Biomedical subjects

Mouldy Sioud

Publications and source records attributed to Mouldy Sioud.

47 records · Page 3Linked to original sources

PubMed2003

Cationic liposome-mediated delivery of siRNAs in adult mice.

RNA interference mediated by small interfering RNAs (siRNAs) is a powerful tool for dissecting gene function and drug target validation. siRNAs can be synthesized in large quantities and thus can be used to analyze a large number of sequences emerging from genome projects in a cost-effective manner. However, the major obstacle to the use of siRNAs as therapeutics is the difficulty involved in effective in vivo delivery. We used a fluorescein-labeled siRNA to investigate cationic liposome-mediated intravenous and intraperitoneal delivery in adult mice. We show that this simple approach can deliver siRNAs into various cell types. In addition, we show that in contrast to mouse cells, siRNAs can activate the non-specific pathway in human freshly isolated monocytes, resulting in TNF-alpha and IL-6 production. Taken together, the data provide a basis for lipid-mediated systemic delivery of siRNAs and indicate that certain siRNA sequences can activate the innate immunity response genes that can be beneficial for the treatment of cancer.

Animals↗

PubMed2003

Systematic identification of sense-antisense transcripts in mammalian cells.

In prokaryotes, a number of endogenous antisense RNAs have been detected and found to exert various biological functions. In eukaryotes antisense RNAs have been found; however, a lack of experimental methodologies that permit the identification of overlapping transcripts in cells presents a barrier to a more systematic identification of antisense RNA. Here we have developed an experimental strategy that allows systematic identification of endogenous mRNAs with long complementary regions to other transcripts. The method was applied to human normal mammary epithelial and breast cancer cells. Experimental validation of the presence of the sense and antisense transcripts by various techniques (e.g., northern blots, RT-PCR) supports the specificity of the method. When the antisense RNAs were specifically targeted, their corresponding mRNA levels significantly altered, a result consistent with a regulatory role for the identified antisense RNAs.

Animals↗

PubMed2003

Gene silencing by systemic delivery of synthetic siRNAs in adult mice.

In mammalian cells, RNA duplexes of 21-23 nucleotides, known as small interfering RNAs (siRNAs) specifically inhibit gene expression in vitro. Here, we show that systemic delivery of siRNAs can inhibited exogenous and endogenous gene expression in adult mice. Cationic liposome-based intravenous injection in mice of plasmid encoding the green fluorescent protein (GFP) with its cognate siRNA, inhibited GFP gene expression in various organs. Furthermore, intraperitoneal injection of anti-TNF-alpha siRNA inhibited lipopolysaccharide-induced TNF-alpha gene expression, whereas secretion of IL1-alpha was not inhibited. Importantly, the development of sepsis in mice following a lethal dose of lipopolysaccharide injection, was significantly inhibited by pre-treatment of the animals with anti-TNF-alpha siRNAs. Collectively, these results demonstrate that synthetic siRNAs can function in vivo as pharmaceutical drugs.

Animals↗

PubMed2003

Generation of an effective anti-tumor immunity after immunization with xenogeneic antigens.

Central and peripheral tolerance mechanisms are expected to hamper the generation of effective immunity against tumors. To break self tolerance against malignant gliomas, we assessed the therapeutic potential of self/foreign antigen cross-reactivity in an immunocompetent rat glioma model. Immunotherapy of tumors using xenogeneic human glioma membrane proteins (HGP) as a vaccine inhibited tumor growth, whereas no significant effect was obtained with rat glioma membrane proteins (RGP). In contrast to RGP, HGP elicited a specific IgG immune response that cross-reacted with RGP. This immune response was found to be mainly a Th1 type response. On tumor sections stained with hematoxylin and eosin, glioma cells are sparse and apoptotic in HGP-immunized rats, whereas control tumors showed condensed and viable cells. Tumor-specific CTL were induced in HGP-immunized rats. Immunohistochemical analysis revealed that a significant number of CD8(+) and CD4(+) cells infiltrated into tumors from HGP-vaccinated rats, whereas RGP vaccination led to only few tumor-infiltrating T cells. Taken together, the data establish the in vivo applicability of the cross-stimulation between self and foreign antigens as an alternative way to break tolerance against the poorly immunogenic gliomas.

Animals↗

PubMed2003

Selective targeting of cancer cells using synthetic peptides.

To establish efficient and reliable therapeutic delivery into cancer cells, a number of delivery agents and concepts have been investigated in the recent years. Among many improvements in targeted and controlled delivery of therapeutics, cell-targeting peptides have emerged as the most valuable non-immunogenic approach to target cancer cells. Peptides can be incorporated into multicomponent gene-delivery complexes for cell-specific targeting. In contrast to larger molecules such as monoclonal antibodies, peptides have an excellent tumor penetration, which make them ideal carriers of therapeutics to the site of primary tumor and the distant metastatic sites. Here we give an update on the progress made during the last two years on the identification and potential of specific synthetic tumor targeting peptides.

Animals↗

PubMed2002

Identification of novel carrier peptides for the specific delivery of therapeutics into cancer cells.

Cancer therapy is currently limited by the difficulty of achieving efficient delivery into target cells. To investigate whether therapeutics can be delivered specifically to cancer cells, we have explored the possibility of selecting small peptides that bind specifically, or preferentially, to breast cancer cell lines. By using random peptide phage libraries and an experimental approach that allows the selection of internalized peptides, cell-specific binding peptides have been identified. The peptides define a major core motif (LTVXPWY) that was not found in negative phages. Phage displaying LTVSPWY peptide sequence exhibited a specific binding to breast cancer cells. None of the selected peptides bound to human primary cells from different tissue origin (e.g., epithelial, endothelial, hematopoetic). The potential of the selected peptides to mediate cellular internalization in the context of phages and recombinant GFP-peptide fusions was demonstrated. By linking the LTVSPWY peptide to an antisense phosphorothioate oligonucleotide against the ErbB2 receptor, specific delivery to cancer cells was achieved. In contrast to free antisense, the peptide-antisense conjugates inhibited ErbB2 gene expression. Thus, efficient delivery of antisense oligonucleotides can be achieved by coupling them to cancer cell-specific peptides, identified by a method that did not require any knowledge about their corresponding receptors.

Amino Acid Sequence↗

PubMed2002

Gene silencing in mammalian cells by preformed small RNA duplexes.

Small interfering RNAs (siRNAs) mediate RNA interference (RNAi), a process in which target mRNAs are degraded. Here, we have investigated the efficacy of preformed siRNAs to modulate the expression of protein kinase Calpha (PKCalpha) and green fluorescent protein (GFP) in mammalian cells. We show that specific inhibition of PKCalpha and GFP can be achieved by in vitro transcribed siRNAs. Interestingly, a transcript harboring two self-complementary siRNAs interrupted by a single-stranded loop region inhibited both PKCalpha and GFP gene expression. These results suggest that the long transcript is processed by single-stranded ribonucleases and/or other proteins into two functional siRNAs. Incubation of the in vitro transcribed bispecific siRNA with protein extracts from HEK 293T cells yielded RNA duplexes similar to the synthetic single siRNA. Taken together, the present data indicate that in vitro transcribed siRNA can be useful for silencing gene expression. Additionally, bi- and perhaps poly-siRNAs may be expressed and processed in mammalian cells.

Base Sequence↗

PubMed2002

Targeting Raf-1 gene expression by a DNA enzyme inhibits juvenile myelomonocytic leukemia cell growth.

Juvenile myelomonocytic leukemia (JMML) is an aggressive childhood disorder with few therapeutic options. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) promote JMML cell growth. A hyperactive function of the ras oncogene is a hallmark of JMML. We therefore targeted the protein kinase Raf-1 downstream of Ras using a DNA enzyme that degrades mRNA-Raf-1. Western blots of JMML cell lysates revealed phosphorylated Raf-1 protein, indicating constitutive activation. Addition of GM-CSF, but not TNF-alpha, increased phosphorylation of both Raf-1 and the mitogen-activated protein kinases (MAPKs) JNK-1 and ERK-1. Depletion of Raf-1 protein markedly impaired activation of MAPKs, induced substantial inhibition of JMML cell colony formation, and virtually abolished GM-CSF hypersensitivity in JMML cells. Exogenous TNF-alpha, but not GM-CSF, restored colony formation of JMML cells pretreated with the enzyme. We could not detect any effect of the enzyme on the proliferation of normal bone marrow cells, indicating its specificity and potential safety. When immunodeficient mice engrafted with JMML cells were treated continuously with the enzyme via a peritoneal osmotic mini-pump for 4 weeks, a profound reduction in the JMML cell numbers in the recipient murine bone marrows was found. We conclude that GM-CSF is a chief regulator of JMML growth and exerts its proleukemic effects primarily via the Ras/Raf-1 signaling cascade. TNF-alpha plays a permissive role, being dependent upon GM-CSF to induce JMML cell proliferation. The DNA enzyme efficiently catabolized mRNA-Raf-1 with subsequent inhibition of JMML cell growth, suggesting its potential as a mechanism-based therapy in this fatal leukemia.

Bone Marrow Cells↗

PubMed2002

[Tyrosine kinase receptor-ras-ERK signal transduction pathway as therapeutic tarfet in cancer].

BACKGROUND: Experimental evidence indicates that various intracellular signalling cascades are altered in tumour cells. Among these, the receptor tyrosine kinase ras-ERK signalling pathway was found to be constitutively active in a significant percentage of human tumours; hence, considerable effort has been directed at finding compounds that inhibit its activation. MATERIAL AND METHODS: We review the recent progress in establishing novel approaches to interference with the constitutive activation of the receptor tyrosine kinase ras-ERK signalling pathway in cancer. RESULTS: Inhibition of the receptor tyrosine kinase ras-ERK signalling pathway activation by various novel agents (e.g. small molecule tyrosine kinase inhibitors, antibodies, FTase inhibitors, SH2/SH3 directed agents, antisense, ribozymes) impaired tumour growth. Some of the developed agents have been tested in clinical trials; promising results were obtained. INTERPRETATION: Inactivation of the receptor tyrosine kinase ras-ERK signalling pathway by small molecular inhibitors has confirmed its involvement in tumour growth. Thus, molecular and/or pharmacological modulation of the components that are critically involved in the constitutive activation of this pathway are expected to improve the treatment of human malignancies.

Antineoplastic Agents↗

PubMed2002

How does autoimmunity cause tumor regression? A potential mechanism involving cross-reaction through epitope mimicry.

BACKGROUND: Although the exact mechanisms mediating the initiation of autoimmune diseases are unknown, sequence similarity between infectious agents and self-proteins (epitope mimicry) has been proposed as the main trigger mechanism. Interestingly, this mechanism of epitope mimicry may also evoke potent tumor immunity. Indeed, experimental data support a beneficial role of autoimmunity in some patients with cancer. Additionally, autoimmunity induced via vaccination with xenogeneic antigens was found to be effective. Thus, the ability to manipulate the immune system via immunologic cross-reactions should have important potential in both preventive and therapeutic strategies for cancer. This strategy may down the friendly established relationship between tumor tissues and the cells of the immune system.

Autoimmunity↗

PubMedDate not supplied

Combination of endostatin and a protein kinase Calpha DNA enzyme improves the survival of rats with malignant glioma.

Malignant gliomas are refractory to conventional therapies, including surgery, radiotherapy and chemotherapy. Thus, a variety of therapies such as the inhibition of angiogenesis and signal transduction pathways have been attempted. In the present study, we have evaluated the combined effect of endostatin, an inhibitor of angiogenesis, and a DNA enzyme targeting the protein kinase Calpha (PKCalpha) gene expression. Inhibition of PKCalpha by a nuclease-resistant DNA enzyme eliminated PKCalpha gene expression and induced apoptosis in most glioma cells. To assess the efficacy of endostatin and the PKCalpha DNA enzyme in vivo, rats bearing the intracranial tumor BT(4)C were given a combined treatment of endostatin and the PKCalpha enzyme. Survival was significantly enhanced by continuous delivery of endostatin (P<.0004) and rats treated with a single injection of the active DNA enzyme lived significantly longer than those treated with the inactive form (P<.045). Interestingly, a single injection of the PKCalpha DNA enzyme in combination with continuous delivery of endostatin significantly improved animal survival compared with PKCalpha (P<.0009) or endostatin (P<.025) alone. Thus, the combined treatment may represent an attractive therapeutic strategy against malignant gliomas.

Angiogenesis Inhibitors↗