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. Biotinylated poly(isobutylcyanoacrylate) nanoparticles were conceived as a platform to design targeted nanoparticles with biotinylated ligands and using the biotin-streptavidin coupling method. The targeted nanoparticles were validated in vivo for the targeted delivery of siRNA after systemic administration to mice bearing a tumor model of the Ewing's sarcoma. The expression of the gene responsible of Ewing's sarcoma was inhibited at 78% ± 6% by associating the siRNA with the cd99-targeted nanoparticles compared with an inhibition of only 41% ± 9% achieved with the nontargeted nanoparticles.

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. As will be explained, the part of the in vitro studies provided useful data about the intracellular delivery of the formulated RNA interfering agents. Investigations performed in vivo have considered animal models of different relevant diseases. Results from these investigations have clearly demonstrated the interest of several polymer nanocarriers tested so far to deliver active RNA interfering effectors in vivo making possible their administration by the intravenous route.

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). The siRNA was administered every 3 days at a dose of 150 or 1500 microg/kg body weight in nude mice. This treatment inhibited the growth of tumors by 90% in the 150-microg group and by even more in the 1500-microg group. Necrotic areas were observed in tumors from animals treated with anti-RhoA siRNA at 1500 microg/kg, resulting from angiogenesis inhibition. In addition, this therapy was found to be devoid of toxic effects, as evidenced by similarities between control and treated animals for the following parameters: body weight gain; biochemical markers of hepatic, renal, and pancreatic function; and macroscopic appearance of organs after 30 days of treatment. Because of its efficacy and the absence of toxicity, it is suggested that this strategy of anti-RhoA siRNA holds significant promise for the treatment of aggressive cancers.

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. One method, among others, to solve the stability problem is the use of colloïdal carriers such as nanoparticles. Nanoparticles have already been applied with success to in vitro drug delivery to particular types of cells and in vivo in several experimental models. Many membrane and intracellular processes deal with nanosized structure (typically 100 nm) which are processed further through the recognition sites of receptors and enzymes. Thus non-viral nanoparticles are interesting candidates to present biochemical molecules such as nucleic acids and proteins to cells as well as to protect them in vivo during delivery. This review focuses on the recent developments in the design of nanotechnologies to improve the delivery of antisense oligonucleotides and siRNAs.

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. Two transfections of either RhoA or RhoC siRNA (8.5 nM) into MDA-MB-231 human breast cancer cells or HMEC-1 endothelial cells induced extensive degradation of the target mRNA and led to a dramatic decrease in synthesis of the corresponding protein. In vitro, these siRNAs inhibited cell proliferation and invasion more effectively than conventional blockers of Rho cell signaling. Finally, in a nude mouse model, intratumoral injections of anti-RhoA siRNA (100 microl at 85 nM) every 3 days for 20 days almost totally inhibited the growth and angiogenesis of xenografted MDA-MB-231 tumors. One may infer from these observations that specific inhibition of the Rho-signaling pathway with siRNAs represents a promising approach for the treatment of aggressive breast cancers.

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. The pAntp-(43-58) forms a complex with ODN through electrostatic interaction resulting in profound changes in the conformation of both the peptide and the ODN. In the case of FHV-gamma2 peptide the complex formation takes place without altering the structure of ODN, and the decreased ratio of deltaepsilon208/deltaepsilon222 reflects the insertion of the complexed peptide into the micelle.

Covalent coupling of a PIM-1 oncogene targeted PNA with an antennapedia derived peptide.

Peptide nucleic acids (PNA) are promising antisense molecule for blocking gene expression in cell culture or in vivo. Nevertheless because they are poor efficient to pass the cellular membrane, it is necessary to use a vectorisation agent to observe an inhibitory effect. We describe the coupling of the rhodamine labeled 17-mer antisense PNA to a fusogenic peptide from antenapedia via S-S linkage, the studies of the penetration of this complex into fibroblast cells and its inhibitory effect on pim1 targeted protononcogene.

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. Junction oncogenes are found in cancers such as certain leukemias, Ewing sarcoma, and thyroid papillary carcinomas. Ewing sarcoma is a cancer of children and young adults with bone metastasis. It is caused by a chromosomic translocation t(11;22) (q24;q12) creating a fusion gene between the genes EWS and Fli-1 giving rise to a chimeric protein which is an unnatural transcription factor. Immortalized NIH/3T3 cells transfected by the EWS-Fli-1 cDNA under the control of the LTR retroviral promoter--which do not undergo apoptosis and which became tumoral--were used for this study. As a model of Ewing sarcoma in nude mice, we have used permanently expressing human EWS-Fli-1 cells grafted to nude mice. The nanospheres or nanocapsules have been used to deliver two different AON: a phosphorothioate, and a structured chimeric AON, both targeted toward the junction area of EWS-Fli-1. Both types of AON-loaded nanoparticles inhibited the growth of the xenografted tumor after intratumoral injections into nude mice, whereas similar nanoparticles with control oligonucleotides had no effect. With AON in nanospheres, we have shown after 24 hours that the mRNA of EWS-Fli-1 was specifically down-regulated, confirming the antisense activity of the targeted AON.

A two step model aimed at delivering antisense oligonucleotides in targeted cells.

To be efficient in vivo antisense oligonucleotides must reach the targeted cells and then cross the cellular membrane. We propose a two step system where the oligonucleotide is first electrostatically bound to a peptide coupled to a ligand of a cellular receptor. A complex is formed which allows the oligonucleotide to be bound to the membrane of the targeted cells. These oligonucleotides are then delivered inside the cells by the subsequent use of a transfection agent. As a reductionist model of peptide coupled to a ligand we have used a lipopeptide and characterized by a filter elution assay the stoichiometry between the peptide and the oligonucleotide in the complexes. Using HeLa cultured cells we have shown that addition of these complexes to the cells triggers the oligonucleotide binding to the cell membrane. The subsequent addition of dendrimers allows these antisense oligonucleotides to inhibit a reporter gene inside the cells.

EWS fli-1 antisense nanocapsules inhibits ewing sarcoma-related tumor in mice.

EWS Fli-1, a fusion gene resulting from a t(11;22) translocation is found in 90% of both Ewing's sarcoma and primitive neuroectodermal tumor (PNET). In the present study, we show that recently developed polyisobutylcyanoacrylate nanocapsules with an aqueous core were able to encapsulate efficiently high amounts of phosphorothioate oligonucleotides (ODN) directed against EWS Fli-1 chimeric RNA. Release of these ODN in serum medium was shown to be biphasic which was explained by the presence of two types of nanocapsules able to release ODN with different kinetics. In addition, nanocapsules were found to provide protection of these oligonucleotides from the degradation in serum. These ODN nanocapsules permitted to obtain inhibition of Ewing sarcoma-related tumor in mice after intratumoral injection of a cumulative dose as low as 14.4 nanomoles. This new type of non viral vector shows great potential for in vivo administration of oligonucleotides.

Modified alkaline elution allows the measurement of intact apurinic sites in mammalian genomic DNA.

The presence of apurinic/apyrimidinic (AP) sites in cell genomes is known to be toxic and mutagenic. These lesions are therefore repaired in cells by efficient enzymatic systems. However, a report (Nakamura and Swenberg, Cancer Res. 59 (1999) 2522-2526) indicates an unexpected high rate of endogenous apurinic/apyrimidinic (AP) sites in genomic DNA in mammalian tissues. The technology used does not allow the authors to distinguish between intact AP sites and 3'cleaved AP sites. The corresponding values range between 2 and 4 sites per million of nucleotides in various human and rat tissues. Using a modified alkaline elution method we show here that the stationary level of intact AP sites is about 0.16 per million of nucleotides in leukemic mouse L1210 cells.

Targeting of single-stranded DNA and RNA containing adjacent pyrimidine and purine tracts by triple helix formation with circular and clamp oligonucleotides.

The aim of this work was to construct an anti-messenger targeted to the pim-1 oncogene transcript, based on circular or clamp oligodeoxyribonucleotides. The formation of bimolecular triplexes by clamp or circular oligonucleotides was investigated using single-stranded targets of both DNA (5'-CCCTCCTTTGAAGAA-3') and RNA type (5'-CCCUCCUUUGAAGAA-3'). The third, 'Hoogsteen' strand of the triplex was represented by G,T-rich sequences. The secondary structures of the complexes were determined by thermal denaturation, circular dichroism and gel mobility shift experiments and shown to depend on the nature of the target strand. With DNA as target, the sequence of a clamp (or circular) oligonucleotide that formed the triple helix was 3'-GGGAGGAAACTTCTTTT-TTGTTGTTT-TT-GGTGGG-5', where the first TT dinucleotide (in italics) is a linker and the second TT (bold) represents the bridge through which the 'Hoogsteen' strand switches from one strand of the Watson-Crick duplex to the other, once the duplex is formed by the corresponding portion of the anti-messenger (underlined). The portion of the 'Hoogsteen' sequence of the triplex between the two TT dinucleotides binds to the 3' extremity of the target strand and runs parallel to it. The portion situated at the 5' end of the oligonucleotide switches to the purine tract of the complementary strand of the duplex and is antiparallel to it. In contrast, with RNA as target, for a branched clamp oligonucleotide that formed a triple helix over its entire length (5'-TTCTTCAAAGGAGGG-3' 3'-GGGTGGTTT-T-GTTGTT-5') the portion of the 'Hoogsteen' sequence that bound to the 3' extremity of the target strand had to be antiparallel to it.

Physico-chemical and biological properties of antisense phosphodiester oligonucleotides with various secondary structures.

The influence of the secondary structure of oligonucleotides having a natural phosphodiester backbone on their ability to interact with DNA and RNA targets and on their resistance to the nucleolytic digestion is investigated. Oligonucleotides having hairpin, looped and snail-like structure are found to be much more stable to nuclease degradation in different biological media and inside cells than the linear ones. The structured oligonucleotides can also hybridise with their DNA and RNA targets.

Uptake and intracellular distribution of oligonucleotides vectorized by a PAMAM dendrimer.

We studied the uptake and intracellular distribution of an FITC labelled phosphodiester oligodeoxynucleotide (ODN) vectorized by a dendrimeric structure in cell culture.

Cell cycle-dependent distribution and specific inhibitory effect of vectorized antisense oligonucleotides in cell culture.

Factors limiting the use of antisense phosphodiester oligodeoxynucleotides (ODNs) as therapeutic agents are inefficient cellular uptake and intracellular transport to RNA target. To overcome these obstacles, ODN carriers have been developed, but the intracellular fate of ODNs is controversial and strongly depends on the means of vectorization. Polyamidoamine dendrimers are non-linear polycationic cascade polymers that are able to bind ODNs electrostatically. These complexes have been demonstrated to protect phosphodiester ODNs from nuclease degradation and also to increase their cellular uptake and pharmacological effectiveness. We studied the intracellular distribution of a fluorescein isothiocyanate-labeled ODN vectorized by a dendrimer vector and found that intracellular ODN distribution was dependent on the phase of the cell cycle, with a nuclear localization predominantly in the G2/M phase. In addition, in order to evaluate the relevance of ODN vectors in enhancing the inhibition of the targeted genes' expression, we developed a rapid screening system which measures the transient expression of two reporter genes, one used as target, the other as control and vice versa. This system was validated through investigating the effect of the dendrimer vector on ODN biological activity. Antisense sequence-specific inhibition of more than 70% of one reporter gene was obtained with a chimeric ODN containing four phosphorothioate groups, two at each end.

Stability of G,A triple helices.

In this work we selected double-stranded DNA sequences capable of forming stable triplexes at 20 or 50 degrees C with corresponding 13mer purine oligonucleotides. This selection was obtained by a double aptamer approach where both the starting sequences of the oligonucleotides and the target DNA duplex were random. The results of selection were confirmed by a cold exchange method and the influence of the position of a 'mismatch' on the stability of the triplex was documented in several cases. The selected sequences obey two rules: (i) they have a high G content; (ii) for a given G content the stability of the resulting triplex is higher if the G residues lie in stretches. The computer simulation of the Mg2+, Na+and Cl-environment around three triplexes by a density scaled Monte Carlo method provides an interpretation of the experimental observations. The Mg2+cations are statistically close to the G N7 and relatively far from the A N7. The presence of an A repels the Mg2+from adjacent G residues. Therefore, the triplexes are stabilized when the Mg2+can form a continuous spine on G N7.

Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides.

Antisense oligonucleotides with base sequences complementary to a specific RNA can, after binding to intracellular mRNA, selectively modulate the expression of a gene. However, these molecules are poorly stable in biological fluids and are characterized by a low intracellular penetration. In view of using oligonucleotides as active molecules, the development of polymeric particulate carriers was considered. Oligonucleotides were associated with biodegradable polyalkylcyanoacrylate nanoparticles through the formation of ion pairs between the negatively charged oligonucleotides and hydrophobic cations. Oligonucleotides bound to these nanoparticles were found to be protected from nuclease attack in cell culture media and their cellular uptake was increased as the result of the capture of nanoparticles by an endocytotic/phagocytotic pathway. The in vivo pharmacokinetic profile of oligonucleotides free or associated with nanoparticles has been investigated after intravenous administration to mice and the stability of these molecules has been evaluated by original methodology based on the use of polyacrylamide gel electrophoresis (PAGE) followed by multichannel radioactivity counting. Stability in vivo in the plasma and in the liver was shown to be improved when the oligonucleotides were adsorbed onto the nanoparticles. These results obtained both in vitro and in vivo open exciting perspectives for the specific delivery of oligonucleotides to the liver, thus considering this approach for the treatment of liver diseases (e.g. liver metastasis or hepatitis).

Analysis of various sequence-specific triplexes by electron and atomic force microscopies.

Sequence-specific interactions of 20-mer G,A-containing triple helix-forming oligonucleotides (TFOs) and bis-PNAs (peptide nucleic acids) with double-stranded DNA was visualized by electron (EM) and atomic force (AFM) microscopies. Triplexes formed by biotinylated TFOs are easily detected by both EM and AFM in which streptavidin is a marker. AFM images of the unlabeled triplex within a long plasmid DNA show a approximately 0.4-nm height increment of the double helix within the target site position. TFOs conjugated to a 74-nt-long oligonucleotide forming a 33-bp-long hairpin form extremely stable triplexes with the target site that are readily imaged by both EM and AFM as protruding DNA. The short duplex protrudes in a perpendicular direction relative to the double helix axis, either in the plane of the support or out of it. In the latter case, the apparent height of the protrusion is approximately 1.5 nm, when that of the triplex site is increased by 0.3-0.4 nm. Triplex formation by bis-PNA, in which two decamers of PNA are connected via a flexible linker, causes deformations of the double helix at the target site, which is readily detected as kinks by both EM and AFM. Moreover, AFM shows that these kinks are often accompanied by an increase in the DNA apparent height of approximately 35%. This work shows the first direct visualization of sequence-specific interaction of TFOs and PNAs, with their target sequences within long plasmid DNAs, through the measurements of the apparent height of the DNA double helix by AFM.

Recruitment of transcription factors to the target site by triplex-forming oligonucleotides.

Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a hairpin-TFO able to recruit transcription factors to a target DNA. The designed oligonucleotide contains a triplex-forming sequence, linked through a nucleotide loop to a double-stranded hairpin including the SRE enhancer of the c-fos gene promoter. We show here that this oligonucleotide can specifically recognise its DNA target at physiological salt and pH conditions. The stability of the triplex formed under these conditions is very high: >90% of the triplex remains intact after 24 h of incubation. Bound to the double-stranded target DNA, the oligonucleotide retains its ability to interact specifically with transcription factors, recruiting them to the proximity of the target DNA. Our results suggest that this type of oligonucleotide may prove useful in the design of new tools for artificial modulation of gene expression.