Download Methods for Silencing Gene expression

‫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/
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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
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.
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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
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.
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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
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.
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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
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).
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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
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.
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