to facilitate efficient siRNA delivery by causing pore formation in the cell membranes through which either free or complexed siRNA is able to diffuse. This nonspecific form of entry into the cell cytosol may prove useful when trying to deliver siRNA to cells which have proven to be difficult to transfect.     

Enhanced membrane disruption and antibiotic action against pathogenic bacteria by designed histidine-rich peptides at acidic pH

The histidine-rich amphipathic cationic peptide LAH4 has antibiotic and DNA delivery capabilities. Here, we explore the interaction of peptides from this family with model membranes as monitored by solid-state (2)H nuclear magnetic resonance and their antibiotic activities against a range of bacteria. At neutral pH, the membrane disruption is weak, but at acidic pH, the peptides strongly disturb the anionic lipid component of bacterial membranes and cause bacterial lysis. The peptides are effective antibiotics at both pH 7.2 and pH 5.5, although the antibacterial activity is strongly affected by the change in pH. At neutral pH, the LAH peptides were active against both methicillin-resistant and -sensitive Staphylococcus aureus strains but ineffective against Pseudomonas aeruginosa. In contrast, the LAH peptides were highly active against P. aeruginosa in an acidic environment, as is found in the epithelial-lining fluid of cystic fibrosis patients. Our results show that modest antibiotic activity of histidine-rich peptides can be dramatically enhanced by inducing membrane disruption, in this case by lowering the pH, and that histidine-rich peptides have potential as future antibiotic agents.