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Antoine Kichler

Publications and source records attributed to Antoine Kichler.

13 recordsLinked to original sources
PubMed2006

Characterization of the gene transfer process mediated by histidine-rich peptides.

Peptides have an interesting potential as gene transfer agents not only because they are biodegradable but also due to their reduced size and the facility of product characterization and large-scale production. Recently, we have shown that the cationic amphipathic 26-mer peptide LAH4 possesses high DNA delivery capacities. To better understand its way of functioning and to further improve its efficiency, we synthesized and tested various LAH4 derivatives. The evaluation of their DNA delivery capacity, as well as their transfection efficiency, makes accessible valuable information about structure-function correlations. In particular, our results indicate the threshold peptide concentration for endosomal escape and conformational preferences for the peptide. Furthermore, the results indicate that a fine balance of peptide-DNA interactions is responsible for the high transfection activity of LAH4. Taken together, the data also suggest that the peptides efficiently transport DNA into the cytoplasm and that the creation of more potent transfection compounds probably needs improvement of other steps during the process.

Cell Line↗
PubMed2006

Cationic amphipathic histidine-rich peptides for gene delivery.

Besides being a useful tool in research, gene transfer has a high potential as treatment for a variety of genetic and acquired diseases. However, in order to enable a gene to become a pharmaceutical, efficient and safe methods of delivery have to be developed. We recently found that cationic amphipathic histidine-rich peptide antibiotics can efficiently deliver DNA into mammalian cells. Our lead compound, LAH4 (KKALLALALHHLAHLALHLALALKKA), demonstrated in vitro transfection efficiencies comparable to those of commercially available reagents. Synthesis and evaluation of LAH mutants provided evidence that the transfection efficiency depends on the number and positioning of histidine residues in the peptide as well as on the pH at which the in-plane to transmembrane transition takes place. Moreover, recent results suggest that binding of the DNA complexes to the plasma membrane is mediated by heparan sulfate proteoglycans and that anionic phospholipids may be involved in the endosomal destabilization process. Finally, we also describe in this review the rationale that led to the development of LAH4 as a DNA carrier as well as the biophysical methods that have allowed us to propose a model which could explain the way this peptide destabilizes the endosomal bilayer.

Amino Acid Sequence↗
PubMed2006

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.

Amino Acid Sequence↗
PubMed2005

The antibiotic and DNA-transfecting peptide LAH4 selectively associates with, and disorders, anionic lipids in mixed membranes.

The histidine-rich amphipathic peptide LAH4 has antibiotic and DNA delivery capabilities. The peptide has a strong affinity for anionic lipids found in the outer membrane of bacterial membranes. A role for anionic lipids in release of cationic plasmid-containing complexes has been proposed previously, and disruption of membrane asymmetry and presentation of phosphatidylserine (PS) in the membrane outer leaflet is a general feature observed in diseased mammalian cells. Therefore, to understand the peptide-lipid interactions in more detail, solid-state NMR experiments on model membranes have been performed. 31P MAS NMR on mixed phosphatidylcholine (PC)/PS and PC/phosphatidylglycerol (PG) membranes has been used to demonstrate a strong interaction between LAH4 and anionic lipids. By using deuterated lipids and wide-line 2H NMR when probing lipid chain order, it is demonstrated that LAH4 preferentially interacts with PS over PC and effectively disorders the anionic PS lipid fatty acyl chains. In addition, we demonstrate that the efficiency of gene transfer in vitro to different cell lines is closely related to the degree of disruption of PS acyl chains for four isomers of LAH4. This work suggests a mechanism of selective destabilization by LAH4 of anionic lipids in the membranes of cells during transfection with implications for nucleic acid delivery in vivo.

Amino Acid Sequence↗
PubMed2005

Cationic steroid antibiotics demonstrate DNA delivery properties.

Recently, cationic steroids have been developed that display broad-spectrum antibacterial activity. These compounds, characterized by the presence of several amino groups, present a facially amphiphilic morphology. Formulations containing such steroids were tested for their ability to facilitate the uptake of a reporter plasmid into various cell lines. The results show that, when associated with the naturally occurring zwitterionic lipid dioleoyl-phosphatidylethanolamine (DOPE), cationic steroid antibiotics allow for transfection levels comparable to those obtained with DOTAP. The activity of the amphiphilic mixture was nearly unaffected by bafilomycin A1 and chloroquine treatment, suggesting a mechanism that is independent of the acidification process associated with endocytosis. Collectively, our results show that DNA delivery agents possessing strong antibacterial properties can be obtained by conjugating amino groups to a steroid nucleus.

Anti-Bacterial Agents↗
PubMed2005

Dilution of reporter gene with stuffer DNA does not alter the transfection efficiency of polyethylenimines.

BACKGROUND: Polyethylenimines (PEIs) are among the most efficient non-viral gene transfer agents developed so far. However, transfections with these polymers were shown to require a very high copy number of plasmid DNA per cell to achieve gene expression. Here, we investigate whether it is possible to reduce the amount of plasmid DNA while keeping a high transfection efficiency. METHODS: Transfection experiments were performed under various conditions in order to study the interdependence between the amount of reporter DNA, the amine-to-phosphate ratio and the transfection efficiency. RESULTS: When suboptimal amounts of linear PEI 22 kDa/DNA complexes were used for transfection, a severe reduction in reporter gene expression was observed. On the other hand, for optimal amounts of PEI/DNA complexes more than half of the reporter gene can be replaced by carrier DNA or polyglutamic acid without substantially decreasing the transfection efficiency of the polymer both in cultured cells and after systemic administration in mice. When used under the same in vitro experimental conditions, the lipospermine DOGS, but not the monocationic lipid DOTAP, gave similar results. CONCLUSIONS: Taken together, our data suggest that the activity of compounds with endosome-buffering capacities, such as PEIs and lipospermines, requires a threshold amount of transfection agent. In addition, our results indicate that, in many gene transfer situations, it will be possible to lower the dose of active plasmid thus reducing costs and the risk of immune stimulation triggered by bacterial DNA.

Animals↗
PubMed2004

Gene transfer with modified polyethylenimines.

Branched and linear polyethylenimines (PEIs) have proven to be efficient and versatile agents for gene delivery in vitro. In addition, systemic administration of positively charged DNA/PEI complexes results in significant reporter gene expression in lungs. However, re-targeting of complexes to organs other than the lung is hampered by non-specific interactions of polyplexes with blood components and non-target cells. Thus, despite considerable transfectional activity, the properties of PEIs need to be further improved. Therefore, various modifications of PEIs have been explored in recent years. For example, to increase the circulation half-life of the DNA complexes, the surface charge of the particles was shielded by grafting hydrophilic polymers such as polyethylene glycols (PEGs) onto their surface. Alternatively, incorporation of certain ligands into the DNA complexes also resulted in charge shielding even without PEGylation. Herein, I review the most recent PEI derivatives, with a special focus on PEGylated and targeted polymers.

Animals↗
PubMed2003

Preparation and evaluation of a new class of gene transfer reagents: poly(-alkylaminosiloxanes).

We report the evaluation of poly(-alkylaminosiloxane) as a novel class of polycationic DNA carriers. Controlled hydrolysis of mono- and di-aminoalkylmethyldimethoxysilane provided a wide range of defined oligomeric mixtures. Basic hydrolysis conditions yielded mixtures composed mainly of cyclic and long linear oligomers, while under acidic conditions mainly short-linear oligomers were derived. They all efficiently interacted with plasmid DNA as revealed by electron microscopy and DNA retardation assays. However, only diamine-based oligomers prepared under basic conditions were able to mediate substantial levels of DNA transfection in human HeLa cells. SiDA1b, prepared by basic hydrolysis of 3-(2-aminoethylamino)propyl-methyl-dimethoxysilane, was found to be at least as efficient as the frequently used cationic transfection agents DOTAP and polyethylenimine (PEI). The transfection activity was sensitive to bafilomycin A1, suggesting a mechanism that depends on proton capture during the acidification process associated with endocytosis.

Animals↗
PubMed2003

The cationic amphipathic alpha-helix of HIV-1 viral protein R (Vpr) binds to nucleic acids, permeabilizes membranes, and efficiently transfects cells.

Viral protein R (Vpr) is a small protein of 96 amino acids that is conserved among the lentiviruses human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. We recently sought to determine whether the karyophilic properties of Vpr, as well as its ability to bind nucleic acids, could be used to deliver DNA into cells. We have found that the C-terminal domain of Vpr-(52-96) is able to efficiently transfect various cell lines. Here, we show that the shortest active sequence for gene transfer corresponds to the domain that adopts a alpha-helix conformation. DNA binding studies and permeabilization assays performed on cells demonstrated that the peptides that are efficient in transfection condense plasmid DNA and are membranolytic. Electron microscopy studies and transfection experiments performed in the presence of inhibitors of the endocytic processes indicated that the major entry pathway of Vpr-DNA complexes is through endocytosis. Taken together, the results show that the cationic C-terminal alpha-helix of Vpr has DNA-condensing as well as membrane-destabilizing capabilities, both properties that are indispensable for efficient DNA transfection.

Cell Line↗
PubMed2003

Histidine-rich amphipathic peptide antibiotics promote efficient delivery of DNA into mammalian cells.

Gene delivery has shown potential in a wide variety of applications, including basic research, therapies for genetic and acquired diseases, and vaccination. Most available nonviral systems have serious drawbacks such as the inability to control and scale the production process in a reproducible manner. Here, we demonstrate a biotechnologically feasible approach for gene delivery, using synthetic cationic amphipathic peptides containing a variable number of histidine residues. Gene transfer to different cell lines in vitro was achieved with an efficiency comparable to commercially available reagents. We provide evidence that the transfection efficiency depends on the number and positioning of histidine residues in the peptide as well as on the pH at which the in-plane to transmembrane transition takes place. Endosomal acidification is also required. Interestingly, even when complexed to DNA these peptides maintain a high level of antibacterial activity, opening the possibility of treating the genetic defect and the bacterial infections associated with cystic fibrosis with a single compound. Thus, this family of peptides represents a new class of agents that may have broad utility for gene transfer and gene therapy applications.

Amino Acid Sequence↗
PubMed2002

Intranasal gene delivery with a polyethylenimine-PEG conjugate.

Polyethylenimines (PEIs) are among the most efficient synthetic DNA carriers. High levels of reporter gene expression can be obtained with these agents on a variety of cells. Nevertheless, the gap between their efficiency and that required for therapeutic approaches is still important. With the aim to improve the in vivo transfection properties of PEIs, we have synthesized a conjugate consisting of the linear polymer of 22 kDa covalently modified with polyethyleneglycol (PEG) residues. The resulting conjugate was able to complex DNA and allowed the preparation of highly concentrated polyplexes, in contrast to non-modified PEIs. Administration by nasal instillation of PEI-PEG/DNA complexes in mice resulted in significant levels of transgene expression. Luciferase activity was greatest 24 h after delivery and decreased thereafter. Our results show that the grafting of PEGs can improve some of the properties of PEIs.

Administration, Intranasal↗
PubMedDate not supplied

Synthesis of linear polyethylenimine derivatives for DNA transfection.

A series of linear polymers containing varying amounts of ethylenimine or N-propylethylenimine units were synthesized by hydrolysis and/or reduction of polyethyloxazolines. The pK(a)s of the polyamines were determined potentiometrically. Gel mobility shift assay showed that the efficiency of DNA complexation was related to the fraction of amino groups that are protonated at neutral pH. The effects of cationic charge density and molar weight of the polymers on the transfection efficiency were evaluated on HepG2 cells. The results obtained with different copolymers show that the transfection efficiency primarily depends on the fraction of ethylenimine units included in the polymer albeit the molar weight is also of importance. On the basis of the results obtained with poly(N-propylethylenimines), we also demonstrate that the high transfection efficiency of polyethylenimines does not solely rely on their capacity to capture protons which are transferred into the endo-lysosomes during acidification.

Cell Line, Tumor↗