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תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Tel Hashomer Medical Research Infrastructure and Services Ltd. Biological Tools Contact : Sylvie Luria PhD. Technology Transfer and Business & Development Manager Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Cell: 052-6667277 [email protected] http://research.sheba.co.il/e/ 1 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Methods for Silencing Gene expression A novel approach and tools to gene silencing to target cancer therapy. The study involves the synthesis of siRNAs targeted to the promoter sequences of genes that are essential for cell survival and proliferation, IGF-1R. Short interfering RNA (siRNA), is typically 19-21 bp long, with two nucleotides overhanging at each 3’ end. Alternatively, 27-mer blunt-ended nucleotides may be used. Presently there are no valid rules that predict the preferential selection of promoter targeted sequences. We have targeted the IGF-1R promoter via GTS, the transcription of two genes playing important roles in the malignant transformation IGF-1R. The importance of insulin-like growth factor 1 receptor (IGF-1R) signalling in malignant behaviour of tumor cells is well established. Its involvement in malignant transformation was first recognized in fibroblasts derived from homozygous IGF-1R null mice embryos. Mouse embryo fibroblasts are prone to malignant transformation; however, in the absence of IGF-1R they become resistant to transformation by several oncogenes (e.g. Simian Virus 40T antigen, Ewing Sarcoma fusion protein). Re-expression of the IGF-1R restored susceptibility to transformation in these cells. In population studies, high serum levels of IGF-1 have been associated with an increased risk on prostate cancer and premenopausal breast cancer. An increased incidence of colorectal adenomas and cancer was observed in acromegaly, in which hypersecretion of GH is accompanied by elevated IGF-1 levels. The tumor promoting functions of IGF-1R are embedded in the multi-stage process of cancer development and progression. Increased IGF-1R activation, by IGF-1 or other mechanisms, may create an anti-apoptotic environment, thereby favoring cell survival and malignant transformation. A number of approaches have been explored to interfere in IGF1R signaling, such as reducing ligand availability by GH antagonists, IGF-1- and IGF-2-antibodies, the use of recombinant human IGF-1 binding proteins, reducing IGF-1R expression by anti-sense and RNA interference or inhibiting IGF-1R activation by IGF-1R antibodies and small molecule tyrosine kinase inhibitors. In vitro and in vivo inhibition of IGF-1R signaling in tumor cells resulted in striking apoptosis in malignant cells growing in anchorage independent conditions and dramatic inhibition of tumor formation after injection in nude mice . Our novel concept demonstrated the silencing of IGF-1R expression with siRNA targeting the mRNA. This silencing lasted for at list 72 hours. This silencing was specific as the siRNA seems not to affect other mRNA. We could also silence the IGF-1R expression with siRNA targeting to promoter of IGF-1R, Our results suggested that targeting essential transcriptional factor binding site would be favourable to achieve inherited long lasting gene silencing. 2 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 In vivo Tool to Identify Novel Targets in Health and Disease useful for Drug Screening The post-transcriptional modification of mammalian transcripts by Adenosine to inosine (Ato-I) RNA editing has been recognized as an important mechanism for the generation of molecular diversity and also regulates protein function through recoding of genomic information. As the molecular players of editing are characterized and an increasing number of genes become identified that are subject to A-to-I modification, the potential impact of editing on the etiology or progression of human diseases is realized. In a few known examples, A-to-I RNA editing recodes an amino acid in the translated protein, and may result in an altered function. Altered editing patterns are associated with several pathological conditions, mainly to central nervous system-related abnormalities such as amyotrophic lateral sclerosis (ALS), epilepsy, major depression disorder (MDD) and glioblastoma multiforme (GBM) and cancer. Editing therefore could have physiological significance, but its precise role is still speculative. Up until now in order to detect editing in-vivo the RNA had to be sequenced. For that the animal had to be scarified for the RNA purification. Since editing plays an important role both in normal development and different disease we are developing tools that will enable us to monitor the levels of A-I RNA editing both in tissue culture and In-vivo. We have developed a genetic tool that can mark A-I RNA editing both in cell both in tissue culture and In Vivo. 3 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Dynamic contrast MRI for assessment of Blood Brain Barrier functioning and its applications The blood-brain barrier (BBB) is the specialized system of capillary endothelial cells that protects the brain from harmful substances in the blood stream, while supplying the brain with the required nutrients for proper function. Unlike peripheral capillaries that allow relatively free exchange of substance across / between cells, the BBB strictly limits transport into the brain through both physical (tight junctions) and metabolic (enzymes) barriers. The BBB is an essential physiological barrier for the maintenance and regulation of the neural microenvironment. Two elements underlie the barrier function of the brain capillary endothelium: 1) a physical barrier comprised of tight junctions, which form an effective seal to intercellular diffusion, and the cells themselves, which exhibit a low rate of endocytosis, and 2) a metabolic/active barrier, comprised of specific membrane transporters expressed by the endothelial cells. Thus the BBB is often the rate-limiting factor in determining permeation of therapeutic drugs into the brain. Additionally, BBB breakdown is theorized to be a key component in central nervous system (CNS) associated pathologies. . We have developed an innovated MRI-based methodology for clinical assessment of BBB functioning. The methodology is based on a combination of dynamic contrast MRI together with a special analysis methodology. The results consist of several calculated parameters and high resolution brain maps, providing quantification of local/spread BBB abnormalities. This Novel technology enables real time BBB permeability assessment for diagnosis of various CNS disorders as well as staging and treatment monitoring. In addition, the methodology may also be used for developing/monitoring therapeutic procedures involving increased perfusion, BBB opening for drug delivery or increased clearance of hazardous substances from the brain as well as for drug discovery and development. The innovation can monitor medical devices that induce BBB opening or restriction enables the delivery of therapeutic drugs directly into the brain. In addition, the technology can predict BBB opening induced toxicity. 4 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Convection-Enhanced Drug Delivery: Increased Efficacy and MRI Monitoring Fluid convection (bulk flow) is a promising technique for the distribution of drugs into brain/tumor tissue. Convection, established by creating an infusion-mediated pressure gradient through intracranial catheters, greatly enhances the distribution of various molecules in the brain. Sustained in situ drug concentrations several orders of magnitude is greater than those achieved by systemic administration can be achieved in large brain volumes at various time intervals. Application of convection-enhanced drug delivery (CED) to brain pathology is an emerging therapeutic discipline. Most of the studies described so far have focused on the treatment of brain tumors. We have recently conducted a phase I/II clinical trial in which patients with recurrent glioblastoma multiforme (GBM) received CED of Taxol with a very high tumor response rate exceeding 70% of the tumors. In clinical trial using CED of immunotoxins (a conjugate protein of diphtheria toxin with a point mutation linked by a thioester bond to human transferrin) the response rate was 35%. Clinical trials have also been conducted with other drugs, such as TP-38 and interleukin (IL)-4, PE38KDEL, which provided evidence of some clinical activity, and with IL13-PE38QQR, which yielded no advantage over the best standard of care in a large-scale clinical study. Our group has been involved in the design, conduct and steering of many of the above studies. A new application of MRI for real-time monitoring of convection and early detection of response to therapy was developed. The depiction of early cytotoxic tissue response to treatment has been expanded beyond the response to CED-based treatments. In addition, we have developed means to increase CED efficacy by modifying the physical characteristics of the infusate. This application has been extended to enable efficient CED of nanoparticles (such as iron oxide, liposoms, gene therapy related particles etc), as well as the application of CED to other extra-cranial organs (such as the prostate). 5 תל השומר מחקרים תשתיות ושירותים רפואיים בע"מ Tel Hashomer Medical Research, Infrastructure and Services Ltd. Tel: +972-3-5305998 Fax: +972-3-5305944 Small Scale Affinity Method for Biomarker Enrichment in Human Serum Biomarkers are the most important diagnostic tools for human disease management, for diagnostic, disease prognosis and for selecting appropriate therapies. Many important biomarkers are fluid borne biomarkers, which can be detected in a fluid sample taken from a subject. Typically, such fluid is blood; the biomarkers may be detectable in whole blood or in serum. However, in many cases, the biomarkers are present at a relatively low level in the blood sample, which increase the difficulty of their detections, especially when we look at multiplex biomarkers. Enriching the biomarkers in the analyzed sample simplified the analysis, enhances dramatically its efficacy and enables their detections when exist in small quantities. Currently, most methods mainly implement depletion of albumin and Immunoglobulins from the serum, with no protein enrichment protocol. Enrichment methods are not widely used, as they are difficult and time consuming. Such methods are typically more suitable for a research laboratory than for a hospital laboratory and for patient care. Protein separation or enrichment is one of the rate-limiting steps in proteomic studies. Specific capture and removal of highly-abundant proteins with large sample-handling capacities are in great demand for enabling detection and analysis of low-abundant proteins. We have developed a novel strategy that involves mini purification steps of a group of protein and combines enrichment and depletion approaches in a format of small scale and multi samples preparation. Our affinity subtraction and enrichment method is compatible with the context of proteomic and systems biology and provides a simple, very accurate and repetitive tool for biomarker analysis in blood sample. 6