Two cationic lipids, bis-guanidiniumspermidine-cholesterol (BGSC) and bis-guanidinium-trencholesterol (BGTC)-cholesterol derivatives bearing two guanidinium groups-have been synthesized and tested as artificial vectors for gene transfer. They combine the membrane compatible features of the cholesterol subunit and the favorable structural and high pKa features of the guanidinium functions for binding DNA via its phosphate groups. Reagent BGTC is very efficient for transfection into a variety of mammalian cell lines when used as a micellar solution. In addition, both BGTC and BGSC present also a high transfection activity when formulated as liposomes with the neutral phospholipid dioleoylphosphatidyl ethanolamine. These results reveal the usefulness of cholesterol derivatives bearing guanidinium groups for gene transfer.

Synthetic gene delivery vectors are gaining increasing importance in gene therapy as an alternative to recombinant viruses. Among the various types of non-viral vectors, cationic lipids are especially attractive as they can be prepared with relative ease and extensively characterised. Further, each of their constituent parts can be modified, thereby facilitating the elucidation of structure-activity relationships. In this forward-looking review, cationic lipid-mediated gene delivery will mainly be discussed in terms of the structure of the three basic constituent parts of any cationic lipid: the polar headgroup, hydrophobic moiety and linker. Particular emphasis will be placed on recent advances in the field as well as on our own original contributions. In addition to reviewing critical physicochemical features (such as headgroup hydration) of monovalent lipids, the use of headgroups with known nucleic-acid binding modes, such as linear and branched polyamines, aminoglycosides and guanidinium functions, will be comprehensively assessed. A particularly exciting innovation in linker design is the incorporation of environment-sensitive groups, the intracellular hydrolysis of which may lead to more controlled DNA delivery. Examples of pH-, redox- and enzyme-sensitive functional groups integrated into the linker are highlighted and the benefits of such degradable vectors can be evaluated in terms of transfection efficiency and cationic lipid-associated cytotoxicity. Finally, possible correlations between the length and type of hydrophobic moiety and transfection efficiency will be discussed. In conclusion it may be foreseen that in order to be successful, the future of cationic lipid-based gene delivery will probably require the development of sophisticated virus-like systems, which can be viewed as "programmed supramolecular systems" incorporating the various functions required to perform in a chronological order the different steps involved in gene transfection.

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We used a fluorescence assay to identify molecules that stabilize G-quadruplexes. Intramolecular folding of an oligonucleotide with four repeats of the human telomeric sequence into a G-quadruplex structure led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5 and 3 ends of the oligonucleotide, respectively. The melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the fluorescence emission of the donor. A series of compounds (pentacyclic crescent-shaped dibenzophenanthroline derivatives) was shown to increase the melting temperature of the G-quadruplex by 2-20°C at 1 M dye concentration. This increase in Tm value was well correlated with an increase in the efficiency of telomerase inhibition in vitro. The best telomerase inhibitor showed an IC 50 value of 28 nM in a standard telomerase repeat amplification protocol assay. Fluorescence energy transfer can thus be used to reveal the formation of four-stranded DNA structures, and its stabilization by quadruplex-binding agents, in an effort to discover new potent telomerase inhibitors.telomere ͉ DNA structure ͉ G-quartet T elomerase was first identified in ciliates (1). This enzyme is an essential factor in immortalization and tumorigenesis (2-4). Furthermore, telomerase is active in most human tumor cells and inactive in most somatic cells and is therefore an attractive target for the design of anticancer agents. Most human telomeric DNA is double-stranded and contains (TTAGGG͞CCCTAA) n repeats except for the extreme terminal part where the 3Ј region of the G-rich strand is singlestranded (5). For human cells, these 3Ј overhangs are surprisingly long (averaging 130-210 bases in length), exist during most of the cell cycle, and are present on all chromosomal ends. The G-rich single-stranded DNA can adopt an unusual four-stranded DNA structure involving G-quartets (6-8) (see Fig. 1) or it might fold back and displace one strand of a telomeric duplex to form a so-called T-loop (9). Optimal telomerase activity requires the nonfolded single-stranded form of the primer and G-quartet formation has been shown to directly inhibit telomerase elongation in vitro (10). Therefore, a drug that stabilizes quadruplexes could interfere with telomerase and telomere replication (11-13).The peculiar geometry of the quadruplex structure should allow its specific recognition by small synthetic ligands, and previous experiments have shown that this assumption is correct (14). Many of the G4 ligands were shown to have antitelomerase activity in vitro, with IC 50 in the low micromo...

RNA interference requires efficient delivery of small doublestranded RNA molecules into the target cells and their subsequent incorporation into RNA-induced silencing complexes. Although current cationic lipids commonly used for DNA transfection have also been used for siRNA transfection, a clear need still exists for better siRNA delivery to improve the gene silencing efficiency. We synthesized a series of cationic lipids characterized by head groups bearing various aminoglycosides for specific interaction with RNA. siRNA complexation with such lipidic aminoglycoside derivatives exhibited three lipid/siRNA ratio-dependent domains of colloidal stability. Fluorescence and dynamic light-scattering experiments showed that cationic lipid/siRNA complexes were formed at lower charge ratios, exhibited a reduced zone of colloidal instability, and had smaller mean diameters compared with our previously described guanidinium-based cationic lipids. Cryo-transmission electron microscopy and x-ray-scattering experiments showed that, although the final in toto morphology of the lipid/siRNA complexes depended on the aminoglycoside type, there was a general supramolecular arrangement consisting of ordered lamellar domains with an even spacing of 67 Å. The most active cationic lipid/siRNA complexes for gene silencing were obtained with 4,5-disubstituted 2-deoxystreptamine aminoglycoside derivatives and were characterized by the siRNA being entrapped in small particles exhibiting lamellar microdomains corresponding to siRNA molecules sandwiched between the lipid bilayers. These results clearly show that lipidic aminoglycoside derivatives constitute a versatile class of siRNA nanocarriers allowing efficient gene silencing.gene silencing ͉ gene transfer vectors ͉ transfection R NAi has become widely used for knocking down the expression of a specific target gene by a posttranscriptional silencing mechanism and thereby it allows phenotypic analysis of gene function in cells (1, 2). Therapeutic approaches involving RNAi are also actively investigated (3, 4). To achieve gene silencing, sequence-specific double-stranded small interfering RNA (siRNA) molecules have to be delivered efficiently into the cytoplasm of cells (5, 6). Various methods have already been used for siRNA delivery, in particular, cationic lipids developed for plasmid DNA transfection (see ref. 7). These cationic lipids are composed of a hydrophobic moiety linked (by a spacer) to a cationic head group bearing either a quaternary ammonium [such as the lipids DOTMA {N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride} and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane)], a polycation [such as the lipid DOGS (dioctadecylamidoglycylspermine) (8) and lipopolyamine RPR120535 (9)], or guanidinium groups [such as the lipid BGTC, bis(guanidinium)-tris(2-aminoethyl)amine-cholesterol (10, 11)]. A colipid such as dioleoyl phosphatidylethanolamine (DOPE) is usually combined with the cationic lipids to form liposomes. The electrostatic interactions between the plasmid D...