Search

Despite its considerable interest in human therapy, in vivo siRNA delivery is still suffering from hurdles of vectorization. We have shown recently efficient gene silencing by non-vectorized cationic siRNA. Here, we describe the synthesis and in vitro evaluation of new amphiphilic cationic siRNA. C 12 -, (C 12 ) 2 - and cholesteryl–spermine x –siRNA were capable of luciferase knockdown at nanomolar concentrations without vectorization ( i.e. one to two orders of magnitude more potent than commercially available cholesteryl siRNA). Moreover, incubation in the presence of serum did not impair their efficiency. Finally, amphiphilic cationic siRNA was pre-loaded on albumin. In A549Luc cells in the presence of serum, these siRNA conjugates were highly effective and had low toxicity.

Photochemical internalization (PCI) technology has been used for PEI-mediated p53 gene transfer in mice bearing head and neck squamous cell carcinoma (HNSCC) xenografts. Using luciferase as a reporter gene, PCI led to a 20-fold increase in transgene expression 48 h after transfection and sustained transgene expression for 7 days. Therefore, iterative p53 gene transfer was performed by means of a weekly single injection of PEIGlu4/p53 complexes alone or with PCI for 5 (group A) or 7 (group B) weeks. The efficiency of p53 gene therapy was evaluated by following tumor growth and expression of P53-related downstream proteins (P21, MDM2, Bcl2, Bax). Apoptosis induction was evidenced through caspase-3 activation and PARP cleavage. Using PCI, tumor growth inhibition was observed in all transfected animals. Further, successful tumor cure was achieved in 17% (group A) and 83% (group B) of animals. PCI-mediated p53 gene transfer led to higher P53 protein expression that was correlated with induction of Bax and P21 proapoptotic proteins, repression of Bcl2 as well as activation of caspase-3, and cleavage of PARP. The present study demonstrates that PCI enhances the in vivo efficiency of PEI-mediated p53 gene transfer and can be proposed for p53 gene therapy in HNSCC.

BACKGROUND: Efficient in vivo vectors are needed to exploit the enormous potential of RNA interference (RNAi). Such methods require optimisation for specific delivery routes, tissues and usages. We tested the capacity of different non-viral vectors and formulation methods for inhibition of exogenous (luciferase) gene expression when used to introduce small interfering RNA (siRNA) into the mouse brain in vivo. METHODS: Polyethylenimine (PEI)-based polyplexes and JetSI (a mixture of cationic lipids)-based lipoplexes were used to vectorise plasmid DNA encoding the firefly Photinus pyralis luciferase gene and picomolar amounts of siRNA directed against this gene. Two controls were used, DNA encoding an unrelated luciferase from Renilla reniformis and a mutated siRNA sequence. RESULTS: First, we found that linear PEI, although efficient for delivering nucleic acids to cells, did not permit development of siRNA activity within the dose range tested (

Protein delivery is emerging as an interesting alternative to gene delivery. We have used our experience with transfection to develop a technique for efficient delivery of anionic proteins such as antibodies into the cytoplasm of cells. As for DNA, when complexed with cationic lipids, large amounts of proteins are shown to enter adherent cells via ubiquitously expressed syndecans. However, protein surface area rather than electric charge ratio governs the delivery characteristics. Delivery of anti-beta-actin and anti-alpha-tubulin IgG's leads to fiber depolymerization. Intracellular delivery of an antibody could thus be regarded as another method for interfering with gene activity.

Controlling the size of condensed DNA particles is a key determinant for their diffusion in vivo as well as for gene delivery to target cells. Towards this goal, DNA molecules were compacted individually by cationic thiol-detergents into discrete nanometric entities. These particles were then stabilized by air-induced dimerization of the detergent into a disulfide lipid on the template DNA. Using a tetradecane-cysteine-ornithine (C 14 COrn) detergent, a solution of 5.5 Kb plasmid DNA was thus converted into a monodisperse population of 35-nm particles. The stability of the complexes, as well as their size, morphology and transfection efficiencies were investigated. Surprisingly, the electrophoretic mobility of the quasi-neutral condensed DNA was found higher than that of the extended DNA polyanion. The diameters of particles resulting from the condensation of DNA of various sizes was measured by dynamic light scattering and found to vary as the cubic root of the DNA size. In an attempt to extend their biodistribution and to target tumour cells, we have prepared folate-poly(ethylene oxide)-coated particles that were shown to bind to the cell-surface folate receptor.

International audience; Polyethylenimine (PEI) derivatives are potent polycationic nonviral vectors for gene transfer. The gene transfer efficiency of glucosylated and galactosylated PEI derivatives was optimized using green fluorescent protein gene as reporter gene in FaDu and PANC3 human carcinoma cell lines. Glucosylated or galactosylated PEI derivatives were found to be slightly less cytotoxic than unsubstituted PEI. Gene transfer efficiency was found to be related to DNA/cell number ratio and optimal gene transfer efficiency was achieved at 4 microg DNA/10(5) cells. PEI-DNA complexes were found to enter cells rapidly and were detected into cytoplasmic vesicles 2 hours post-transfection. Green fluorescent protein gene expression was detected 4-6 hours after transfection and reached maximal value 24 hours post-transfection. The results achieved demonstrated that glucosylated PEI yield higher and longer gene transfer efficiency than unsubstituted PEI. Using glucosylated PEI allowed to achieve significant gene transfer in more than 10% of the total cell population for more than 4 days. These data were then applied to p53 gene transfer in PANC3 cells bearing p53 gene deletion and consequently unable to initiate apoptosis. Using glucosylated PEI, p53 gene transfer was successfully achieved with subsequent recovery of p53 mRNA expression and transient P53 protein expression. P53 protein functionality was further demonstrated because transfected cells underwent apoptosis.

As a prerequisite to nonviral gene therapy approaches of ovarian carcinoma, we evaluated the possibility of transfecting established tumor cell lines (SKOV3, IGROV1) as well as primary mesothelial and tumor cells by various polyethylenimine (PEI) derivatives. Several PEI-based vectors were able to effectively transfect these cells, as shown by high luciferase expression levels (10(8) to 10(9) relative light units per milligram of cell protein) that corresponded with 25-50% of green fluorescent protein-positive cells after 24 hours. However, unpredictable differences were observed among the vectors and cell types that a posteriori justified the screening procedure. We also showed that cells that were not transfected after the first experiment remained transfectable in a subsequent transfection experiment to a level similar to that of the initial population. This experiment does not support the emergence of a transfection-resistant cell population and opens the door to multiple therapeutic gene deliveries. Although efficacy and cell targeting still remain to be improved, PEI derivatives appear to be promising molecules for the development of nonviral gene therapy of ovarian carcinoma.

Background Several nonviral vectors including linear polyethylenimine(L-PEI) confer a pronounced lung tropism to plasmid DNA when injected into the mouse tail vein in a nonionic solution. Methods and results We have optimized this route by injecting 50 µg DNA with excess L-PEI (PEI nitrogen/DNA phosphate=10) in a large volume of 5% glucose (0.4 ml). In these conditions, 1–5% of lung cells were transfected (corresponding to 2 ng luciferase/mg protein), the other organs remaining essentially refractory to transfection (1–10 pg luciferase/mg protein).β-Galactosidase histochemistry confirmed alveolar cells, including pneumocytes, to be the main target, thus leading to the puzzling observation that the lung microvasculature must be permeable to cationic L-PEI/DNA particles of ca 60 nm. A smaller injected volume, premixing of the complexes with autologous mouse serum, as well as removal of excess free L-PEI, all severely decreased transgene expression in the lung. Arterial or portal vein delivery did not increase transgene expression in other organs. Conclusions These observations suggest that effective lung transfection primarily depends on the injection conditions: the large nonionic glucose bolus prevents aggregation as well as mixing of the cationic complexes and excess free L-PEI with blood. This may favour vascular leakage in the region where the vasculature is dense and fragile, i.e. around the lung alveoli. Cationic particles can thus reach the epithelium from the basolateral side where their receptors (heparan sulphate proteoglycans) are abundant. Copyright © 2000 John Wiley & Sons, Ltd.

International audience; Background The ideal non‐viral vector should be cell‐type directed and form complexes with DNA that are physically stable, small and electrically neutral. Methods We have synthesized several PEI derivatives that coat the PEI/DNA complexes with water‐soluble residues able to stabilize the particles, to mask their surface charge and eventually to direct them to a particular tissue. The morphologies and sizes of the complexes were observed by TEM and DLS techniques, and their apparent surface charge was quantitated by zeta potential measurements; in vitro transfection efficacies were determined in serum‐containing cell culture medium. Results When compared to DNA complexes formed with the unmodified PEI, extensive grafting with maltose (15–25% of the amine functions) led to beneficial electrostatic shielding of the particle surface, but was unable to prevent aggregation in physiological salt concentration. More extended hydrophilic residues were therefore explored as a mean of physical repulsion between the particles. Low grafting (2.7%) with a linear dextran nonasaccharide led to small and stable toroids having no apparent surface charge, yet still reaching effective transfection levels. Electron microscopy of complexes with a higher extent of grafting showed worm‐like structures unsuited for cell entry. Conjugation of PEI with as little as 0.5% of a terminally galactose‐derivatized polyethyleneglycol (PEG)‐3400 also gave neutral complexes of another worm‐like structure that failed to transfect receptor‐expressing hepatocytes. Conclusion These results show that conjugation of large and flexible hydrophilic residues to PEI, while protecting the complexes from parasitic interactions also interfere with DNA condensation. PEG conjugation after PEI/DNA complex formation may avoid this problem, provided intracomplex reorganization is slow. Finally an anti‐GD2 antibody (mAb) grafted with PEI was synthesized. The corresponding protein‐coated DNA complexes were compact and small (50–60 nm), yet did not enhance transfection of GD2 ganglioside‐expressing cells.

International audience; Several non-permanent polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyethylenimines, are efficient transfection agents per se, i.e. without the addition of lysosomotropic bases, or cell targeting or membrane disruption agents. These vectors have been shown to deliver genes as well as oligonucleotides both in vitro and in vivo. Our hypothesis is that their efficiency relies on extensive endosome swelling and rupture that provides an escape mechanism for the polycation/DNA particles.

International audience; Synthetic gene transfer vectors could be an attractive alternative to biological vehicles for gene therapy. Polyamine lipids, such as lipospermines are among the most powerful synthetic vectors. This high efficiency is probably related to their endosome buffering ability that could allow DNA to escape from enzymatic degradation. Indeed, this led us recently to discover a comparable transfection efficacy for a multicationic polymer, polyethyleneimine (PEI), that can also buffer the intra-endosomal space. We discuss here the transfection mechanism and the potential uses of these vectors.

Optimal in vitro gene delivery with (poly)cationic amphiphiles requires an excess of cationic charges with respect to DNA phosphates. We have developed targeted transfection systems based on electrically neutral lipospermine/DNA particles, to which synthetic tri-antennary galactose ligands were conjugated to provide an interaction with cells, such as HepG2 cells, that express Gal/GalNAc receptors at their surface. Transfection, which was cell specific, increases ≪ 1000-fold with 25% neogalactolipid, i.e. approaching the value observed with optimized positively charged transfection complexes. Unexpectedly, neutral particles containing thiol-reactive phospholipids, were also efficient gene delivery systems, although non cell specific.

Non-natural oligonucleotides were synthesized by modified phosphoramidite procedure and tested in triple helix-mediated DNA recognition.

A 2-sperminoguanosine nucleotide has been synthesized and incorporated into oligonucleotides which showed increased duplex melting termperature.

Two new non-natural nucleosides bearing an amide (8) or an amidine (9) function have been synthesized. Their properties and the geometry of the exocyclic double bond have been studied.

This review describes new perspectives offered by the synthesis of non-natural nucleosides to overcome current limitations and extend the triplex-mediated DNA recognition scheme to any sequence. Alternate strand purine binding, direct pyrimidine recognition, and binding to the whole base-pair are described. The review highlights structural requirements to the design of modified nucleosides, as well as perturbing events such as tautomeric ambiguity and intercalation for the extended heterocyclic bases.

The complexation of primary ammonium salt substrates by macrocyclic polyether receptor molecules provides a general method for studying the nature and stereochemistry of intermolecular interactions. The substrates and receptors are fitted each with one of the interacting units and the resulting effects in the complex are analyzed. The method is used to study the biologically important indole-pyridinium donor-acceptor interaction. The complexes between macrocycles, bearing an indole group in side chains, and pyridinium-ammonium salts display a characteristic charge-transfer band. The absorption coefficients and stability constants have been determined. Competition experiments also provide a new method for measuring the stability constants of macrocycle-ammonium complexes in organic solvents.

R,3R,11R,12R)-l,4,7,10,13,16-Hexaoxa- cyclooctadecane-2,3,11,12-tetracarboxylic acid mono- hydrazinium monotetramethylammonium salt mono- + + 2-- hydrate, CaHI2N.HsN 2.C16HEEOI4.H20, Mr= 563.6, monoclinic, P2,, a = 12.075 (I), b = 14.436 (4), c = 8.018.(1)A, fl= 95.45 (1) °, U= 1391 ./k 3, Z= 2, D x = 1.35 g cm -3, 2 = 1.5418/~, g(Cu Kct) = 9.5 cm -1, F(000) = 604, T-- 292 K, R = 0.079 and wR -- 0.118 for 2155 observed data. The macrocyclic dianionic ligand is in a relaxed conformation, with a rectangular array of ether O atoms in the mean plane of the ring, the two other O atoms being out of the plane in the direction towards the complexed cation. The hydrazinium monocation is anchored to the ligand by three N-H...O hydrogen bonds. Introduction. The conjunction of a macrocyclic poly- ether core and anionic groups surrounding this molecular cavity makes the tetracarboxy-18-crown-6 ligand (Behr, Girodeau, Hayward, Lehn & Sauvage, 1980) a very powerful complexing agent for cations (Lehn, 1978; Behr, Lehn & Vierling, 1982). With the idea of taking advantage of this property to stabilize unusual cations (Behr, Dumas & Moras, 1982), we crystallized an adduct with hydrazine from water at pH -- 4. We hoped that, although partly protonated, the ligand would still be a strong enough complexing agent to stabilize the H3~-NH 3 dication which normally exists only in strong acid. Attempts to characterize this complex by indirect physical techniques (NMR, pH-- metric titration) were unsuccessful since the sequential protonation of a tetracarboxylate ligand/hydrazine mixture leads to ambiguous interpretations of whether protonation occurs on a carboxylate or an amino group. We therefore undertook the X-ray analysis of a complex crystallized in medium acidic conditions. Experimental. A single crystal with approximate dimen- sions 0.34 × 0.29 x 0.23 mm was sealed in a Linde- mann-glass capillary and optically aligned on a Nonius CAD-4 diffractometer. Preliminary study and data 0108-2701/87/112134-04501.50

The stability constants of the complexes formed by the polyfunctional macro-cyclic receptor molecules of type 1 with cationic substrates have been determined and analyzed in terms of structural factors. The binding strength is dominated by electrostatic interactions; the tetracarboxylate 1 (O−) forms by far the most stable complexes of all known macrocyclic polyethers. Lipophilic groups also significantly enhance stabilities, stressing the role of such residues in biological receptor sites. The complexation selectivity of organic ammonium cations is determined by two main factors: (a) central discrimination, resulting from the macrocyclic cavity, strongly favors binding of primary ammonium cations with respect to more highly substituted ones and also distinguishes among (R-NH3+)-substrates differently substituted on the C(α)-atom; (b) lateral discrimination arises from interactions between the substrate and the side chains borne by the macrocycle; again electrostatic and lipophilic group effects play the major role; diammonium cations are especially strongly bound. Complexation of biogenic amines follows the same trends; for instance noradrenaline and norephedrine are selectively bound with respect to adrenaline and ephedrine, pointing towards potential applications in selective binding and transport of biologically active substances.

A number of functionalized and chiral macrocyclic polyethers have been synthesized by condensation of the dithallium alcoholate of (R,R)-(+)-tartaric acid derivatives with α, ω-dihalides. In this way for instance, the tetracarboxylic [18]-O6 macrocycle 3c and its derivatives become readily available. They form complexes with various cationic substrates. NMR. and crystal-structure data provide information about the orientation of the side chains X in 3 with respect to the macrocycle. It is concluded that in the secondary amides like 3b and in their complexes the four X-groups are preferentially in an axial orientation on the average. This property is of much significance for the design of molecular receptors and catalysts based on this macrocyclic structure. The preparation of a number of other macrocycles is also described.

The crystal and molecular structure of the complex formed by the ionized bistartro-[18]-crown-6 receptor molecule 1 with the ethylenediammonium cation, is described. The macrocycle is roughly planar, the carboxy groups of each tartaric acid residue being in a diaxial relationship and extending above and below this plane. This conformation allows ‘lateral’ interactions with bound species to occur. The substrate is sandwiched between two macrocycles, with one of its NH heads anchored to the polyether core, and the other one in contact with the two carboxy groups of the neighboring molecule.