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.

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.

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.

Physicians' practices and opinions regarding prenatal screening for human immunodeficiency virus and other sexually transmitted diseases.

BACKGROUND AND OBJECTIVES: Early prenatal diagnosis of sexually transmitted diseases (STDs), particularly human immunodeficiency virus (HIV), is critical for maternal and infant health. We conducted a survey to assess physicians' prenatal STD screening practices and opinions. STUDY DESIGN: A random sample of obstetricians and family physicians was selected from the Minnesota Medical Association directory to complete a standardized telephone survey. RESULTS: Eighty-three (86%) of 96 eligible obstetricians and 94 (95%) of 99 eligible family physicians completed the survey. Nearly all physicians recommend universal prenatal screening for syphilis (97%) and hepatitis B (99%); fewer physicians recommend prenatal screening for HIV (43%), chlamydia (26%), and gonorrhea (24%). Adjusting for physicians' specialty, female physicians were more likely than male physicians to recommend universal prenatal HIV screening (odds ratio [OR] = 2.1; 95% confidence interval [CI] = 1.1-4.2). Adjusting for physicians' ages, physicians with more than 20% uninsured/Medical Assistance patients were more likely than other physicians to recommend prenatal gonorrhea screening (OR = 3.1; 95% CI = 1.4-6.8); similar factors were associated with chlamydia screening. Although 89% of physicians supported universal prenatal HIV counseling and voluntary screening, the median percentage of prenatal patients screened for HIV was only 10%. CONCLUSIONS: Most physicians reported routinely screening prenatal patients for syphilis and hepatitis B. Although many physicians agreed with recommendations for universal prenatal HIV screening, their reported screening practices varied considerably from this approach.

Automatic garage door openers: hazard for children.

OBJECTIVES: Despite significant advances in automatic garage door opener design, automatic garage door openers continue to severely injure or kill children. In this investigation, we sought to determine the frequency and circumstances of accidents that have caused severe injury or death to children. We also tried to develop a means by which homeowners can evaluate their door openers. METHODS: We present the histories of three children severely injured or killed by automatic garage door openers. We reviewed national data of similar accidents primarily published by the US Product Safety Commission and Underwriters Laboratories. Also, we evaluated 50 automatic door openers for safety of operation. The reversing mechanisms of door openers were tested using a cardiopulmonary resuscitation mannequin, a roll of paper towels, and a block of wood. RESULTS: In the United States, at least 85 children have had permanent brain injury or have died since 1974 as a result of accidents involving automatic door openers. A review of circumstances of the accidents illustrates that accidents are caused both by use of the openers by children and by faults in design. Most accidents have occurred when children have found access to the activation devices and have been entrapped under closing doors that failed to reverse. However, in one case, an adult activated the opener and left the premises before the door completely closed. Our evaluation of 50 garage door openers showed that although 88% percent reversed when encountering a block of wood, 40% failed to reverse when coming down on a supine, child-sized cardiopulmonary resuscitation mannequin. CONCLUSIONS: Automatic garage door openers pose a serious risk of severe injury or death to children. It is probable that many doors would not reverse if they came down on a young child. Therefore, we have devised a way for homeowners to test their door openers that closely mimics our evaluations using the mannequin by using a large roll of paper towels. If the door fails to reverse using this test, we suggest that homeowners disconnect their openers and operate the doors manually until the openers are serviced or replace their automatic openers with one that meets the latest Underwriters Laboratory standards. We also have other recommendations regarding the safe operation of the doors, including improving the safety standards for openers in apartment complexes. Compliance with these recommendations should reduce the number of injuries to children caused by garage door openers.

Investigation of the intracellular stability and formation of a triple helix formed with a short purine oligonucleotide targeted to the murine c-pim-1 proto-oncogene promotor.

In our previous work we have shown that the oligonucleotide 5'-GGGGAGGGGGAGG-3' gives a very stable and specific triplex with the promoter of the murine c-pim-1 proto-oncogene in vitro[Svinarchuk, F., Bertrand, J.-R. and Malvy, C.(1994)Nucleic Acids Res., 22, 3742-3747]. In the present work, we have tested triplex formation with some derivatives of this oligonucleotide which are designed to be degradation-resistant inside the cells, and we show that phosphorothioate and the oligonucleotide with a 3' terminal amino group are still able to form triplexes. Moreover these oligonucleotides, like the 13mer oligonucleotide of similar composition [Svinarchuk, F., Paoletti, J., and Malvy, C. (1995) J. Biol. Chem., 270, 14068-14071], are able to stabilize the targeted duplex. In vivo DMS footprint analysis after electroporation of the pre-formed triplex into the cell have shown the presence of the triple helix inside the cells. This triplex structure partially blocks c-pim-1 promotor activity as shown by transient assay with a c-pim-1 promoter-luciferase gene construct. To our knowledge these data are the first direct evidence that conditions inside cells are favorable for triplex stability with non-modified oligonucleotides. However we were unable to show triplex formation inside living cells using various methods of oligonucleotide delivery. We suppose that this may be due to the oligonucleotide being sequestered by cellular processes or proteins. Further work is needed to find oligonucleotide derivatives and ways of their delivery to overcome the problem of triplex formation inside the cells.

In vitro inhibition of the pim-1 protooncogene by chimeric oligodeoxyribonucleotides composed of alpha- and beta-anomeric fragments.

We show that oligodeoxyribonucleotides (oligos) composed of alpha- and beta-anomeric sections can be used as antisense compounds. An octamer has been chosen as an effector domain to form a substrate for RNaseH. This octamer is complementary to the translation start site of the pim-1 protooncogene mRNA. Chimeric alpha-beta oligos and their beta-analogs have a similar binding affinity for their target. These oligos also direct efficient RNaseH-mediated cleavage of target mRNA. Among all alpha-beta oligos studied, one with an alpha-fragment bound by its 3'-end to the 3'-end of the beta-octamer is the most resistant to nucleolytic digestion in biological media. The alpha-beta oligos have been found to inhibit in vitro translation of pim-1 RNA with specificity.

A short purine oligonucleotide forms a highly stable triple helix with the promoter of the murine c-pim-1 proto-oncogene.

A homopurine-homopyrimidine region of murine c-pim-1 proto-oncogene was chosen as a target for triple-helix-forming oligonucleotide. Oligonucleotide 5'-GGG-GAGGGGGAGG-3' was shown to bind to its target sequence in the presence of 50 mM Na+ or K+, 10 mM MgCl2 and 20 mM Tris-acetate, pH 7.5. This oligonucleotide is bound in an antiparallel orientation with respect to the homopurine sequence. As was shown by co-migration assay the triplex is stable up to 65 degrees C. At 37 degrees C it was practically irreversible: after 24 hours of co-migration assay there was no traces of triplex dissociation. The rate of triplex formation was highly accelerated with increase of temperature and Mg2+ concentration. This rate was higher for superhelical DNA when compared to the linear and circular ones and the preference was dependent from temperature and Mg2+ concentration. The precision of this interaction is extremely high: sequences in c-pim-1 promoter region with only one substitution when compared to the target gave negligible triplex formation under investigated conditions. These data suppose that natural triplex structures could play an important role in eukaryotic gene regulation and/or chromatin structure formation.

Sequence-specific interaction of alpha-beta-anomeric double-stranded DNA with the p50 subunit of NF kappa B: application to the decoy approach.

The potential use of alpha-beta-anomeric duplex oligonucleotides to inhibit transcription factor activity by the decoy approach is investigated in this report. Indeed, several alpha-beta-anomeric heteroduplexes display a sequence-specific interaction with the p50 subunit of the transcription factor NF kappa B. Used in a decoy approach, these duplexes interact strongly enough with this transcription factor to modulate the expression of a reporter gene, under the control of NF kappa B. However, all the alpha-beta-anomeric heteroduplexes do not interact with the p50 subunit; the sequence of the chirally natural beta-anomeric strand may explain the different recognition properties of the protein. The analysis of the appropriate beta-anomeric sequences is consistent with a preferential interaction of the p50 subunit with one strand of double-stranded DNA.

Alpha beta chimeric antisense oligonucleotides: synthesis and nuclease resistance in biological media.

A new type of chimeric oligonucleotides composed of alpha- and beta-sections is described. The sequence of eight beta-nucleotides flanked at 3'- or/and 5'-ends by nuclease-resistant alpha-oligonucleotides has been chosen as an effector domain to form a substrate for RNase H. The synthesized oligonucleotides are complementary to the translation initiation site of the pim protooncogene mRNA. We used the chemical ligation method to prepare the chimeric oligonucleotides. The thermal stability of heteroduplexes formed by the alpha beta oligonucleotides with a complementary strand is not significantly altered compared to that of their beta-analogs. These oligonucleotides promote efficient RNase H-mediated cleavage of pim mRNA. Among the alpha beta oligonucleotides studied, one with an alpha-fragment bound by its 3'-end to the 3'-end of the beta-octanucleotide proved to be the most resistant to nucleolytic digestion in human plasma, calf serum, and murine fibroblast lysate. This alpha beta oligonucleotide directs more specific RNA cleavage by RNase H than its beta beta counterpart.

9-Aminoellipticine-derivatized alpha- and beta-oligodeoxyribonucleotides targeted to the cap of beta-globin mRNA: hybridization to natural and engineered mRNA, inhibition of translation, and improved effect of tandem chains.

We studied the duplex stability and the antimessenger activity of 9-aminoellipticine-5'-functionalized alpha- and beta-anomeric DNA sequences complementary to the first 14 nucleotides of the rabbit beta-globin mRNA. The duplex formed by the beta-conjugate with the natural mRNA target possessed a marginally better stability to that of the duplex formed by the unfunctionalized compound, as measured by the thermal elution. The alpha-conjugate did not anneal to native mRNA, possibly due to the interference of the 9-aminoellipticine with the cap structure and, unlike the beta-adduct, was practically inactive as inhibitor of translation in a cell-free system. However, it did hybridize to an RNA construction containing the beta-globin mRNA plus an additional 50 bases in 5'. Surprisingly, translation from this construction was inhibited by the alpha-species in spite of the nonvicinity of the target to the cap. Both alpha and beta conjugates hybridized to a DNA 14-mer of the same sequence as that targeted onto the mRNA. Thermal denaturation and fluorescence spectroscopy showed that the drug brought no considerable stabilization to the duplex, the linker presumably being unfavorable to intercalation. An increased stability of the complex and a higher inhibitory effect on cell-free beta-globin translation were obtained with two contiguous beta-oligomers of which one was functionalized.

Molecular modelling of 9-aminoellipticine interactions with abasic oligonucleotides.

We have used molecular mechanics to study the insertion of the DNA intercalating agent 9-aminoellipticine (9AE) into single and double stranded abasic oligonucleotides containing abasic sites in the aldose or furanose conformations. 9AE-abasic oligonucleotide complexes have been considered with 9AE bound at abasic sites as a covalent complex, a reversible complex or a Schiff base. Results are in good agreement with experimental data available on abasic oligonucleotides (melting temperature measurement, NMR results) and allow an analysis of different possible structures for 9AE-abasic oligonucleotide complexes. Hypotheses concerning the role of 9AE-abasic site complexes in enzymatic inhibition are formulated from these data.

Comparison of anti-RNA properties of normal and ellipticine functionalized alpha and beta-oligonucleotides.

RNA is not cleaved as a consequence of the binding of RNase H to the duplex between RNA and a complementary alpha-oligodeoxyribonucleotide (oligo). In consequence targets have been selected which do not a priori require the action of RNase H to inhibit genetic expression. Two models have been used: The Friend Murine Leukemia Virus (F-MuLV) and the synthesis of rabbit beta globin.alpha-oligos trigger specific inhibitions in both systems. The functionalisation in 5' with the intercalating agent 9-NH2-ellipticine renders the oligos resistant to degradation and allows a direct action on cells.