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post Posted: Mar 28 2004, 04:49 PM
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post Posted: Mar 28 2004, 08:09 AM
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For some additional background information for those interested in the concepts behind the technology.


The Scientist Volume 18 | Issue 6 | 12 | Mar. 29, 2004

5-Prime | A Powerful Tool in the Silencing Trade

Courtesy of Sirna Therapeutics

1. What is this powerful tool? RNA interference (RNAi) is a type of posttranscriptional genetic regulation that occurs naturally in the cytoplasm to protect the cell against excess and foreign RNAs. Double-stranded RNA (dsRNA), an unusual type of nucleic acid encoded in viral genomes and transposable elements, triggers a process that regulates gene expression without touching the genome.

2. What do we know about it? Scientists know that RNAi protects the cell against viral infections and genomic instability, but no one knows exactly how dsRNA triggers RNAi's defense mechanism. Scientists do know the chain of events that the dsRNA initiates, which ultimately rids the cell of mRNA.

3. How does it work? Dicers, a class of RNase III enzymes, recognize dsRNA molecules and cut them into short (~21-25) nucleotide segments called small interfering RNAs (siRNAs). These segments hook up with an RNA-induced silencing complex (RISC), and together they hunt for homologous mRNA substrates, which they then degrade, effectively silencing gene expression.

4. Where does RNAi normally occur? So far, researchers have found RNAi in every organism they have studied, from fungi to humans; the proteins involved are highly conserved. RNAi regulates flower color in petunias and developmental timing in nematodes, and it acts as an antiviral agent in mammals.

5. Why are researchers excited about RNAi? Partly because it is simple (no genome manipulation is necessary), and partly because it's so specific. Investigators can use it as an experimental tool for sequence-specific silencing and high-throughput screening: By introducing a particular dsRNA, scientists can inactivate specific mRNAs and quickly assess gene function. UK researchers have produced entire libraries of dsRNAs for Caenorhabditis elegans, and others are working on a similar library for humans. Other studies in plants, worms, and mice have shown that RNAi's silencing effects can spread throughout an entire organism during development. Now drug companies are investigating RNAi's potential clinical applications, such as a treatment for macular degeneration and other diseases, an antiviral agent, and a possible gene therapy.

--Maria W. Anderson

post Posted: Mar 26 2004, 11:49 AM
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Benitec Initiates Patent Infringement Lawsuit to Protect RNAi Gene Silencing Technology

post Posted: Mar 24 2004, 04:13 PM
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IN REPLY TO A POST BY andyc, Tue 23/03/04 08:45pm

I think BLT is unusual as such a large proportion of the shareholders (~3000 from memory?) are long term and rusted on. The bulk of the stock is simply not traded because the major shareholders are expecting big things by years end and have supported the stock for some time. I suspect they will snap up the small offerings if thin trade dropped below $1.

post Posted: Mar 23 2004, 08:45 PM
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Don't like the look of the chart at the moment... SP seems to be drifting out of the channel which began August 2002 and the failure to make a new high around 19/02/04 doesn't look encouraging... Its currently hovering around its $1.00 support level which it has been bouncing off recently ranging between $1 and around $1.15....

I traded this support a couple times but don't think it will hold this time... Should be interesting to watch nonetheless...

post Posted: Mar 4 2004, 07:10 AM
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IN REPLY TO A POST BY jarm, Thu 26/02/04 04:45pm

Further background to the science behind BLT. There are 75 companies currently involved. A royalty stream will find its way back to BLT in almost all cases.

RNAi - technologies, markets and companies


RNA interference (RNAi) or gene silencing involves the use of double stranded RNA (dsRNA). Once inside the cell, this material is processed into short 21-26 nucleotide RNAs termed siRNAs that are used in a sequence-specific manner to recognize and destroy complementary RNA. The report compares RNAi with other antisense approaches using oligonucleotides, aptamers, ribozymes, peptide nucleic acid and locked nucleic acid.

Various RNAi technologies are described, along with design and methods of manufacture of siRNA reagents. These include chemical synthesis by in vitro transcription and use of plasmid or viral vectors. Other approaches to RNAi include DNA-directed RNAi (ddRNAi) that is used to produce dsRNA inside the cell, which is cleaved into siRNA by the action of Dicer, a specific type of RNAse III. MicroRNAs are derived by processing of short hairpins that can inhibit the mRNAs. Expressed interfering RNA (eiRNA) is used to express dsRNA intracellularly from DNA plasmids.

Delivery of therapeutics to the target tissues is an important consideration. siRNAs can be delivered to cells in culture by electroporation or by transfection using plasmid or viral vectors. In vivo delivery of siRNAs can be carried out by injection into tissues or blood vessels or use of synthetic and viral vectors.

Because of its ability to silence any gene once the sequence is known, RNAi has been adopted as the research tool to discriminate gene function. After the genome of an organism is sequenced, RNAi can be designed to target every gene in the genome and target for specific phenotypes. Several methods of gene expression analysis are available and there is still need for sensitive methods of detection of gene expression as a baseline and measurement after gene silencing. RNAi microarray has been devised and can be tailored to meet the needs for high throughput screens for identifying appropriate RNAi probes. RNAi is an important method for analyzing gene function and identifying new drug targets that uses double-stranded RNA to knock down or silence specific genes. With the advent of vector-mediated siRNA delivery methods it is now possible to make transgenic animals that can silence gene expression stably. These technologies point to the usefulness of RNAi for drug discovery.

RNAi can be rationally designed to block the expression of any target gene, including genes for which traditional small molecule inhibitors cannot be found. Areas of therapeutic applications include virus infections, cancer, genetic disorders and neurological diseases. Side effects can result from unintended interaction between an siRNA compound and an unrelated host gene. If RNAi compounds are designed poorly, there is an increased chance for non-specific interaction with host genes that may cause adverse effects in the host.

The markets for RNAi are difficult to define as no RNAi-based product is in clinical development yet. The major use of RNAi reagents is in research but it partially overlaps that of drug discovery and therapeutic development. It is estimated to be $300 million currently and will increase to $400 million in 2005 and $850 million by the year 2010. The value of the drug discovery market based on RNAi can be assessed at $500 million currently with increase to $650 million in the year 2005 and further doubling to $1 billion in the year 2010. Even if a few products get into the market by the year 2010, this market will expand to $3.5 billion based on revenues from sales of RNAi-based drugs.



post Posted: Feb 26 2004, 04:45 PM
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IN REPLY TO A POST BY AgentCooper, Tue 24/02/04 09:18pm   [READ POST]

This seems to be a greatly undervalued stock. For those interested in the technology here is some background you might find useful with respect to therapeutic potential.
Source: Public Library of Science, Biology - Originally Published January 20, 2004

RNAi Therapeutics: How Likely, How Soon?

02/24/04 -- RNA interference (RNAi) has been called "one of the most has exciting discoveries in biology in the last couple decades," and since it was first recognized by Andrew Fire et al. in 1998, it has quickly become one of the most powerful and indispensable tools in the molecular biologist's toolkit. Using short double-stranded RNA (dsRNA) molecules, RNAi can selectively silence essentially any gene in the genome. It is an ancient mechanism of gene regulation, found in eukaryotes as diverse as yeast and mammals, and probably plays a central role in controlling gene expression in all eukaryotes. In the lab, RNAi is routinely used to reveal the genetic secrets of development, intracellular signaling, cancer, infection, and a full range of other phenomena. But can the phenomenon hailed by the journal Science as the "Breakthrough of the Year" in 2002 break out of the lab and lead to novel therapies as well? Pharmaceutical giants are hoping so, and several biotech companies have bet their futures on it, but not everyone is sanguine about the future of RNAi therapy.

At the heart of its promise as a powerful therapeutic drug lies the exquisite selectivity of RNAi: like the fabled "magic bullet," an RNAi sequence seeks out and destroys its target without affecting other genes. The clinical applications appear endless: any gene whose expression contributes to disease is a potential target, from viral genes to oncogenes to genes responsible for heart disease, Alzheimer's disease, diabetes, and more.

But for all its promise, RNAi therapy is a long way from entering the clinic. While it is a proven wunderkind in the lab, to date no tests have been done in humans, and only the most modest and circumscribed successes have been demonstrated in animals. The road to clinical success is littered with great ideas that have come a cropper along the way, including two other RNA-based therapies, antisense and ribozymes, both of which showed promise at the bench but have largely stumbled before reaching the bedside. Is RNAi also likely to fall short? Or is it different enough to make this third try the charm?

Clinical Naïveté, Mysterious Mechanisms

To be a successful drug, a molecule must overcome a long set of hurdles. First, it must be able to be manufactured at reasonable cost and administered safely and conveniently. Then, and even more importantly, it must be stable enough to reach its target cells before it is degraded or excreted; it must get into those cells, link up with its intracellular target, and exert its effect; and it must exert enough of an effect to improve the health of the person taking it. And, finally, it must do all this without causing significant toxic effects in either target or nontarget tissues. No matter how good a compound looks in the lab, if it fails to clear any one of these hurdles, it is useless as a drug.

For RNA-based therapies, manufacture has been seen as a soluble problem, while delivery, stability, and potency have been the most significant obstacles. "There was a lot of clinical naïveté" in the early days of antisense and ribozymes, according to Nassim Usman, Vice President for Research and Development at Sirna Therapeutics in Boulder, Colorado. "Compounds were pushed into the clinic prematurely." Sirna began as the biotech startup Ribozyme Pharmaceuticals, which tried to develop ribozymes to treat several conditions, including hepatitis C. A ribozyme is an RNA molecule whose sequence and structure allow it to cleave specific target RNA molecules (see Figure 1). "The initial results with hepatitis C were not that inspiring," says Usman, because the molecule they used had low potency and a short half-life once in the body. Despite "enormous doses," the viral load was not significantly affected. "It just didn't have the characteristics to be a drug," he says. No ribozyme has yet been approved for use by the United States Food and Drug Administration (FDA).

Similarly, despite much initial enthusiasm, attempts to develop antisense drugs have been largely disappointing. Antisense is a single strand of RNA or DNA, complementary to a target messenger RNA (mRNA) sequence; by pairing up with it, the antisense strand prevents translation of the mRNA (see Figure 2). At least that was the theory, and early clinical results seemed to support the theory: antisense drugs effectively reduced tumor sizes in anticancer trials and viral loads in antiviral trials. But closer inspection revealed these results were largely due to an increase in production of interferons by the immune system in response to high doses of the foreign RNA, rather than to specific silencing of any target genes. (The relatively high proportion of C-G sequences in antisense mimics bacterial and viral genes, thus triggering the immune response.) When the antisense dose was lowered to prevent the interferon response, the clinical benefit largely disappeared as well. Thus, rather than being a highly specific therapy, antisense seemed to be a general immune system booster.

But as long as patients were getting better, does it matter what the mechanism was? "It doesn't matter if you are a patient, but it does matter if you are trying to develop the next drug," says Cy Stein, Associate Professor of Medicine and Pharmacology at Columbia University College of Physicians and Surgeons in New York City. Stein has researched antisense for more than a decade. "If you get the mechanism wrong, you're not going to be able to do it."

To date, only one antisense drug has received FDA approval. Vitravene, from Isis Pharmaceuticals in Carlsbad, California, is used to treat cytomegalovirus infections in the eye for patients with HIV. Vitravene is actually a DNA antisense drug, which binds to viral DNA, though, says Usman, "it's unclear whether it actually works by an antisense mechanism." Stein expresses a similar skepticism about the mechanism of a second antisense drug, Genasense. Genasense is a DNA-based treatment that targets Bcl-2, a protein expressed in high levels in cancer cells, which is thought to protect them from standard chemotherapy. The FDA is currently reviewing an application for Genasense, based on promising results in the treatment of malignant melanoma.

RNAi: A Natural Alternative

Growing disillusionment with antisense and ribozymes coincided with the discovery of RNAi and the realization that it was a far more potent way to silence gene expression. RNAi uses short dsRNA molecules whose sequence matches that of the gene of interest. Once in a cell, a dsRNA molecule is cleaved into segments approximately 22 nucleotides long, called short interfering RNAs (siRNAs) (see Figure 3). siRNAs become bound to the RNA-induced silencing complex (RISC), which then also binds any matching mRNA sequence. Once this occurs, the mRNA is degraded, effectively silencing the gene from which it came. (Details of the structure and function of the RISC are still largely unknown, but it is thought to act as a true enzyme complex, requiring only one or several siRNA molecules to degrade many times that number of matching mRNAs.)

The extraordinary selectivity of RNAi, combined with its potency - in theory, only a few dsRNAs are needed per cell - quickly made it the tool of choice for functional genomics (determining what a gene product does and with what other products it interacts) and for drug target discovery and validation. By "knocking down" a gene with RNAi and determining how a cell responds, a researcher can, in the course of only a few days, develop significant insight into the function of the gene and determine whether reducing its expression is likely to be therapeutically useful. But does RNAi have a better chance to succeed as a drug than antisense or ribozymes?

"The fundamental difference favoring RNAi is that we're harnessing an endogenous, natural pathway," says Nagesh Mahanthappa, Director of Corporate Development at Alnylam Pharmaceuticals in Cambridge, Massachusetts, the second of two major biotech company developing RNAi-based therapy. The exploitation of a pre-existing mechanism, he says, is the main reason RNAi is orders of magnitude more potent than either of the other two types of RNA strategies.

Delivery, Delivery, Delivery

More potent in the test tube, at least. But stability and delivery are also the major obstacles to successful RNAi therapy, obstacles that are intrinsic to the biochemical nature of RNA itself, as well as the body's defenses against infection with foreign nucleotides. "For the strongest reasons, you can't get away from this," says Stein. "The problem is that a charged oligonucleotide will not pass through a lipid layer," which it must do in order to enter a cell. John Rossi, Director of the Department of Molecular Biology at City of Hope Hospital in Duarte, California, who has worked on RNA-based therapies for 15 years, concurs. "The cell doesn't want to take up RNA," he says, which makes evolutionary sense, since extracellular RNA usually signifies a viral infection. Injected into the bloodstream, unmodified RNA is rapidly excreted by the kidneys or degraded by enzymes.

To solve the problem of cell penetration, most researchers have either complexed the RNA with a lipid or modified the RNA's phosphate backbone to minimize its charge. Mahanthappa thinks the complexing approach is unlikely to be a simple solution, since drug approval would require independent testing of the lipid delivery system as well. Instead, Alnylam is pursuing backbone modification. "Some minimal modification is going to be necessary" to increase cell uptake and to improve stability in the blood stream, Mahanthappa says. "What we have learned from the antisense field is that even without other delivery strategies, when you administer RNA at sufficient doses, if it's stable, it gets taken up by cells."

"Anything that can be done to increase half-life in circulation would improve delivery," says Judy Lieberman, a Senior Investigator at the Center for Blood Research and Associate Professor of Pediatrics at Harvard Medical School in Cambridge, Massachusetts. But that may not be the only problem, she cautions. Lieberman's lab recently demonstrated the ability of RNAi to silence expression of the Fas gene in mice, protecting them from fulminant hepatitis. Fas triggers apoptosis, or programmed cell death, in response to a variety of cell insults. In her experiment, Lieberman delivered the RNA by high-pressure injection into the tail. The RNA got to the liver, silenced Fas, and protected the mice from hepatitis. However, a significant fraction of animals died of heart failure, brought on because the injection volume was about 20% of the mouse blood volume. Such a delivery scheme simply will not work in humans. "Delivery to the cell is still an obstacle," Lieberman explains. "Unless you really focus on how to solve that problem, you're not going to get very far."

Unanswered Questions

Even assuming delivery problems can be solved, other questions remain, including that of whether therapeutic levels of RNAi may be toxic. Mahanthappa says, "The conservative answer is we just don't know. The more aggressive answer is there's no reason to think so." Rossi isn't so sure. "The target of interest may be in normal cells as well as cancer cells," he says. "That's where you get toxicity."

But if small RNAs can be delivered to target cells efficiently and without significant toxicity, will they be effective medicines? Usman of Sirna is confident they will be. "If you can get it there, and if it's in one piece, there no doubt in our minds that it will work," he says. To date, numerous experiments in animal models suggest RNAi can downregulate a variety of target genes effectively. However, there are still two unanswered questions about whether that will translate into effective therapy.

The first is whether RNAi's exquisite specificity is really an advantage beyond the bench. "It's unclear whether highly specific drugs give you a big therapeutic effect," says Cy Stein. For instance, he says, "most active antitumor medicines have multiple mechanisms of action. The more specific you make it, the less robust the therapeutic activity is likely to be." Rossi agrees: "Overspecificity has never worked," he says.

The second question is what effect an excess of RNA from outside the cell will have on the normal function of the RISC, the complex at the heart of the RNAi mechanism. The number of RISCs in the cell is unknown, and one concern is that the amount of RNA needed to have a therapeutic effect may occupy all the available complexes. "We are usurping a natural cell system that is there for some other purpose, for knocking out endogenous gene function," says Rossi. With the introduction of foreign RNA, will the system continue to perform its normal function as well, or will it become saturated? "That's the big black box in the field," he says.

Looking Ahead to the Clinic

Despite the questions and unsolved problems, Sirna, Alnylam, and several other companies are moving ahead to develop RNAi therapy; indeed, some outstanding questions are probably only likely to be answered in the process of therapeutic development. The first applications are likely to be in cancer (targeting out-of-control oncogenes) or viral infection (targeting viral genes). To avoid some of the problems of delivery, initial trials may deliver the RNA by direct injection into the target tissue (for a tumor, for instance) or ex vivo, treating white blood cells infected with HIV, for example.

Having spent a decade trying to develop ribozymes, says Usman, Sirna is prepared for the rough road it faces. "We haven't solved all the problems, but we know how to proceed to work through them. It's no surprise to us - we've seen this movie before." Usman expects Sirna to file an Investigational New Drug Application with the FDA by the end of 2004 and to have a human clinical trial in progress in 2005. "Without a doubt, there will be an RNAi-based drug within ten years," Usman predicts.

Stein isn't so sure and thinks that too much is still to be learned about RNAi and the body's reaction to it to be confident that RNA-based therapies will ultimately be successful. "The whole field was founded on the belief it was rational, but there are huge gaps in our knowledge, and so you need a bit of luck to be successful," he says. "I think people are surprised at how complicated it is, but why should it be any other way? It's an intellectual conceit to think that nature is simple."

Further Reading

Couzin J (2002) Breakthrough of the year: Small RNAs make big splash. Science 298: 2296-2297.

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811.

Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA (1995) CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374: 546-549.

Song E, Lee SK, Wang J, Ince N, Ouyang N (2003) RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 9: 347-351.

Vitravene Study Group (2002) A randomized controlled clinical trial of intravitreous fomivirsen for treatment of newly diagnosed peripheral cytomegalovirus retinitis in patients with AIDS. Am J Ophthalmol 133: 467-474.

Written by Richard Robinson

post Posted: Feb 24 2004, 09:18 PM
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IN REPLY TO A POST BY jarm, Fri 20/02/04 05:11am   [READ POST]

Oh, oh!

Now listed on German exchanges, including Berlin exchange from next week.

Could be good for the share price once these punters get a hold of it.

Look out!


post Posted: Feb 20 2004, 05:11 AM
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IN REPLY TO A POST BY theadder, Wed 18/02/04 07:40am   [READ POST]

This group own all the meaningful patents. Their proposal to float on the NASDAQ allied with the likelihood of an exponential rise in licensing apps over the medium term should drastically affect the SP. Later direct therapeutic apps will appear and will enable an overall reduction in the background work needed to reach human trials so the future appears very bright for them. However, with a share register that is largely locked in and with only 2000 in all and 20 million locked up by institutions, a sizeable number by other major backers and general ignorance, the SP is seemingly incapable of altering sensibly on such thin local trade. NASDAQ listing will change all that.

post Posted: Feb 18 2004, 07:40 AM
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[SIZE=1]Serologicals' Research Division, Chemicon International, Inc. and Promega Corporation Enter into an Agreement for DNA-directed RNAi Product Development and Applications

full article at:


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