published 03/03/2005

It started out with an elegantly simple concept: By designing a single stranded oligonucleotide that binds to a gene’s messenger RNA (mRNA), it should be possible to inhibit its translation into protein. If the presence of that protein caused or contributed to a disease state, then eliminating it should exert a therapeutic effect. And, indeed, antisense technology does work. There’s even a drug on the market. But there are other ways to influence mRNA – especially through RNA interference (RNAi) technology, by which short pieces of double-stranded RNA, constructed to match a particular gene sequence, bind to a nuclease complex that chews up the mRNA. Different means, same end. This technology works, too – at least in animal models. Given the fact that two separate clinical trials are now underway using RNAi (also known as gene silencing), it shouldn’t be too long before we get the first hint at whether this newer technology is a winning way to produce new drugs.



Since the turn of the year, companies involved in developing RNA-based therapies – either directly or indirectly – have been flooding the newswires with one announcement after another. And a quick scan of the headlines gives one an instant appreciation of just how active the field is at the moment. By digging a little deeper, it also become obvious that companies dedicated to developing drugs that act on RNA – whether they employ antisense or interference technology – have made substantial progress in understanding just how to turn a research-based phenomenon into a clinically relevant disease treatment.

Most recently, Australian firm Benitec Ltd., which specializes in RNA interference (RNAi) technology, signed a second deal with the City of Hope to develop a radical sort of therapy for AIDS. In particular, the collaborators will explore the safety and efficacy of RNA-mediated inhibition of HIV replication in patients for whom whole highly active anti-retroviral therapy (HAART) hasn’t worked. Patients will be given their own blood stem cells that have been genetically modified via DNA-directed RNA interference to generate HIV resistance. If everything goes as planned, an RNA-based HIV drug could be in the clinic in 2006.

Alnylam Pharmaceuticals Inc. – one of the heavy hitters in the RNA interference arena – and its collaborators at the Ludwig Maximilian University Munich just published data on the mechanisms by which short interfering RNAs (siRNAs) stimulate the immune system. It’s been known for some time that long double-stranded RNA triggers a fatal interferon response in mammalian cells. As reported in the Feb. 20, 2005 online version of Nature Medicine, researchers have now identified the responsible sequences, on siRNAs, making it possible to design siRNAs that avoid interferon induction while still targeting the sequence of interest.

You might expect a deluge of news from companies that have staked their claims in RNA interference, also known as gene silencing, because it’s been one of the hottest technologies around for the last three or four years. (You can find background on gene silencing in the Signals articles, “Shoot The Messenger” and “RNAi Firms Stake Their Claims.”)

But you might not predict that the handful of older companies that built their business plans around antisense technology would have anything new to say – especially because there were several very high profile clinical failures in 2004. Chief among those was the blow up of Genta Inc.’s Genasense apoptosis-targeting cancer therapy, which was rejected by an FDA advisory panel in May as a treatment for melanoma and subsequently failed a Phase III trial in multiple myeloma.

The firm received yet another hit when its Genasense partner Aventis (part of the Sanofi-Aventis Group) pulled out of the relationship. But Genta hasn’t given up, at least not yet, and is still conducting clinical trials of the second-generation compound (in combination with chemotherapy) in other indications, including lymphoma, leukemia and solid tumors.

Isis Pharmaceuticals Inc., too, suffered a severe clinical setback last year, when its first-generation antisense drug alicaforsen failed to outperform the placebo in a Phase III trial in Crohn’s disease. Yet, the company is continuing the development of this drug candidate – because it also found that alicaforsen, which inhibits ICAM-1, worked well in three Phase II trials in ulcerative colitis, where it is delivered by way of an enema.

As well, Isis is in early clinical trials with a second-generation inhibitor of apoB-100, for treating high cholesterol, and another second generation compound, which targets protein tyrosine phosphatase, for treating Type II diabetes. And those are just the firm’s unpartnered programs. It’s also got product development alliances with (among others) Eli Lilly & Co. and OncoGenex Technologies Inc. (both in cancer).

Obviously, then, not only is antisense still alive and kicking, but also it remains an appealing technology platform for a number of recent startups and continues to attract venture capital, as detailed in the tables in this article.

Isis has actually played a big role in start-up activity. In mid-February, it licensed an anti-cancer antisense drug candidate to Sarissa Inc., a new biotech firm spun out of the University of Western Ontario, Canada. This product, which was developed via Isis’ second-generation chemistry, inhibits thymidylate synthase, a well-known drug target that protects cancer cells from the ravaging effects of various chemotherapeutic agents.

In December 2001, Isis joined forces with Circadian Technologies Ltd. to form the publicly traded Australian company Antisense Therapeutics Ltd. specifically to further the application of Isis’ antisense platform into new markets. Isis originally owned a 14 percent stake in the Australian firm and also entered into a five-year antisense drug discovery and development agreement with it.

Antisense Therapeutics is already in Phase II clinical trials with its inhibitor of VLA-4, an immune system protein that’s known to play a key role in the onset and progression of multiple sclerosis. The company also garnered an exclusive license from Murdoch Childrens Research Institute, based in Melbourne, to patents covering antisense directed against the IGF-1 receptor for dermatological indications, including psoriasis. A product candidate is now in a proof-of-concept clinical trial.

Company	Technology	Disease Focus	Programs/ Partnered	Programs/Not Partnered	Comments
Acuity Pharmaceuticals (Philadelphia, PA)	RNA interference	Ophthalmics: age-related macular degeneration and diabetic retinopathy	-----	Wet AMD; siRNA that targets VEGF	Phase I in AMD initiated 10/04; raised $2.4M in series A round in 2/04 and $15M series B round in 10/04
Aegera Therapeutics (Montreal, Canada)	Antisense	Apoptosis in oncology	-------	Second-generation antisense drug AEG35156; enhances sensitivity of cancer cells to apoptosis and chemotherapy by lowering XIAP protein levels	Phase Ib trial being conducted by Cancer Research-UK; raised US$15M in series C round in 2/04
Antisense Pharma (Regensburg, Germany)	Antisense	Malignant tumors	------	Lead product AP 12009 blocks production of TGF-beta 2	AP 12009 currently in Phase IIb trials in glioma and Phase I/II trials in pancreatic cancer and melanoma; raised $19.7M in 3rd round financing in 4/04
atugen (Berlin, Germany)	RNA interference	Metabolic diseases; epithelial cancers; ocular diseases	------	------	Early-stage discovery & research in siRNA therapeutics; also performs gene target validation for partners; raised $6.1M in 3rd round financing in 3/04

And OncoGenex Technologies, a privately held firm based in Vancouver, British Columbia, was already developing its own antisense inhibitor to the target clusterin, a cell survival protein whose expression limits the effectiveness of standard chemotherapy.

By partnering with Isis, the Canadian firm got access to Isis’ second-generation antisense chemistry. As part of the collaboration, OncoGenex is responsible for conducting the clinical trials on the clusterin compound (OGX-011) plus an additional anti-cancer compound, called OGX-225, which targets the messages for insulin-like growth factor binding proteins 2 and 5, each of which is involved in regulating growth signals.

Why did the company pick antisense technology to develop its first drug candidate? According to Scott Cormack, OncoGenex’ president and CEO, clusterin is an intracellular and extracellular protein, so the protein per se is non-druggable – making the mRNA a desirable target. And, the company chose antisense over RNAi because antisense is more advanced and provided an immediate clinical opportunity. “We did the work and filed the patents for these targets under the belief that at some point RNAi will be a good therapy,” Cormack explained. “We don’t believe at this point that RNAi is usable in systemically administered human therapeutics.”

And OncoGenex is off to a great start in the clinic. The results from the Phase I trial of OGX-011 were especially encouraging. In this study, prostate cancer patients who were candidates for a prostatectomy were given the antisense compound plus standard hormone therapy. The data showed that the antisense compound not only achieved a high concentration in its target tissue but also inhibited clusterin in prostate cancer cells from these patients by greater than 90 percent and inhibited clusterin expression in lymph nodes by greater than 95 percent.

With all those irons in the fire, Isis is bound to come up with at least one approved drug in the not-too-distant future. If so, it would probably become only the second antisense drug to make it through the regulatory approval process. The first, Isis’ Vitravene, was approved in 1998 for treating AIDS-related cytomegalovirus infection.

The fact that only one antisense drug has been approved for sale speaks volumes about the challenges faced by researchers in the field. For one thing, delivery of a nucleotide-based compound has proven tricky – it’s chewed up by enzymes in the circulation or removed by the liver and kidneys. For another, scientists had to figure out how to combat immune reactions. Plus, they needed to modify the chemical structure of the antisense compounds per se. Because the phosphodiester backbone of natural DNA didn’t stand up to bloodstream nucleases, researchers have used medicinal chemistry to alter the backbone in various ways that increase the compound’s stability in the circulation. As well, so-called second generation molecules tend to be far more potent. (For a detailed description of these dilemmas, see the Signals article, “Antisense: Poised To Strike.”)

Isis Pharmaceuticals’ second-generation chemistry.
Image courtesy of Isis Pharmaceuticals.

According to Stanley Crooke, Isis’ chairman and CEO, “After 15 years and around $2 billion invested in RNA-based drug discovery, we are at the end of the beginning. We’ve answered all the basic questions to know whether RNA-based drug discovery would have value.” For instance, “We needed to understand where the drugs go, how they get there and how they are cleared. We had to invent medicinal chemistry for oligonucleotides. We needed to understand the mechanisms by which they worked and how they behave as drugs,” Crooke explained.

And, even though there have been failures, “it’s a given that we would have drugs fail,” he added. But those failures were not due to toxicity: “We’ve treated about 4,000 patients and we’ve never yet run into dose limiting toxicity, even with the first generation drugs,” Crooke said.

All in all, he said, “The progress of the technology has been pretty linear. What’s fluctuated wildly is peoples’ opinion of the technology.” Indeed, antisense technology has fallen in and out of favor a number of times over the last 15 years. That’s not the case with RNA interference technology, though – at least not yet. It’s still wildly popular – and several products are already in the clinic.


Obviously, drug candidates that are based on gene silencing technology are similar enough to antisense compounds that one might predict they would face similar challenges on the long road from bench research to clinical trials in humans. And they have – but then, the RNAi companies had the advantage of learning from the history of antisense drug development.

Indeed, these days antisense and RNAi companies are often joining forces – either directly through R&D collaborations or indirectly by licensing or cross-licensing patents. Here again, Isis plays an important role: It owns or exclusively licenses more than 1,400 issued patents worldwide that cover many aspects of RNA-based drug discovery and development. And that includes various chemistries -- the reason that Isis licensed several patents to Eyetech Pharmaceuticals Inc. for the development of Macugen, which is an aptamer (a non-antisense short synthetic oligonucleotide). Isis’ IP also covers RNA interference – which explains why Isis has partnered with Alnylam Pharmaceuticals to develop and commercialize RNAi therapeutics.

But Alnylam’s patent estate also covers antisense motifs and mechanisms and oligonucleotide chemistry, so Isis gained nonexclusive licenses to Alnylam’s relevant patents. Thus, both parties strengthened their positions in their respective fields and will also share in each other’s success.

Alnylam’s also forged numerous alliances focused on specific product opportunities. It’s got an agreement with Merck & Co. Inc. to develop RNAi therapeutics for spinal cord injury and a separate agreement with Merck that covers wet age-related macular degeneration.

The RNA interference process.
Image courtesy of Alnylam Pharmaceuticals.

This project actually represents Alnylam’s most advanced program – a chemically modified RNAi therapeutic that targets vascular endothelial growth factor (VEGF). It’s got other programs in Parkinson’s disease (a collaboration with the Mayo Clinic) and respiratory syncytial virus infection – an unpartnered program that the firm intends to get into the clinic by mid-2006. All three of those programs will deliver the drug directly – to the eye, the brain and the lung, respectively. In its most recent deal, Alnylam joined forces with Medtronic Inc. to develop drug-device combinations for use in neurodegenerative disorders. “This takes RNAi technology to a new frontier,” said John Maraganore, Alnylam’s president and CEO.

Systemic administration of an RNAi therapeutic in humans has yet to come – although company researchers have demonstrated its feasibility through in vivo silencing of the gene for apolipoprotein B in animal models in a paper published in Nature in November 2004.

Alnylam has chosen to chemically modify its siRNAs to increase their stability and enhance their ability to resist nuclease degradation. According to Maraganore, “siRNA is rapidly degraded in the blood and tissues if it’s not chemically modified.” The particular modifications vary depending on the route of administration, too: “For systemic delivery, there is additional chemistry to achieve delivery into cells,” he explained. The company’s achieved this in its mouse model system, but is still “optimizing the technology.”

When choosing siRNAs to be used as therapeutics, it’s also important to identify those that are the most biologically active, Maraganore said. However, “We don’t yet know how to predict the most potent sequences in silico.” Therefore, company researchers “start with a gene of interest (one that has good biological validation) and then use bioinformatics to get down to 50-200 sequences,” added Barry Greene, Alnylam’s COO. After that, the scientists actually construct these 50-200 sequences and test them empirically to pick the most potent, he said. Only then do they engineer in the various chemical modifications.

Thus, in about a year Alnylam researchers have deduced how to pick a potent sequence, how to stabilize it, and how to get it into cells. That’s much quicker than small molecule development programs, which can take 2-4 years to get a compound worthy of animal studies, Greene said. But then, “Progress has been remarkably fast in the whole field” of RNA interference, Maraganore added.


Indeed it has. In fact, two companies have already started clinical trials with sRNAs – and both have gone after the same indication. Privately held Acuity Pharmaceuticals holds the honor of being the very first company to take an siRNA-based therapeutic into human clinical trials. In October 2004, Acuity began testing its lead compound Cand5, which targets VEGF mRNA, in patients with wet age-related macular degeneration (AMD). (VEGF promotes the growth of blood vessels and leakage that lead to vision loss in this disease.) And the firm managed to accomplish this feat less than two years after its founding.

Because a single siRNA drug molecule is able to stop the production of hundreds or even thousands of VEGF protein molecules, Acuity believes that it will prove to be a very potent therapy. As well, the eye is the perfect place to test an siRNA-based drug – especially because it’s an enclosed system, so delivery (via intravitreal injection) is localized and the probability of systemic toxicity is practically nil. As well, “the back of the eye has retinal epithelial pigment cells that take up the drug, after which RNA interference is activated,” explained Dale Pfost, Acuity’s president and CEO. And, while the primary endpoint of this Phase I trial is safety, the company hopes to gain some insight into efficacy. It will be some time before the results of this early trial are known, however, probably towards the end of the year, Pfost said.

Less than two months after Acuity started its clinical trial, Sirna Therapeutics Inc. dosed its first patient in a Phase I trial of its RNAi therapeutic Sirna-027. Like Acuity, Sirna targeted wet AMD for its first indication. But Sirna’s drug candidate is chemically modified (which Acuity’s is not) and it targets the VEGF receptor rather than VEGF per se.

Sirna and Acuity aren’t the only firms developing drugs for AMD – which has become a very attractive product opportunity for companies large and small. The competition is fierce for new therapies, and the first to make it to market was Macugen, an aptamer that also targets VEGF, developed by Eyetech Pharmaceuticals in partnership with Pfizer Inc. (For more information on therapies in development for back-of-the-eye diseases, see the Signals article, “The Eyes Have It.”)

Private Antisense And RNAi Companies
That Raised Venture Capital in 2004

Company	Technology	Disease Focus	Programs/ Partnered	Programs/Not Partnered	Comments
Divergence (St. Louis, MO)	RNAi-based functional genomics	Parasite-specific molecular targets; novel nematocidal chemistries (agricultural applications)	-----	-----	Raised $4.1M in series B round in 1/04
Genable Technologies (Dublin, Ireland)	RNA interference	Gene based therapies for inherited diseases, especially dominant negative diseases such as retinitis pigmentosa	-----	-----	Start-up; raised $1.3M in first round in 9/04
InDex Pharmaceuticals (Stockholm, Sweden)	Antisense	Drugs and diagnostics for inflammation and cancer	Ulcerative colitis drug candidate Kappaproct (antisense inhibitor of p65 protein) partnered with Serono (2/04)	------	Phase II trial results could not confirm that a single dose of Kappaproct was able to induce remission; partners intend to pursue development (2/05); raised $4.7M in 4th round financing in 5/04
Nucleonics (Horsham, PA)	RNA interference	Viral diseases HBV, HCV & HIV	------	------	Plans to file INDs for HBV and HCV in 2005; raised $49.2M in series B round in 4/04-6/04

While Acuity plans to limit its product development programs to ocular diseases (including diabetic retinopathy), Sirna has spread its net further. It’s inked an alliance with Eli Lilly in oncology and another with Targeted Genetics Corp. in Huntington’s disease. In this latter collaboration, the partners intend to use Targeted Genetics’ adeno-associated viral vectors for the local delivery of siRNAs targeting Huntington’s protein.

Sirna’s also developing putative drugs for asthma – targeting Th2 cytokines via local delivery to the airways -- and diabetes – by silencing the expression of phosphatase 1B via systemic delivery -- in-house, and expects to file INDs in 2006 and 2007, respectively.

Plus, in the most unusual application of RNA interference technology to date, Sirna has jumped into the hair removal game through its December 2004 acquisition of Skinetics Biosciences Inc. Sirna Dermatology has targeted a critical gene essential for hair growth and has demonstrated that reduction of this gene's expression by a topically administered chemically modified siRNA permanently disrupts hair follicle integrity in an animal model. The company anticipates initiating human trials in 2006.

“It’s important to understand that ribozymes and antisense were early attempts at what has become RNA interference,” explained Martin Schmieg, Sirna’s SVP and CFO. “They laid the groundwork, so to speak.” That’s certainly true at Sirna: The company got its start as Ribozyme Pharmaceuticals Inc. and consequently has amassed a wealth of knowledge and skills appropriate to working with RNA-based drugs.

However, “ribozymes, antisense and siRNAs are three different beasts,” added Barry Polisky, Sirna’s SVP of research. “All operate by different mechanisms.” The advantage of siRNA is that it “engages a natural highly stable intracellular machine that cells use for their own purposes.” In the end, then, “siRNA really is the gene silencing approach of choice.”

“We feel there are four challenges to making siRNAs into therapeutics,” Schmieg said. These include serum stability; circulation time; target specificity and cellular activity. “We believe that with our chemistry knowledge and our ability to modify siRNA, we have accomplished all four.”

Time will tell. The results of Sirna’s first clinical trial – in patients with wet AMD – should be available by the third quarter of 2005. That’s about the same time that Acuity plans to report its early findings – and between the two of them, we may get our first glimpse as to how straightforward the path will be for RNAi-based therapeutics going forward.