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We report the evaluation of 18-mer 2′-O-methyl-modified ribose oligonucleotides with a full-length phosphorothioate backbone chemically conjugated at the 5′ end to the oligospermine units (Sn-: n = 5, 15, 20, 25, and 30 [number of spermine units]) as splice switching oligonucleotides (SSOs). These conjugates contain, in their structure, covalently linked oligocation moieties, making them capable of penetrating cells without transfection vector. In cell culture, we observed efficient cytoplasmic and nuclear delivery of fluorescein-labeled S20-SSO by fluorescent microscopy. The SSO conjugates containing more than 15 spermine units induced significant carrier-free exon skipping at nanomolar concentration in the absence and in the presence of serum. With an increasing number of spermine units, the conjugates became slightly toxic but more active. Advantages of these molecules were particularly demonstrated in three-dimensional (3D) cell culture (multicellular tumor spheroids [MCTSs]) that mimics living tissues. Whereas vector-complexed SSOs displayed a drastically reduced splice switching in MCTS compared with the assay in monolayer culture, an efficient exon skipping without significant toxicity was observed with oligospermine-grafted SSOs (S15- and S20-SSOs) transfected without vector. It was shown, by flow cytometry and confocal microscopy, that the fluorescein-labeled S20-SSO was freely diffusing and penetrating the innermost cells of MCTS, whereas the vector-complexed SSO penetrated only the cells of the spheroid’s outer layer. Keywords: cationic oligonucleotide-oligospermine conjugate, carrier-free oligonucleotide delivery, multicellular tumor spheroids, MCTS, splice switching oligonucleotides, SSO, exon skipping

Oligospermine-siRNA conjugates are able to induce efficient luciferase gene silencing upon carrier-free transfection. These conjugates are readily accessible by a versatile automated chemistry that we developed using a DMT-spermine phosphoramidite reagent. In this article, we used this chemistry to study a wide range of structural modifications of the oligospermine-siRNA conjugates, i.e., variation of conjugate positions and introduction of chemical modifications to increase nuclease resistance. At first we examined gene silencing activity of a series of siRNA-tris(spermine) conjugates with and without chemical modifications in standard carrier assisted conditions. The three spermine units attached at one of the two ends of the sense strand or at the 3'-end of the antisense strand are compatible with gene silencing activity whereas attachment of spermine units at the 5'-end of the antisense strand abolished the activity. 2'-O-Methylated nucleotides introduced in the sense strand are compatible while not in the antisense strand. Thiophosphate links could be used without activity loss at the 3'-end of both strands and at the 5'-end of the sense strand to conjugate oligospermine. Consequently a series of oligospermine-siRNA conjugates containing 15 to 45 spermines units in various configurations were chosen, prepared, and examined in carrier-free conditions. Attachment of 30 spermine units singly at the 5'-end of the sense strand provides the most potent carrier-free siRNA. Longevity of luciferase gene silencing was studied using oligospermine-siRNA conjugates. Five day long efficiency with more than 80% gene expression knockdown was observed upon transfection without vector. Oligospermine-siRNA conjugates targeting cell-constitutive natural lamin A/C gene were prepared. Efficient gene silencing was observed upon carrier-free transfection of siRNA conjugates containing 20 or 30 spermine residues grafted at the 5'-end of the sense strand.

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.

siRNA delivery to cells offers a convenient and powerful means of gene silencing that bypasses several barriers met by gene delivery. However, nonviral vectors, and especially polymers, form looser complexes with siRNA than with plasmid DNA. As a consequence, exchange of siRNA for larger polymeric anions such as proteoglycans found outside cells and at their surface may occur and lower delivery. We show here that making siRNAs “gene-like,” via short complementary A 5–8 /T 5–8 3′ overhangs, increases complex stability, and hence RNase protection and gene silencing in vitro up to 10-fold. After decomplexation in the cytoplasm, sticky siRNA (ssiRNA) concatemers fall apart. ssiRNAs are therefore not inducing antiviral responses, as shown by the absence of IFN-β production. Finally, transfection experiments in the mouse lung show that ssiRNA should be particularly suited to silencing with linear polyethylenimine in vivo .

Oligonucleotide delivery is a crucial issue for therapeutical purposes and is often addressed by conjugation to short cationic peptides although with controversial results. To further examine this mechanism, a 15-mer anionic oligonucleotide was conjugated to a cationic peptide in order to obtain a diblock compound with an overall positive charge with aggregation properties. These microaggregates were efficiently internalized in cells via the expeditious pathway used by commercial gene delivery systems. Moreover, stability of the duplex formed with the complementary sequence increased without inhibiting oligonucleotide enzyme recognition as shown by the properties of the conjugate to prime chain elongation by Taq DNA polymerase in a linear amplification/sequencing process.

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 (

Suicide gene therapy could be an attractive addition to the treatment of ovarian carcinomas, for which acquired chemoresistance frequently results in treatment failure. Here we show that transfection of the HSV-tk gene, followed by incubation with up to 1 mM ganciclovir fails to induce cell death in SKOV3 chemoresistant human ovarian carcinoma cells. However, co-transfection of HSV-tk with Cip1/Waf1 encoding the p21(cip1/waf1) inhibitor of cdks, allows 100 microM ganciclovir to eradicate the population of tumor cells. Potentiation of a drug by co-transfer of HSV-tk with Cip1/Waf1could thus represent another therapeutic approach for tumours that are resistant to conventional therapy.

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.

International audience; With the aim of developing dendrimer nanovectors with a precisely controlled architecture and flexible structure for DNA transfection, we designed PAMAM dendrimers bearing a triethanolamine (TEA) core, with branching units pointing away from the center to create void spaces, reduce steric congestion, and increase water accessibility for the benefit of DNA delivery. These dendrimers are shown to form stable nanoparticles with DNA, promote cell uptake mainly via macropinocytosis, and act as effective nanovectors for DNA transfection in vitro on epithelial and fibroblast cells and, most importantly, in vivo in the mouse thymus, an exceedingly challenging organ for immune gene therapy. Collectively, these results validate our rational design approach of structurally flexible dendrimers with a chemically defined structure as effective nanovectors for gene delivery, and demonstrate the potential of these dendrimers in intrathymus gene delivery for future applications in immune gene therapy.

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.

The size of condensed DNA particles is a key determinant for in vivo diffusion and gene delivery to cells. Gene molecules can be individually compacted by cationic thiol detergents into nanometric particles that are stabilized by oxidative conversion of the detergent into a gemini lipid. To reach the other goal, gene delivery, a series of cationic thiol detergents with various chain lengths (C(12)-C(16)) and headgroups (ornithine or spermine) was prepared, using a versatile polymer-supported synthetic strategy. Critical micelle concentrations and thiol oxidation rates of the detergents were measured. The formation and stability of complexes formed with plasmid DNA, as well as the size, xi-potential, morphology, and transfection efficiency of the particles were investigated. Using the tetradecane/ornithine detergent, a solution of 5.5 Kpb plasmid DNA molecules was converted into a homogeneous population of 35 nm particles. The same detergent, once oxidized, exhibited a typical lipid phase internal structure and was capable of effective cell transfection. The particle size did not increase with time. Surprisingly, the gel electrophoretic mobility of the DNA complexes was found to be higher than that of plasmid DNA itself. Favorable in vivo diffusion and intracellular trafficking properties may thus be expected for these complexes.

A novel technology combining replication- and integration-defective human immunodeficiency virus type 1 (HIV-1) vectors with genetically modified dendritic cells was developed in order to induce T-cell immunity. We introduced the vector into dendritic cells as a plasmid DNA using polyethylenimine as the gene delivery system, thereby circumventing the problem of obtaining viral vector expression in the absence of integration. Genetically modified dendritic cells (GMDC) presented viral epitopes efficiently, secreted interleukin 12, and primed both CD4+and CD8+HIV-specific T cells capable of producing gamma interferon and exerting potent HIV-1-specific cytotoxicity in vitro. In nonhuman primates, subcutaneously injected GMDC migrated into the draining lymph node at an unprecedentedly high rate and expressed the plasmid DNA. The animals presented a vigorous HIV-specific effector cytotoxic-T-lymphocyte (CTL) response as early as 3 weeks after a single immunization, which later developed into a memory CTL response. Interestingly, antibodies did not accompany these CTL responses, indicating that GMDC can induce a pure Th1 type of immune response. Successful induction of a broad and long-lasting HIV-specific cellular immunity is expected to control virus replication in infected individuals.

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.

Several limitations to nonviral gene delivery have been overcome. Small nanometric particles have been obtained by condensation ofDNA with a polymerizable cation followed by DNA template-directed homopolymerization of the vector. Targeting of specific cell types has been achieved by using polyethylenimine (PEI), a cationic polymer, coupled to cell ligands such as galactose or small RGD peptide that allows cell entry by a receptor-mediated endocytosis pathway. Escape of the DNA complexes from the endosomes is favored by the ‘proton sponge’ effect as a consequence of the high buffering capacity of PEI. The last barrier to gene delivery, i.e. the nuclear membrane, can be crossed at the level of the nuclear pore complexes, by using nuclear localization signal DNA molecule conjugates.

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.

Before being considered for a cystic fibrosis (CF) gene therapy trial, any gene delivery agent must be able to show that it produces low levels of toxicity as well as being able to protect the DNA from nuclease degradation. Here we show that complexes of linear polyethylenimine (L-PEI) and DNA can repeatedly be administered to animals (up to 21 consecutive days) without eliciting an immune response against PEI/DNA particles or inducing toxic side effects due to accumulation of PEI in the lungs. However, the host response to the exogenous protein resulted in some decrease of expression. PEI-mediated transfection was unaffected by treatment of the complexes with DNase (frequently used to reduce the viscosity of lung secretions in CF patients). Taken together, these properties make L-PEI a valuable vector for gene therapy of CF.

A series of pyrimidine nucleosides modified at the C-4 position by different donor/acceptor groups has been sythesized. Since binding properties rely on the conformation of the exocyclic moiety, their structure has been determined by 2D NMR experiments and X-ray crystallography.

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.

Recognition of DNA sequences by triple helix-forming oligonucleotides is restricted to homopurine sequences. Based on geometrical considerations, a single nucleoside, 4-guanidinocytidine 1, was designed to recognize both guanosine (as the natural 1β anomer) and cytidine (as the 1α anomer) in a new triple helix context where the third strand is centered in the major groove of the target duplex. Several methods of synthesis of this nucleoside are described.

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.

An improved synthesis of the deoxy-2-fluoroinosine nucleoside is described, that makes use of mild fluorination (polyvinylpyridinium polyhydrogenfluoride) and O-silyl deprotection (triethylamine trihydrofluoride) reactions. The derived 5′-dimethoxytrityl-2-fluoroinosine-3′-phosphoramidite was incorporated into 10-, 15- and 20- mer oligonucleotides containing up to 7 non-natural bases. © 1997 Published by Elsevier Science Ltd.

To explore whether endosomal release presents a major barrier to lipospermine-mediated gene delivery, acidic membrane-active peptides derived from influenza virus or artificial sequences were incorporated into DNA/dioctadecylamidoglycylspermine (= Transfectam) complexes. Depending on the cell line used, gene expression levels are approximately 3-30-fold higher than those obtained by applying DNA complexed to optimal amounts of Transfectam alone. In addition, gene transfer efficiency of DNA complexes with lower amounts of Transfectam (1.5-2 charge equiv) is increased by a factor of up to 1000 by peptides INF6 (influenza virus derived sequence) and INF10 (artificial sequence). The helper lipids 1,2-dioleoylphosphatidylethanolamine, egg phosphatidylethanolamine, and 1,2-dioleoyl-racglycerol also can enhance the gene transfer. Thus, endosomal escape seems to be only a moderate barrier for optimized, positively charged DNA/Transfectam complexes, but a substantial bottleneck for less positively charged complexes.

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.

Background Melanoma represents one of the most aggressive and therapeutically challenging malignancies as it often gives rise to metastases and develops resistance to classical chemotherapeutic agents. Although diverse therapies have been generated, no major improvement of the patient prognosis has been noticed. One promising alternative to the conventional therapeutic approaches currently available is the inactivation of proteins essential for survival and/or progression of melanomas by means of RNA interference. Survivin and cyclin B1, both involved in cell survival and proliferation and frequently deregulated in human cancers, are good candidate target genes for siRNA mediated therapeutics. Methods We used our newly developed sticky siRNA-based technology delivered with linear polyethyleneimine (PEI) to inhibit the expression of survivin and cyclin B1 both in vitro and in vivo, and addressed the effect of this inhibition on B16-F10 murine melanoma tumor development. Results We confirm that survivin and cyclin B1 downregulation through a RNA interference mechanism induces a blockage of the cell cycle as well as impaired proliferation of B16-F10 cells in vitro. Most importantly, PEI-mediated systemic delivery of sticky siRNAs against survivin and cyclin B1 efficiently blocks growth of established subcutaneaous B16-F10 tumors as well as formation and dissemination of melanoma lung metastases. In addition, we highlight that inhibition of survivin expression increases the effect of doxorubicin on lung B16-F10 metastasis growth inhibition. Conclusion PEI-mediated delivery of sticky siRNAs targeting genes involved in tumor progression such as survivin and cyclin B1, either alone or in combination with chemotherapeutic drugs, represents a promising strategy for melanoma treatment.

RNA interference allows the design of new inhibitors that target deregulated pathways in cancer. However systemic delivery of siRNA for the treatment of solid tumors still remains an issue. In our study, in order to suppress the progression of lung cancer metastasis in mice, we developed sticky siRNA (ssiRNA) to inhibit survivin and cyclin B1, two candidates involved in cell survival and proliferation. We exploited the linear polyethylenimine (PEI) as potent non-viral carrier to efficiently deliver our inhibitors. As a proof of concept, we have chosen a very aggressive mammary adenocarcinoma model (TSA-Luc cells), which forms lung metastases upon systemic cell injection. We confirmed in vitro , that the ssiRNAs delivered with PEI are not only able to inhibit our target genes at the mRNA and protein levels, but are also able to block the cell cycle and cell proliferation through a mechanism of RNA interference. More importantly, we showed in vivo by luciferase dosage, bioimaging and tissue section, an inhibition of lung tumor metastases after systemic delivery of cyclin B1 and survivin ssiRNA complexed with PEI. Alternating treatment with cisplatin and ssiRNA/PEI showed an additive effect between the two anticancer drugs on lung tumor inhibition leading to a significant increase in animal survival. Moreover a promising window between activity (IC 50 ) and toxicity (LD 50 ), essential for therapeutic application, was observed. Our data show that systemic delivery of ssiRNA/PEI complexes targeting the cell cycle is a valuable strategy for the treatment of lung tumor metastasis and that it can be combined with chemotherapy.

Triple helix-forming oligonucleotides could bind directly to natural B-DNA provided the sequence-recognition groups were directly linked to the ribose backbone. For adenine recognition, the H bonding network of a deoxyrihosylamide is required. To this end, deoxyribosylurea and deoxyribosylformamide were obtained by oxydative degradation of thymine. The former compound was shown to be an untractable slow exchange mixture of anomers. β-Deoxyribosylformamide (F), a 7/3 cis:truns mixture of isomers, was tritylated. phosphitylated and an oligonucleotide T(F) 8 T was synthesized and characterized.

Thyroid hormone 3,5,3'-triiodo-L-thyronine (T3) is required for normal brain development in vertebrates. T3 acts through two classes of nuclear receptors (TR alpha and TR beta) that have distinct developmental spatial and temporal distributions suggesting different functions during neuronal development. One possibility is that TR alpha, which is expressed early in embryogenesis, is involved in neuroblast proliferation. To test this hypothesis we used the embryonic chick optic lobe, as we found that T3 stimulates [3H]thymidine incorporation in this tissue both in vivo and in vitro during embryonic days 6-9. We applied oligonucleotides (ODNs) against TR alpha and TR beta to primary cultures of chick optic lobes. By employing a cationic lipid vector we could use very low ODN concentrations (< 150 nM). Antisense ODNs against TR alpha significantly inhibited [3H]thymidine incorporation, whereas antisense TR beta had no significant effect. However, both ODNs inhibited expression of TRs, as they blocked transcription from a T3-activated reporter gene. Random ODNs used as controls had no significant effect on [3H]thymidine incorporation or on T3-dependent transcription. These observations suggest that TR alpha is implicated in neuroblast proliferation and add credence to the hypothesis that the multiplicity of nuclear receptors allows for specific actions of T3 during development.

International audience; The presence of thiol-reactive phospholipid derivatives, such as N-4-(p-maleimidophenyl)butyryl) dipalmitoylphosphatidylethanolamine (MPB-DPPE), in electrically neutral lipospermine/DNA particles results in more than a 100-fold increased transfection efficiency of human hepatoma HepG2 cells and murine 3T3 fibroblasts. These effects could be ascribed to the presence of thiol-reactive functions, such as maleimide, bromoacetamide and dithiopyridyl linkage, on the transfecting particles. We propose that such particles react with thiol groups present at the surface of the cells, leading to their covalent anchoring a process that is probably followed by an endocytosis of the complex.

A modified 11-mer oligodeoxyribonucleotide 1 * of sequence ATTG * AATG * TTT, containing two spermine residues linked to the C2 position of inosine (G * ; 2-aminoinosine is guanosine) was synthesized. Its duplex 2·1 * structure with the complementary strand 2 melted 25-15°C higher than the natural 2·1 DNA, depending on the ionic composition of the medium. Furthermore, polyacrylamide gel electrophoresis showed 1 * to be able to replace 1 from 2·1 in a physiologically relevant medium and some 30°C below the melting temperature of 2·1 .

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.

An amphiphilic dendrimer bearing a hydrophobic alkyl chain and hydrophilic poly(amidoamine) dendrons is able to combine the advantageous features of lipid and dendrimer vectors to deliver a heat shock protein 27 siRNA and produce potent gene silencing and anticancer activity in vitro and in vivo in a prostate cancer model. This dendrimer can be used alternatively for treating various diseases.

DNA ligation is the weak link in the chain of gene cloning. We have developed a straightforward nonenzymatic alternative to this reaction that employs easily available commercial reagents. The method uses the affinity of distamycin for the minor groove to join DNA ends together. Phosphodiester bonds are formed after cyanoimidazole-promoted phosphate activation in the presence of manganese(II) cations. When transfected into eukaryotic cells, the chemically ligated plasmid is transcribed even more efficiently than after enzymatic ligation. At present this technique compares favorably with T4 ligation for AT-rich cohesive termini, but in principle it could be extended to any restriction site.

Gene transfer into mammalian cells is a prerequisite to gene therapy. Designed synthetic DNA carriers could be attractive alternatives to presently used viral vectors. Toward this end, lipopolyamines have been developed, which spontaneously condense DNA and coat it with a cationic lipid layer. The resulting nucleolipidic particles transfect efficiently various eukaryotic cells.

Gene transfer into mammalian cells is a prerequisite to gene therapy. Designed synthetic DNA carriers could be attractive alternatives to presently used viral vectors. Toward this end, lipopolyamines have been developed, which spontaneously condense DNA and coat it with a cationic lipid layer. The resulting nucleolipidic particles transfect efficiently various eukaryotic cells.

Gene transfer into mammalian cells is a prerequisite to gene therapy. Designed synthetic DNA carriers could be attractive alternatives to presently used viral vectors. Toward this end, lipopolyamines have been developed, which spontaneously condense DNA and coat it with a cationic lipid layer. The resulting nucleolipidic particles transfect efficiently various eukaryotic cells.

An improved synthesis of the deoxy-2-fluoroinosine nucleoside is described, that makes use of mild fluorination (polyvinylpyridinium polyhydrogenfluoride) and O-silyl deprotection (triethylamine trihydrofluoride) reactions. The derived 5′-dimethoxytrityl-2-fluoroinosine-3′-phosphoramidite was incorporated into 10-, 15- and 20- mer oligonucleotides containing up to 7 non-natural bases. © 1997 Published by Elsevier Science Ltd.

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.

Recognition of DNA sequences by triple helix-forming oligonucleotides is restricted to homopurine sequences. Based on geometrical considerations, a single nucleoside, 4-guanidinocytidine 1, was designed to recognize both guanosine (as the natural 1β anomer) and cytidine (as the 1α anomer) in a new triple helix context where the third strand is centered in the major groove of the target duplex. Several methods of synthesis of this nucleoside are described.

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.

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

Prostate cancer is at present the most common malignancy in men in the Western world. When localized to the prostate, this disease can be treated by curative therapy such as surgery and radiotherapy. However, a substantial number of patients experience a recurrence, resulting in spreading of tumor cells to other parts of the body. In this advanced stage of the disease only palliative treatment is available. Therefore, there is a clear clinical need for new treatment modalities that can, on the one hand, enhance the cure rate of primary therapy for localized prostate cancer and, on the other hand, improve the treatment of metastasized disease. Gene therapy is now being explored in the clinic as a treatment option for the various stages of prostate cancer. Current clinical experiences are based predominantly on trials with adenoviral vectors. As the first of a trilogy of reviews on the state of the art and future prospects of gene therapy in prostate cancer, this review focuses on the clinical experiences and progress of adenovirus-mediated gene therapy for this disease.