Turning Squalene into Cationic Lipid Allows a Delivery of siRNA in Cultured Cells.

Covalent binding of squalene to siRNA has already been shown to be an interesting way of delivering siRNA in vivo. Whether squalene derivatives could also be used to deliver siRNA in cells without covalent binding similar to usual transfection with cationic lipids is the question addressed in this article. Accordingly, we investigated the activity of two squalene derivatives bearing a quaternary ammonium head group and a guanidinium group, respectively.

Plasma hydrogenated cationic detonation nanodiamonds efficiently deliver to human cells in culture functional siRNA targeting the Ewing sarcoma junction oncogene.

The expression of a defective gene can lead to major cell dysfunctions among which cell proliferation and tumor formation. One promising therapeutic strategy consists in silencing the defective gene using small interfering RNA (siRNA). In previous publications we showed that diamond nanocrystals (ND) of primary size 35 nm, rendered cationic by polyethyleneimine-coating, can efficiently deliver siRNA into cell, which further block the expression of EWS/FLI-1 oncogene in a Ewing sarcoma disease model.

siRNA associated with immunonanoparticles directed against cd99 antigen improves gene expression inhibition in vivo in Ewing's sarcoma.

Ewing's sarcoma is a rare, mostly pediatric bone cancer that presents a chromosome abnormality called EWS/Fli-1, responsible for the development of the tumor. In vivo, tumor growth can be inhibited specifically by delivering small interfering RNA (siRNA) associated with nanoparticles. The aim of the work was to design targeted nanoparticles against the cell membrane glycoprotein cd99, which is overexpressed in Ewing's sarcoma cells to improve siRNA delivery to tumor cells.

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.

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.

Presence of divalent cation is not mandatory for the formation of intramolecular purine-motif triplex containing human c-jun protooncogene target.

Modulation of endogenous gene function, through sequence-specific recognition of double helical DNA via oligonucleotide-directed triplex formation, is a promising approach. Compared to the formation of pyrimidine motif triplexes, which require relatively low pH, purine motif appears to be the most gifted for their stability under physiological conditions. Our previous work has demonstrated formation of magnesium-ion dependent highly stable intermolecular triplexes using a purine third strand of varied lengths, at the purine•pyrimidine (Pu•Py) targets of SIV/HIV-2 (vpx) genes (Svinarchuk, F.

Effects of silencing RET/PTC1 junction oncogene in human papillary thyroid carcinoma cells.

Background: RET/PTC1 rearrangement is the most common genetic alteration identified to date in papillary thyroid carcinomas (PTC) and represents an interesting target for small interfering RNA (siRNA) strategies because it is present only in the tumor cells and not in the normal cells. Our aims were (i) to target the RET/PTC1 oncogene by siRNAs, (ii) to assess the knockdown effects on cell growth and cell cycle regulation, and (iii) to identify genes affected by the RET/PTC1 silencing.

Methods: Three efficient siRNAs previously designed in our laboratory in a model of murine PTC (RP-1 cells) were used to knockdown RET/PTC1 in the TPC-1 cells.

New core-shell nanoparticules for the intravenous delivery of siRNA to experimental thyroid papillary carcinoma.

Purpose: Development 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.

Methods: Nanoparticles were prepared by redox radical emulsion polymerization of isobutylcyanoacrylate and isohexylcyanoacrylate with chitosan.

Antitumor vectorized oligonucleotides in a model of ewing sarcoma: unexpected role of nanoparticles.

Oligonucleotides (ONs) such as antisense oligonucleotides (AS-ON) and siRNAs are used as experimental tools to study gene function and are currently being tested in clinical trials for use as therapeutic anticancer agents. However, their therapeutic use has been limited by their low physiological stability and their slow cellular uptake. The systemic delivery of sequence-specific AS-ON targeting the EWS/FLI1 gene product by a targeted, nonviral delivery system dramatically inhibits tumor growth in a murine model of Ewing's sarcoma.

Synthesis and studies on cell-penetrating peptides.

The ability of different synthetic cell penetrating peptides, as Antennapedia (wild and Phe(6) mutated penetratins), flock house virus, and integrin peptides to form complexes with a 25mer antisense oligonucleotide was compared and their conformation was determined by circular dichroism spectroscopy. The efficiency for oligonucleotide delivery into cells was measured using peptides labeled with a coumarin derivative showing blue fluorescence and the fluorescein-labeled antisense oligonucleotide showing green fluorescence. Fluorescence due to the excitation energy transfer confirmed the interaction of the antisense oligonucleotide and cell-penetrating peptides.

Polymer nanocarriers for the delivery of small fragments of nucleic acids: oligonucleotides and siRNA.

The success of the application of new therapeutic methods based on RNA interfering strategies requires the in vivo delivery of active ODN or siRNA down to the intracellular compartment of the target cells. This article aims to review the studies related to the formulation of RNA interfering agents in polymer nanocarriers. It will present the different types of polymer nanocarriers used as well as the biological activity of the resulting ODN and siRNA loaded nanocarriers.

Delivery of small interfering RNA. A review and an example of application to a junction oncogene.

RNA interference strategies using small interfering RNA is one of the most important discoveries in biology in recent years. This technology alongside antisense oligonucleotides is very promising and our group has focused its work on the targeting of junction oncogenes with these molecules. We have taken, as first example, papillary thyroid carcinoma.

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.

Intravenous delivery of anti-RhoA small interfering RNA loaded in nanoparticles of chitosan in mice: safety and efficacy in xenografted aggressive breast cancer.

Overexpression of RhoA in cancer indicates a poor prognosis, because of increased tumor cell proliferation and invasion and tumor angiogenesis. We showed previously that anti-RhoA small interfering RNA (siRNA) inhibited aggressive breast cancer more effectively than conventional blockers of Rho-mediated signaling pathways. This study reports the efficacy and lack of toxicity of intravenously administered encapsulated anti-RhoA siRNA in chitosan-coated polyisohexylcyanoacrylate (PIHCA) nanoparticles in xenografted aggressive breast cancers (MDA-MB-231).

N-terminal acylation of the SV40 nuclear localization signal peptide enhances its oligonucleotide binding and membrane translocation efficiencies.

Octanoyl and palmitoyl groups were coupled to the N-terminus of an analog of the SV40 nuclear localization signal peptide, SV126-133(Ser128), to study the effect of the fatty acid chain length on the complex formation with a single-stranded antisense oligodeoxynucleotide (ODN) and on the cellular uptake of the complex. The strongest binding affinity was observed for the palmitoylated peptide, indicating the better accessibility of the positively charged lysyl and arginyl side-chains to the phosphate groups due to the turn structures stabilized by the palmitoyl group. On increase of the peptide to ODN molar ratio (rM), gradual unstacking of the bases was observed, the maximal rate being reached at rM=10.

Innovative nanotechnologies for the delivery of oligonucleotides and siRNA.

One way to reach intracellular therapeutic targets in cells consists in the use of short nucleic acids which will bind specifically to on targets thanks to either Watson-Crick base pairing or protein nucleic acids recognition rules. Among these short nucleic acids an important class of therapeutic agents is antisense oligonucleotides and siRNAs. However, the major problem of nucleic acids is their poor stability in biological media.

Antisense oligonucleotide nanocapsules efficiently inhibit EWS-Fli1 expression in a Ewing's sarcoma model.

The cytogenetic abnormality of Ewing's sarcoma is related to the presence of a balanced t(11;22) translocation expressing the EWS-Fli1 chimeric fusion protein. Oligonucleotides (ODNs) are specific compounds that inhibit gene expression at the transcriptional level. They possess a poor bioavailability and are degraded by nucleases very rapidly.

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.

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.

Oncogene-targeted antisense oligonucleotides for the treatment of Ewing sarcoma.

The genetic hallmark of the Ewing sarcoma family of tumours (ESFT) is the presence of the t(11;22)(q24;q12) translocation, present in up to 85% of cases of ESFT, which creates the EWS/FLI1 fusion gene and results in the expression of a chimeric protein regulating many other genes. The inhibition of this protein by antisense strategies has shown its predominant role in the transformed phenotype of Ewing cells. In addition, the junction point at the mRNA level offers a target for short therapeutic nucleic acids that is present only in the cancer cells and not in the normal tissues of a patient.

Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDA-MB-231 breast cancer cells in vitro and in vivo.

Overexpression of RhoA or RhoC in breast cancer indicates a poor prognosis, due to increased tumor cell proliferation and invasion and tumor-dependent angiogenesis. Until now, the strategy of blockage of the Rho-signaling pathway has used either GGTI or HMG-CoA reductase inhibitors, but they are not specific to RhoA or RhoC inhibition. In this study, a new approach with anti-RhoA and anti-RhoC siRNAs was used to inhibit specifically RhoA or RhoC synthesis.

The solution synthesis of antisense oligonucleotide-peptide conjugates directly linked via phosphoramide bond by using a fragment coupling approach.

To improve antisense oligonucleotide penetration inside cells, conjugates of oligonucleotides and cell-penetrating peptides, covalently linked through a phosphoramide bond, were prepared by a fragment coupling approach in the liquid phase. Two methods were used for this synthesis, i.e.

Short double-stranded ribonucleic acid as inhibitor of gene expression by the interference mechanism.

The rapid development of the small interfering ribonucleic acid (siRNA)-induced inhibition of the gene expression at the RNA level offers to research groups a new strategy for the understanding of gene functions. The siRNA approach is close to antisense oligonucleotide technology and takes advantage of the progress of chemically synthesized oligoribonucleotides. This approach for the mammalian cells was described by Elbashir et al.

Therapeutic potentialities of EWS-Fli-1 mRNA-targeted vectorized antisense oligonucleotides.

We have used structured antisense oligonucleotides (AON), which are protected against extra and intracellular degradation by their internal structure. We have shown that if correctly designed this structure does not prevent them from hybridizing to the mRNA target. This concept allows reducing the number of thioate groups in the oligonucleotide and therefore the potential toxicity.

Interaction of fusogenic peptides with an antisense oligonucleotide in solution and in the presence of micelles: conformational studies.

The conformational effect of the interaction between various fusogenic peptides and an 18mer single stranded antisense oligonucleotide (ODN), targeted towards the green fluorescent protein mRNA, has been studied by circular dichroism spectroscopy in water and in the presence of anionic lysolipid micelles. The peptides used were the third helix of Antennapedia homeodomain pAntp-(43-58), the flock house virus FHV-gamma-(364-407) peptide, and its N-terminal gamma1-(364-384) and C-terminal gamma2-(390-407) fragments. The most significant conformational changes were observed in ODN-pAntp-(43-58) and ODN-FHV-gamma2 complexes.