Jean-Rémi Bertrand

Nanodiamond as a Vector for siRNA Delivery to Ewing Sarcoma Cells

The ability of diamond nanoparticles (nanodiamonds, NDs) to deliver small interfering RNA (siRNA) into Ewing sarcoma cells is investigated with a view to the possibility of in-vivo anticancer nucleic-acid drug delivery. siRNA is adsorbed onto NDs that are coated with cationic polymer. Cell uptake of NDs is demonstrated by taking advantage of the NDs intrinsic fluorescence from embedded color-center defects. Cell toxicity of these coated NDs is shown to be low. Consistent with the internalization efficacy, a specific inhibition of EWS/Fli-1 gene expression is shown at the mRNA and protein level by the ND-vectorized siRNA in a serum-containing medium.

Efficacy of siRNA nanocapsules targeted against the EWS-Fli1 oncogene in Ewing sarcoma.

The EWS–Fli1 fusion gene encodes for a chimeric oncogenic transcription factor considered to be the cause of the Ewing sarcoma. The efficiency of small interfering RNAs (siRNAs) targeted toward the EWS–Fli1 transcript (at the junction point type 1) was studied, free or encapsulated into recently developed polyisobutylcyanoacrylate aqueous core nanocapsules. Because this mRNA sequence is only present in cancer cells, it therefore constituted a relevant target. Studies of the intracellular penetration by confocal microscopy in NIH/3T3 EWS–Fli1 cells showed that nanocapsules improved the intracellular penetration of siRNA with mainly a cytoplasmic localization. These biodegradable siRNA-loaded nanocapsules were then tested in vivo on a mice xenografted EWS–Fli1-expressing tumor; they were found to trigger a dose-dependant inhibition of tumor growth after intratumoral injection. A specific inhibition of EWS–Fli1 was observed, too. These findings now open new prospects for the treatment of experimental cancers with junction oncogenes.

Optimisation of dendrimer-mediated gene transfer by anionic oligomers

The application of synthetic vectors for gene transfer has potential advantages over virus‐based systems. Their use, however, is limited since they generally lack the efficiency of gene transfer achieved with recombinant viral vectors such as adenovirus. Polyamidoamine (PAMAM) and phosphorus‐containing dendrimers (P‐dendrimers) are specific polymers with a defined spherical structure. They bind to DNA through electrostatic interactions thus forming complexes that efficiently transfect cells in vitro.

siRNA nanoformulation against the Ret/PTC1 junction oncogene is efficient in an in vivo model of papillary thyroid carcinoma

Delivery is a very important concern for therapeutic applications of siRNA. In this study, we have used chitosan-coated poly(isobutylcyanoacrylate) nanoparticles to deliver siRNA with a complementary sequence to the fusion oncogene ret/PTC1. By screening the mRNA junction we have selected a potent siRNA sequence able to inhibit this oncogene in a model of Papillary Thyroid Carcinoma cells. This siRNA sequence has then been validated by a shRNA approach using the same sequence. Furthermore, the high ret/PTC1 inhibition has triggered a phenotypic reversion of the transformed cells. We have designed well-defined chitosan decorated nanoparticles and succeeded to reduce their size. They have allowed to protect ret/PTC1 siRNA from in vivo degradation and leading to significant tumour growth inhibition after intratumoral administration.

New Core-Shell Nanoparticules for the Intravenous Delivery of siRNA to Experimental Thyroid Papillary Carcinoma

PurposeDevelopment of efficient in vivo delivery nanodevices remains a major challenge to achieve clinical application of siRNA. The present study refers to the conception of core-shell nanoparticles aiming to make possible intravenous administration of chemically unmodified siRNA oriented towards the junction oncogene of the papillary thyroid carcinoma.MethodsNanoparticles were prepared by redox radical emulsion polymerization of isobutylcyanoacrylate and isohexylcyanoacrylate with chitosan. The loading of the nanoparticles with siRNA was achieved by adsorption. The biological activity of the siRNA-loaded nanoparticles was assessed on mice bearing a papillary thyroid carcinoma after intratumoral and intravenous administration.ResultsChitosan-coated nanoparticles with a diameter of 60xa0nm were obtained by adding 3% pluronic in the preparation medium. siRNA were associated with the nanoparticles by surface adsorption. In vivo, the antisense siRNA associated with the nanoparticles lead to a strong antitumoral activity. The tumor growth was almost stopped after intravenous injection of the antisense siRNA-loaded nanoparticles, while in all control experiments, the tumor size was increased by at least 10 times.ConclusionThis work showed that poly(alkylcyanoacrylate) nanoparticles coated with chitosan are suitable carriers to achieve in vivo delivery of active siRNA to tumor including after systemic administration.

Influence of the Internalization Pathway on the Efficacy of siRNA Delivery by Cationic Fluorescent Nanodiamonds in the Ewing Sarcoma Cell Model

Small interfering RNAs (siRNAs) are powerful tools commonly used for the specific inhibition of gene expression. However, vectorization is required to facilitate cell penetration and to prevent siRNA degradation by nucleases. We have shown that diamond nanocrystals coated with cationic polymer can be used to carry siRNAs into Ewing sarcoma cells, in which they remain traceable over long periods, due to their intrinsic stable fluorescence. We tested two cationic polymers, polyallylamine and polyethylenimine. The release of siRNA, accompanied by Ewing sarcoma EWS-Fli1 oncogene silencing, was observed only with polyethylenimine. We investigated cell penetration and found that the underlying mechanisms accounted for these differences in behavior. Using drugs selectively inhibiting particular pathways and a combination of fluorescence and electronic microscopy, we showed that siRNA gene silencing occurred only if the siRNA:cationic nanodiamond complex followed the macropinocytosis route. These results have potential implications for the design of efficient drug-delivery vectors.

Comparative activity of α- and β-anomeric oligonucleotides on rabbit β globin synthesis: Inhibitory effect of cap targeted α-oligonucleotides

Abstract α-anomeric oligonucleotides are resistant to nucleases and display parallel annealing to RNA complementary sequences. We compared the effect of α- and β-oligonucleotides targeted against various mRNA regions on the rabbit β globin in vitro synthesis. In order to determine the role of RNase H, experiments were performed in both rabbit reticulocyte lysate and wheat germ extract. As expected β-oligonucleotides were found more efficient in wheat germ extract which is rich in RNase H activity and α-oligonucleotide targeted against the initiation codon or downstream had no effect because they do not induce mRNA cleavage by RNase H. However, we report, for the first time, a specific translation inhibition by α-oligonucleotides. This occurs provided they are targeted against the cap region in 5′ of the mRNA.

α-anomeric DNA: β-RNA hybrids as new synthetic inhibitors of Escherichia coli RNase H, Drosophila embryo RNase H and M-MLV reverse transcriptase

Abstract Nuclease-resistant α-anomeric DNA: β-rna hybrids are inhibitors of Escherichia coli RNase H, and Drosophila embryo RNase H. RNase H activities were measured by polyacrylamide gel electrophoresis, employing a short substrate, (A)12: d[G-G-(T)12-G-G], or by acid-solubility techniques, using a long substrate, poly(A): poly(dT). Strand exchanges which could be responsible for the observed inhibition have been ruled out by S1 nuclease experiments and by using inhibitors which do not allow strand exchange. Our results suggest that RNase H, for which DNA: RNA duplexes are the natural substrates, binds to non-physiological α-DNA: RNA hybrids and is consequently inhibited. These hybrids also inhibit the RNA-dependent DNA polymerase activity of M-MLV reverse transcriptase, therefore appearing as potential inhibitors of at least two reverse transcriptase activities. However, the inhibitory effect of these hybrids with respect to M-MLV reverse transcriptase is also observed with the single-stranded α-DNA itself. Unexpectedly, polymerase activity is highly stimulated by α-oligos, analogous in their sequence to the β primer used at a concentration unable to generate a detectable synthesis. These results suggest that the inhibition of reverse transcriptase activity with the α: β may occur at different levels.

In Vivo Potentialities of EWS-Fli-1 Targeted Antisense Oligonucleotides-Nanospheres Complexes

The EWS/FLI‐1 fusion gene, resulting from a t(11;22) translocation, plays a key role in the pathogenesis of Ewing sarcoma. Previously, we have shown that antisense oligonucleotides designed against EWS‐Fli‐1 inhibited tumor growth in nude mice provided they were delivered intratumorally by nanocapsules or by CTAB‐coated nanospheres. In this study, we have used two types of nanospheres (designated as type 1 and type 2 nanospheres) stabilized with chitosan for both intratumoral and systemic administration of oligonucleotides. Inhibition of the tumor growth in vivo was found to be dependent on the carrier type as well as on antisense oligonucleotide modification. Indeed, whereas both types of nanospheres were efficient in reducing tumor growth after intratumoral injection, we have obtained only with type 2 nanospheres an antitumoral effect after intravenous injection in a preliminary experiment. Additionally, the anticancer efficacy of a localized modification of the EWS‐Fli‐1 phosphodiester/phosphorothioate chimeric antisense oligonucleotide was demonstrated. In cell culture the oligonucleotides inhibit cell growth by their antisense activity. Further investigations are needed in vivo to learn the mechanism of action of the complexes.

Inhibition of moloney murine leukemia virus reverse transcriptase by α-anomeric oligonucleotides

After parallel hybridization to complementary template RNA, alpha-anomeric oligonucleotides are not primers for Moloney murine leukemia virus reverse transcriptase. As can be expected they are competitors of classical primer oligonucleotides (beta-anomeric). They therefore inhibit the RNA dependent DNA polymerase activity of Moloney murine leukemia virus reverse transcriptase with either homopolymeric or heteropolymeric substrates. Non complementary alpha-oligonucleotides display no inhibitory activity. alpha-Oligonucleotides are therefore potential candidates for inhibition of retroviral reverse transcriptases by interference with the primer binding sites.