driver
- 30 Mar 2006 17:03
smiler o
- 20 Nov 2006 08:27
- 568 of 1180
SAR Going well Rns out : )
potatohead
- 20 Nov 2006 11:18
- 569 of 1180
White paper presented at the RNAi for Functional Analysis and Target Validation
Conference in Boston on 9-12 Nov 2004 run by the Cambridge Healthtech Institute in
the US.
The Importance of siRNA Delivery in Cancer Target Validation
It is estimated that an additional 5,000-10,000 new potential drug targets for various diseases will emerge
as a result of the sequencing of the human genome. Given the expense of drug development (approx. $800
million to bring a therapeutic to market), functional drug target validation represents one of the most
important steps in the Drug Discovery pipeline and, as such, requires an efficient, meaningful and highthroughput
evaluation system.
In the past, time consuming genetic approaches such as gene disruption by homologous recombination and
forward genetic mutagenesis strategies were used. Nucleic acid-based approaches that act to silence gene
expression, in particular Antisense Technology seemed to provide a sequence specific, broadly applicable,
time and cost effective alternatives. While Antisense has made some impact on both target validation and
therapeutics, there are several major technical issues that have prevented its widespread application.
The discovery of siRNA in 2001 revolutionized target validation with its clear potential to overcome the
shortcomings of Antisense Technology as described below:
Design: While it is estimated that only 1/8 of Antisense Oligonucleotides (ODNs) are functional,
guidelines in reviews, web sites and commercial algorithms have facilitated relatively easy selection of
functional siRNA sequences.
Stability: In comparison to ODNs, chemically synthesized siRNAs are generally stable in cell lines for up
to 6 generations allowing adequate time for phenotypic evaluations. Endogenous expression of siRNA
molecules from plasmid and viral vectors allows for longer-term analysis.
Toxicity: The high non-specific toxicity associated with ODNs is also believed to be alleviated by the use
of siRNAs as they are functional at very low doses, reducing the possibility of non-specific protein
binding, potential off-target effects and associated toxic side effects. The investigation of several target
specific siRNAs and Missense siRNAs further confirms specificity of the response.
Delivery: Although electroporation is occasionally used, the most commonly used methods for delivery
of charged nucleic acids (including ODNs, ribozymes and siRNAs) are liposomes, polymers and charged
lipids. However protocols generally need to be individually optimized for the efficient delivery of nucleic
acids into different cell lines. In fact, some siRNA transfection reagent suppliers offer kits to establish and
optimize siRNA mediated gene silencing in a new cell line of interest. The necessity of initially
optimizing transfection conditions for each individual cell line in a cancer cell panel, and then having to
run all these different protocols for each target makes high throughput target validation very challenging.
In essence, efficiency of transfection and adaptability of the method constitute the key criteria for the
development of a successful high throughput siRNA delivery system to enable Cancer Target Validation.
Transfection efficiency
Transfection efficiency encompasses several requisites: Universal delivery of siRNAs to a large panel of
cancer cell lines under the same transfection conditions, including adherent and suspension cells. This
would enable evaluation of target knockdown in cell lines representative of the major cancer types,
including the top 8 solid tumors (namely lung, colon, breast, prostate, ovarian, melanoma, gastrointestinal
and liver) as well as leukemias and lymphomas thus facilitating identification of targets that selectively
kill particular tumor types while having no effect on others as well as those that destroy a broad spectrum
of cancer cell types. Transfection of the majority of cells in a population (ie. high transfection rate) is
paramount to long-term cell based assays, as a low transfection rate causes the dilution of the siRNA
effect giving error-prone and difficult to evaluate results. High levels of siRNA transfected per cell allow
for maximum target knockdown by the siRNA in each cell. Any toxicity inherent in the reagent or
protocol would increase the base line of an assay and so make analysis of results of toxicity assays more
difficult to interpret, therefore minimal toxicity associated with transfection reagent and protocol is vital.
High reproducibility would confer credibility on results and allow for comparisons between subsequent
validation experiments and projects.
Adaptability
A quick transfection protocol adaptable to high through put format, eg. 96 well plates, and potentially
automated systems, would enable the simultaneous evaluation of a target in a panel of cell lines or several
targets in a particular cancer cell line or cancer type (encompassing several cell lines) as well as various
controls. Adaptability of the transfection system to a wide range of functional and phenotypic assays
would be vital to the characterization and complete validation of each cancer target. Such validation
requires analysis in a panel of functional assays including short term viability assays using Alamar Blue,
MTT or WST1; long term viability assays (up to 14 days) such as clonagenicity assays; apoptosis assays
including DNA fragmentation (analysis of subG1 populations), cell morphology (size/granulation),
phosphatidyl serine exposure (Annexin/propidium iodide) and mitochondrial membrane depolarization
(JC1). Cancer phenotype assays would include anchorage dependent (in soft agar) and anchorage
independent clonagenicity assays and Boyden chamber migration assays. The majority of these assays are
adaptable to a rapid throughput format with the latter phenotypic assays adaptable to 96 well plate
analysis.
Cancer Target Validation
EiRx Therapeutics Plc. has developed an siRNA delivery system that satisfies all the above criteria to
create a high through put Cancer Target Validation Platform. This has facilitated in-house validation of
more than 100 potential apoptosis modulating anti-cancer targets to date.
The establishment of such efficient siRNA delivery systems is essential to the rapid development and
worldwide use of siRNA-based target validation. Such siRNA delivery processes can be adapted to in vivo
and explant models and can also be adapted to other disease indications. The potential value of such
systems are apparent given the recent prospective figures for the 2010 world RNAi market published by
Frost and Sullivan, UK (World RNAi markets: Current and Future Outlook): it is expected to grow from
an estimated $48 million in 2003 to $328 million in 2010, with almost 50% of that figure ($146.4 million)
being target validation.
potatohead
- 20 Nov 2006 12:11
- 570 of 1180
could break 40 today.. looks promissing, shares mag should do something on this
potatohead
- 20 Nov 2006 12:34
- 571 of 1180
about to tick up again.. bleeding heck, it might just happen today
potatohead
- 20 Nov 2006 13:19
- 572 of 1180
Notable milestones in 2004/5 include:
* flotation on AIM
* licensing of 4 cancer drug targets to OSI Pharmaceuticals, in a deal worth
up to $18.8M depending on results
* collaboration with structure-based drug discovery company Sareum plc, to
advance EiRx kinase targets to Phase I/II clinical trials
* acquisition of oncology company Auvation Ltd
* collaborative research agreement with Merck & Co. Inc. to evaluate EiRx's
siRNA delivery technology
* award of a Marie Curie Programme grant from the European Commission, to
support development of the company's screening, chemistry and efficacy
testing capabilities
Potential new therapies for colorectal and other cancers
A grant from the Marie Curie Programme helped fund the development of EiRx's
EnPAD(TM) technology, which has been designed to identify novel drug candidates
targeted against specific biological pathways. Using its EnPAD(TM) technology
together with its ALIBI(TM) platform, EiRx's scientists have discovered a series
of related compounds with selective activity against transformed cell types
including colorectal and breast cancer cell lines. The company has submitted
patent applications to protect these novel therapeutic candidates in key global
markets.
potatohead
- 20 Nov 2006 15:57
- 573 of 1180
big volume today... news tomorrow ???
laurie squash
- 20 Nov 2006 16:05
- 574 of 1180
4 million buy just went through!
potatohead
- 20 Nov 2006 17:23
- 575 of 1180
I believe we have news this week
potatohead
- 21 Nov 2006 12:48
- 576 of 1180
should see large volumes again this afternoon
we have a big link with BIOFOCUS.. research this heavily.. you will see what i mean
potatohead
- 21 Nov 2006 14:20
- 577 of 1180
http://home.businesswire.com/portal/site/google/index.jsp?ndmViewId=news_view&newsId=20061121005028&newsLang=en
Hematological Cancers Drug Pipeline Update 2006 Lists More Than ...
Business Wire (press release), CA - 10 minutes ago
... to be leading strategies. Small molecule apoptotic inducers and kinase inhibitors are as well in the forefront. There are today ...
Marcel1970
- 21 Nov 2006 14:52
- 578 of 1180
The only volume i can see is selling hence the share price drop.
laurie squash
- 21 Nov 2006 15:00
- 579 of 1180
Link is informative but have to agree with Marcel.
smiler o
- 21 Nov 2006 15:14
- 580 of 1180
agree, positive news is what it needs ! :)
potatohead
- 21 Nov 2006 16:45
- 581 of 1180
been told there is news tomorrow.. hold on tight
smiler o
- 21 Nov 2006 16:54
- 583 of 1180
driver, we may all be caught in a time warp ! :)
kimoldfield
- 22 Nov 2006 10:11
- 584 of 1180
News tomorrow PH?!
kim
potatohead
- 22 Nov 2006 11:57
- 585 of 1180
yes there must be, was told news very very soon.. put it this way, latest by friday.. otherwise the ceo has some explaining to do
potatohead
- 22 Nov 2006 13:46
- 586 of 1180
has anyone got the impression that these 500k sells is someone trying to short the stock..