Oligobenzylethylenimine enriches linear polyethylenimine with a pH-sensitive membrane-disruptive property and leads to enhanced gene delivery activity.

We report here the synthesis of a diblock linear polymer of oligo(benzylethylenimine)-b-polyethylenimine (OBzEI-PEI) and investigate its gene delivery properties. The linear copolymer OBzEI-PEI was prepared in a straightforward manner by acidic hydrolysis of a diblock polyoxazoline, which had been made by sequential polymerization of 4-benzyl-2-ethyl-2-oxazoline followed by 2-ethyl-2-oxazoline. pH titration and DNA complexation profiles of the new polymer are similar to regular linear PEIs, but with higher gene transfection efficiencies in various cell lines despite a decreased cellular uptake of plasmid DNA. Further experiments suggest that the OBzEI tail complements the intrinsic proton-sponge endosomolytic activities of PEI with an acid pH-sensitive membrane-perturbing activity.

Pyridylthiourea-grafted polyethylenimine offers an effective assistance to siRNA-mediated gene silencing in vitro and in vivo.

Success of synthetic interfering nucleic acids (siRNAs)-based therapy relies almost exclusively on effective, safe and preferably nanometric delivery systems which can be easily prepared, even at high concentrations. We prepared by chemical synthesis various self-assembling polymers to entrap siRNAs into stable polyplexes outside cells but with a disassembly potential upon sensing endosomal acidity. Our results revealed that pyridylthiourea-grafted polyethylenimine (πPΕΙ) followed the above-mentioned principles. It led to above 90% siRNA-mediated gene silencing in vitro on U87 cells at 10 nM siRNA concentration and did not have a hemolytic activity. Assembly of siRNA/πPΕΙ at high concentration was then studied and 4.5% glucose solution, pH 6.0, yielded stable colloidal solutions with sizes slightly below 100 nm for several hours. A single injection of these concentrated siRNA polyplexes into luciferase-expressing human glioblastoma tumors, which were subcutaneously xenografted into nude mice, led to a significant 30% siRNA-mediated luciferase gene silencing 4 days post-injection. Our results altogether substantiate the potential of self-assembling cationic polymers with a pH-sensitive disassembly switch for siRNA delivery in vitro and also in vivo experiments.

Age-associated modifications of Base Excision Repair activities in human skin fibroblast extracts.

Base Excision Repair (BER) is the predominant repair pathway responsible for removal of so-called small DNA lesions such as abasic sites (AP site), uracil (U), 8-oxo-7,8-dihydroguanine (8oxoG), thymine glycol (Tg). In this study, we investigated effect of aging on excision efficacy of several endogenous base lesions and AP sites using an in vitro multiplexed fluorescent approach on support (parallelized oligonucleotide cleavage assay). Human fibroblasts nuclear extracts from 29 donors of different ages were characterized in their ability to simultaneously excise the different lesions. Clearly, three different groups of lesions emerged according to the efficiency of their cleavage: one exhibited very high cleavage efficiency (AP sites and U paired with G), one showed intermediate cleavage efficiency (U paired with A and Tg). The third group included 8oxoG, A paired with 8oxoG, T at CpG site and hypoxanthine (Hx) and displayed poor repair. Aging was significantly associated with modification of excision efficiency for AP sites, uracil, Tg and 8oxoG. Repair rate decreased for the first three lesions and the most drastic effects were observed for repair of U:A. Surprisingly, excision of 8oxoG increased with aging suggesting a completely different regulation or adaptation for the initiation step of this related specific repair pathway.

Oligonucleotide-oligospermine conjugates (zip nucleic acids): a convenient means of finely tuning hybridization temperatures.

Synthesis of oligonucleotide probes and control of their hybridization temperature are key aspects of polymerase chain reaction (PCR)-based detection of genetic sequences. A straightforward means to approach the last goal is to decrease the repulsion between the polyanionic probe and target strands. To this end, we have developed a versatile automated synthesis of oligonucleotide-oligospermine derivatives that gave fast access to a large variety of compounds. Plots of their hybridization temperatures T(m) vs overall charge provided a measure of the impact of interstrand phosphate repulsion (and of spermine-mediated attraction) on the main driving force of duplex formation, i.e., base pairing. It showed that stabilization brought about by excess cationic charges can be of larger absolute magnitude than interstrand repulsion, even in high salt media. Base sequence and conjugation site (3' or 5') hardly influenced the effect of spermine on T(m). In typical PCR probe conditions, the T(m) increased linearly with the number of grafted spermines (e.g., 6.2 degrees C per spermine for a decanucleotide probe). The large data set of T(m) vs number of spermines and oligonucleotide length allowed us to empirically derive a simple mathematical relation that is accurately predicting the T(m) of any oligonucleotide-oligospermine derivative. Zip nucleic acids (ZNA) are thus providing an interesting alternative to locked nucleic acids (LNA) or minor groove binders (MGB) for raising the stability of 8-12-mer oligonucleotides up to ca. 70 degrees C, the level required for quantitative PCR experiments.

Cationic oligonucleotide-peptide conjugates with aggregating properties enter efficiently into cells while maintaining hybridization properties and enzymatic recognition.

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.

omega-Hydrazino linear polyethylenimine: a monoconjugation building block for nucleic acid delivery.

Nonviral gene therapy requires efficient vectors that are able to deliver nucleic acids inside the targeted cell nucleus. Developing new tools for the synthesis of supramolecular vectors with improved transfection efficiency and better biodistribution is therefore a crucial issue. Here we describe the synthesis of a 140-mer linear polyethylenimine (L-PEI) terminated at one end by a highly nucleophilic hydrazine residue. This cationic polymer, whose backbone is well known for its remarkable gene-delivery efficiency, constitutes a building block for omega-regioselective conjugation to molecules through the formation of stable linkages such as the hydrazone bonds. To demonstrate the potential of the omega-hydrazino linear polyethylenimine, human serum transferrin, a ligand that is well know to improve gene-delivery systems, was used as a model of sensitive material. The blood protein was oxidized to generate an aldehyde function and was subsequently conjugated to hydrazino PEI. The new polyethylenimine-transferrin (PEI-Tf) vector was purified and was shown to condense plasmid DNA into compact superstructures compatible with cellular uptake. Finally, the cellular-binding and gene-delivery properties of PEI/DNA polyplexes incorporating different quantities of transferrin were evaluated by FACS analysis and luciferase assay.