RXi Reports on Dicer-insensitive 25bp RNAi Triggers

There is little question that Alnylam enjoys considerable control over arguably the most desirable type of synthetic RNAi triggers: 19-21 base-pair dsRNAs with 3’ overhangs- some of this control, of course, being somewhat subject to the outcome of the Tuschl Tussle and the review of the Tuschl II patent in Europe. In any case, this had caused a number of competitors to try and avoid this IP estate by designing around these Tuschl-type siRNAs. One of the more popular workaround designs are (blunt-ended) 25 base-pair dsRNAs, being developed by the likes of RXi, Intradigm (now part of Silence Therapeutics), and Invitrogen (RXi has a deal with Invitrogen, most likely covering aspects of RXi’s 25mers). RXi now presents data (Salomon et al.: Modified dsRNAs that are not processed by Dicer maintain potency and are incorporated into the RISC) on the performance of these 25mer siRNAs that are consistent with the notion that it is possible to find highly potent siRNAs with such structures, and that, interestingly, modified 25bp duplexes may often not be subject to Dicer processing before being loaded into the RISC effector complex.

One concern with the use of 25mers is that, yes, they, like a lot of other structures can harness the remarkably robust RNAi gene silencing pathway, but is its potency sufficient for its therapeutic translation? Because RNAi triggers have to satisfy a few other requirements besides potency, as a platform, it is not sufficient to find one or two highly potent sequences, but maybe 5-10 to choose from. The importance of such choice arises from the need to minimize off-targeting and immunostimulatory potential, related to this the ability to modify a siRNA without compromising too much activity, and finding sequences that are conserved in pre-clinical animal models etc.

Sufficient potency to me means 50% knockdown (aka IC50) values of at least 300pM in standard tissue culture experiments (Alnylam’s siRNA in TTR01 e.g. has a single-digit pM potency). Salomon and colleagues provide examples of 2 siRNAs with IC50s of 50-150pM and add, but without showing the actual data, that they ‘have identified duplexes with potency in the picomolar range for over 10 genes and at multiple sites per gene’. So while I am convinced that highly potent 25mer siRNAs can be discovered, the question about the ease of finding those remains an open one. Support that they may not be that rare comes from the gene-specific 25mer siRNA patents filed by Intradigm/Silence Therapeutics that show silencing results from relatively detailed siRNA tiling assays.

It should be possible to increase the pool of potent 25mers by applying design rules like those by Zamore and controlled by Silence Therapeutics. Salomon and colleagues for example report that some of the modifications in RXi's particular 25mer design (unmodified guide strand, 4 5' and 3' terminal 2'-O-methyls in the passenger) affected the unwinding and loading of the passenger strand which may therefore be subject to the Zamore end-stability claims. Moreover, the value of the 3’ terminal mismatches of the guide strand for enhancing RISC turnover, as covered under the first issued Zamore patent, may be particularly high for long guide strands (25mers, see below) because such extended base-pair complementarity (=thermodynamic stability) could negatively impact RISC detachment from the cleaved message and therefore the efficiency with which it can be recycled for the next round of target cleavage. In that sense, Intradigm may have been quite clever in realizing the synergy of the 3' terminal mismatches particularly for the long duplexes as practiced by the company.

Another interesting aspect of the study, also relevant for IP purposes, is that the 25mers were loaded onto RISC without prior Dicer processing. 25mers may have been expected to be subject to Dicer processing to generate Tuschl-type siRNAs. Although this particular legal theory remains to be tested, this may not be the ideal patent for the 25mer companies because Alnylam may argue that Dicer substrates just serve as pro-drugs to the Tuschl-type siRNAs and therefore would require a license. Moreover, if they were indeed processed by Dicer, they might conflict with Dicerna which specializes on Dicer-substrate siRNAs (Dicerna typically uses 27mers). It is therefore of interest that this publication demonstrates quite convincingly that 25base-pair duplexes, whether modified or not, are not processed by Dicer prior to loading into cleavage-competent RISC. Accordingly, the 25mers may well steer clear from infringing either Alnylam’s Tuschl-type siRNAs or Dicerna's Dicer substrates.

As I stated, Alnylam’s RNAi trigger estate is scientifically the most desirable in a number of regards. Extending the length of the dsRNA for example creates additional challenges as it relates to innate immune stimulation, and reduced delivery efficiency for certain siRNA-conjugate delivery approaches. On the other hand, it is certainly possible to find therapeutic candidates for workaround designs like the 25mers and the judicial application of design rules may address some of the disadvantages in the efficiency of finding potent duplexes.

As Alnylam’s Chinese Wall in RNAi trigger is being tested and faces key challenges later this year, both in the US (Tuschl Tussle) and in Europe (oral hearing on T-II at the EPO following challenges by the likes of Merck and Silence), it is delivery more than ever that will be key to Alnylam in strengthening its position in deal negotiations. In particular, assuming that Big Pharma cannot afford to wait forever before making their moves in RNAi Therapeutics, SNALP delivery should continue to be their key for commanding the next $100M+ licensing terms. I'm not really sure what is holding them up as the present situation must have been predictable for the company and I still feel that a move coordinated with Tekmira and one Big Pharma company could be the solution.

PS: While this paper has only come to my attention this week via my pubmed alerts, some of the data contained therein has been previously presented by RXi at scientific conferences. Moreover, whereas the paper appeared in the June edition of NAR, it first appeared online already in early 2010.

Afterword

When I started this blog, it was initially from the viewpoint of an academic scientist with a keen interest in RNAi drug development. As such I had a strong bias towards patents built on ground-breaking scientific discoveries and somewhat sneered at workaround technologies. As the years have passed, I have however come to the realize that while an academic scientist might be embarrassed to report to peers that a workaround may come close to the real thing, workarounds are a well accepted, even encouraged business practice and Big Pharma will take this into account in their RNAi trigger deal-making. Moreover, because publishing cutting-edge research as quickly as possible is the objective of academic research, in many cases, despite the ‘more worthy’ nature of such science, it has often proven difficult to build strong patents on many of these discoveries (issues such a pre-mature disclosures etc). Biotech companies, on the other hand, can conduct research in a way to maximize its IP value.

Silence Therapeutics Issued Important Zamore siRNA Design Rule Patent

Silence Therapeutics announced yesterday the issuance of a United States patent related to the design of gene silencing siRNAs. This is the second such patent issuance in little more than a month for which Intradigm, Silence’s merger partner earlier this year, had exclusively licensed from the University of Massachusetts and that is based on fundamental work by the Zamore lab on the enzymology of RNAi. These and other recent patent issuances related to sequence/gene-specific siRNAs address some of my concerns that I had with regard to Silence’s RNAi trigger strategy that I felt was at the risk of being too narrow and, as a consequence, becoming outdated. They should complement Old Silence's lipoplex delivery technology for vascular endothelia with valuable siRNAs in the fields of oncology and retinal disorders. Together, these developments provide evidence that the merger is starting to prove synergistic, with Silence providing the technical know-how and Intradigm complementary IP.

The two Zamore patents that were recently issued in the US relate to guidelines on how to design siRNAs with increased efficacy and selectivity. The first one, announced last month (US patent 7732593) teaches the use of mismatches between the 3’ end of the siRNA guide strand with the target mRNA so as to enhance the ability of the RNAi gene silencing complex RISC to detach from a target mRNA once cleaved and seek out new target mRNAs (note: one of the reasons RNAi is so potent is because one siRNA can destroy many mRNAs). The biochemical experiments on which these claims are based have well stood the test of time. The value of this particular patent for Silence Therapeutics is that it gives them and potential partners optionality for eventually replacing the somewhat restrictive Atu-siRNA design with next-generation siRNA structures based on solid scientific evidence. It has to be said, however, that while many current siRNAs have in fact two such mismatches, or ‘non Watson-Crick base pairs’, namely the classical Tuschl dTdT overhang that was originally conceived as stabilizing the siRNA from exonucleolytic degradation, the claims specify at least three such mismatches in the last five nucleotides of the guide strand. Moreover, the benefit of such mismatches in living cells remains to be determined, as some of the helicases and nucleases that may facilitate RISC detachment from a cleaved target mRNA in living cells may have been missing in the test tube experiments by the Zamore lab.

So while US7732592 is certainly a quite useful patent to have control over, it is the Zamore patent US7750144 of which the issuance was announced yesterday (‘Methods and Compositions for Enhancing Efficacy and Specificity of RNA Silencing’) that should provide for some interesting discussions. This patent is based on one of the classic findings in RNAi molecular biology, namely that the efficacy and selectivity of small duplex RNAs, both siRNAs and miRNAs, is critically determined by the relative thermodynamic stabilities of the two ends of an siRNA (Schwarz et al., 2003: Asymmetry in the assembly of the RNAi enzyme complex). Accordingly, it is the strand of which the 5’ end is less stably base-paired that is preferentially loaded into the RISC gene silencinng complex to become the guide strand, while the non-incorporated strand is discarded as the 'passenger strand'.

This has a few implications for siRNA design. First of all, it increases the absolute loading and consequently silencing efficacy of an siRNA. Secondly, by preferentially loading just the desired strand to become the guide, the potential undesired/off-target activity of the passenger strand can be virtually eliminated. These asymmetry rules have also been extensively validated in living cells and are part and parcel of most siRNA design strategies and algorithms. They are also consistent with structural X-ray crystallographic findings that show that the 5’ end of a guide strand within RISC is unpaired. As a result, it is very likely that a number of siRNA therapeutic candidates currently in the clinic could be interpreted to fall within the scope of this patent. This should also encourage some of the research & reagent companies that sell synthetic siRNAs to take a license from Silence Therapeutics.

The actual issued claims relate to a method of decreasing passenger strand activity by introducing one or more changes in the interaction between the 5’ end of the guide strand with the passenger strand such that the loading of the guide strand is enhanced. The broadest interpretation, and the one that I would expect Silence will take, is that any siRNA containing such a modified nucleotide or mismatch in the specified region (the first 5 nucleotides of a guide strand) would infringe. Most siRNAs should fall within this definition. In some cases, such modifications may have in fact been motivated by other things like stabilizing an siRNA against degradation or reducing innate immune responses, but this remains to be tested, unless the companies involved choose to settle without resorting to yet more exhausting and extremely tedious legal battles. I should note here that when one follows the patent prosecutions especially in Europe, there does not seem to be a single patent that gets issued that will not be opposed by the other party, no matter the scientific merits (my personal opinion). It is enough that some in Big Pharma routinely abuse the patent system to bully smaller competitors into bankruptcy, essentially a way of doing business, and pure-play RNAi Therapeutics companies should be smart enough to know that by adopting these practices they only destroy the size of the shared pie they all depend on: patent-protected innovation. But I digress...

In summary, the recent events have lessened my concerns that it was largely Intradigm that was the beneficiary of the merger as their historical focus on IP is now bearing fruit in the form of some quite fundamental patent issuances and should nicely complement Silence’s strength in the science and clinical translation of RNAi which I had considered to be the stronger of the two. So while I still await data that similarly support the company’s claim that the merger also brought synergies in siRNA delivery, the combined company appears to be quite undervalued here with a market cap of around $20M. The patent issuances should also bode well for the extension of the RNAi trigger-focused part of Silence’s relationship with AstraZeneca. Because of this and because the markets appear to be on summer vacation with the stock virtually unchanged following such strategically important patent news for the company, I’ve decided to buy a few shares of Silence here.

PS: For the various reasons that you can also find in Silence’s regulatory filings, investments in RNAi Therapeutics in general and Silence Therapeutics in particular have to be considered very high risk. Moreover, although I consider myself fairly familiar with the molecular biology of RNAi, I am not trained in intellectual property and my interpretations of the Zamore patents have to be read in this light. Please also read the disclaimer at the bottom of the page.

Silence Therapeutics and Intradigm Merge to Offer Complete RNAi Therapeutics Package

Finally, the long wait has come to an end. The just announced imminent merger of publicly traded Silence Therapeutics (UK/Germany) with privately held Intradigm (USA) illustrates that consolidation has also arrived in RNA(i) Therapeutics. The stated objective is for the financially strapped firms to achieve a size sufficient to be considered a one-stop partner for a Big Pharma/Biotech (BP/P from now on) interested in RNAi Therapeutics, and also partly in a bid to enlarge their investor base. This approach is in part inspired by the successes of Alnylam and Sirna Therapeutics which had and have been very successful in monetizing their broad approach towards RNAi Therapeutics. While there are factors speaking for such an approach, times have somewhat changed in RNAi Therapeutics such that the established players such as the Roches and Novartis' are more likely to take a pick-and-choose approach in their partnering strategies. As such, companies that are focused on fewer technologies, especially in delivery, but then can get it right, may be preferred partners for these BP/Bs.

On the other hand, for a BP/B that views a platform partnership as a way to start testing the RNAi Therapeutics waters somewhat more extensively, I am thinking here of the likes of AstraZeneca, Boehringer Ingelheim and Aventis, an enlarged Silence Therapeutics may be worth a second look at least as a cheaper alternative to the gold-standard Alnylam Pharmaceuticals. The risk, however, that I see is that ‘broad’ could easily equal ‘lack of depth’.

As much as the desire to grow in size, the merger was driven by the desperate need of both companies. It is therefore highly likely that the merger will be approved as described in the Admission Document, also because of the holidays which makes it unlikely for a third party to make a too-good-to-refuse buy-out offer. Net of debt and after parallel fund raisings from existing Intradigm shareholders (about £5M) and a placing led by Nomura (about £9M), the combined company should soon have ~$20M in liquid assets, which will give the company about a year’s time to find a platform partner that is willing to part with some decent upfront cash. Especially with part of their operations now based in the US, it may also make sense to pursue a listing on the US markets for their future cash needs.

While the claim is to be a broadly positioned RNAi Therapeutics company, the combined company clearly has a strong bias towards cancer. Therefore, an alternative headline of this blog post would have been ‘Silence Therapeutics and Intradigm to Form Cancer RNAi Therapeutics Powerhouse’. It is here that I suspect most of the scientific synergies and value drivers of this merger will come from. I do not have to describe here the high unmet medical needs in cancer, and it is the unique promise of RNAi Therapeutics to go after the many thus far undruggable, yet well-validated cancer-related targets that makes it an area of such high value proposition to the industry. Since nanoparticles can be targeted to cancers, delivery should be possible: e.g. via the tumor vasculature, the Achilles heel of solid cancers, and antibodies to haematological malignancies. In this regard, Silence Therapeutics and Intradigm bring to the table cationic lipoplexes as a means to knock down genes in the tumor vasculature directly (Silence Therapeutics), also as supported by the literature, and a less validated cationic peptide polymer-based delivery technology by Intradigm ('PolyTran').

But will the proposed merger address the one area next to viable delivery that has been nagging both companies in the past...intellectual property? Clearly, combining two comparably weak patent estates do not a strong one make. A quick glance over what is issued and pending, however, suggests that the patent estates could synergize to a certain degree.

Silence (as most others) has been mostly troubled by the Tuschl and Kreutzer-Limmer patents that make it very difficult for them to find dsRNA lengths to exploit their issued Atu-siRNA design pattern without having to to get a license from Alnylam: blunt-ended dsRNAs with alternate 2’-O-methyl modified RNAs. Atu-027 for example, the combined company’s lead candidate for solid cancers that has entered phase I studies this summer, is a 23bp dsRNA and Tuschl I should be a formidable challenge here. Intradigm, like RXi/Invitrogen’s non-Dicer dsRNAs before (rxRNA solo/Stealth), chose to minimize Tuschl-KL problems by opting for a 25bp blunt-end design, and Silence’s alternating 2’-O-methylation pattern IP might give those a proprietary touch. As I cannot find any evidence that Intradigm currently has any access to 25bp structure-specific patents aside from Fire-Mello, I wonder how extensive the siRNA design IP access to the Zamore (University of Massachusetts) patent estate really is. Zamore’s seminal discoveries centered around the thermodynamic properties of effective siRNA, and if added to the 25bp dsRNAs could further endow those with a proprietary look. One issued patent relates to the stability of the guide RNA 3’ end with the target mRNA and which has implications for RNAi catalytic turnover. This one, however, would serve more the purpose of a design-around fig-leaf, and not necessarily represent a means to exclude others. The more important discovery by Zamore, however, is related to differential end stabilities of siRNA duplexes themselves, a basic siRNA rule that essentially everybody in the field follows. If Intradigm had indeed access to such IP, it could give them some leverage over other companies [maybe somebody reading this can provide me/the blog with more information on the extent of the Intradigm-Zamore relationship].

An area where there should be synergies is a broader gene target base in oncology. I had been wary of Silence’s focus on the PI3K pathway which, despite the well-known importance of this pathway in cancer biology, appears to be too closely connected to the history of the R&D staff for comfort and may have caused them to oversee more promising targets. On the other hand, Intradigm has been pursuing a large list of 50 largely cancer-related gene targets, and has obtained a sequence-specific patent on one of those which should give the enlarged work-force plenty to work on. Despite all these genes, after burning through almost $40M and noise around their initial VEGF-directed lead candidate subsided, Intradigm alone would appear to be some a long time from the clinic. With Silence Therapeutics’ Atu-027 and lipoplexes, the new company can boast at least boast one candidate undergoing clinical evaluation and possibly more to come.

In summary, given the bleak alternatives, the merger makes sense. For the field of RNAi Therapeutics, it may also be good to have companies with sufficient size to attract the attention of investors. Long gone are the days that the Alnylam-Sirna rivalry kept attention levels high. There is, however, a lot to be sorted out for the new company before it can gain the hoped-for traction.

RNAi Therapeutics: Qiagen High-Throughput RNAi User Meeting San Francisco (Part 2 of 2)

After the coffee break, John Hogenesch illustrated how RNAi has revolutionized our concept of mammalian genetics. Before RNAi, researchers essentially studied two expression states of a gene: when it was fully expressed (+/+), or when both alleles had either lost their function or weren’t expressed at all (-/-), and rarely the heterozygous state (+/-). RNAi, however, now allows the functional output of a gene to be measured in virtually all the states in between, depending on the RNAi dosage used.

Hogenesch’s systems biology team is interested in how the circadian rhythm is genetically wired that that it can be robust and yet adaptable, and consequently set up a beautiful system whereby the activity of master transcription factors of this genetic program could be measured in real-time over days based on the luminescence generated by binding of the transcription factors to a reporter gene. They then knocked down known regulators of this genetic circuit to varying degrees by titrating the siRNA amount and then assessed the correlation between the degrees of knock down and functional outputs. Pleasantly surprising not only Hogenesch, but also all the researchers worrying about how the degree of knockdown affects the phenotype they are seeing in RNAi experiments, in many cases the functional output appeared to almost directly correlate with the remaining expression level of a gene after knockdown.

However, in cases where there exist paralogues, that is highly related genes that have largely retained overlapping functions, the knockdown of one gene is often compensated almost entirely by the increased expression of the corresponding paralogue. You probably may want to avoid targeting these genes with an RNAi Therapeutics, or any other therapeutic for that matter. Finally, there were also cases where the knockdown gave non-linear responses, and this is mostly when enzymatic reactions were affected.

Nevertheless, as everybody in the audience having traveled from across the country for this one-day meeting knew, genetic circuits, long-term, are usually quite robust and can adjust to external fluctuations. It is therefore also of interest to RNAi Therapeutics (and then again, all classes of drugs, too), particularly for chronic applications, that out of the ~4400 gene knockouts in mice that have been generated so far, only 1000 are lethal in the homozygous state, and just 53 in their heterozygous state, and these already are probably biased numbers (scientists like to study genes with apparent phenotypes, lethality being a very obvious one). This has implications both for chronic drug treatment in general (drug effect may diminish) as well as when we worry about adverse consequences of off-targeting (off-targeting may be tolerated better than expected).

Next up was Loren Miraglia from the Genomics Institute of the Novartis Research Foundation (GNF, San Diego), a basic research institute with preclinical capabilities to feed into the Novartis drug development pipeline. The main part of the presentation focused on the technical aspects of establishing a lentiviral RNAi library for the investigation of difficult-to-transfect cells. The upshot was that these libraries once established can be very powerful, however it takes a considerable infrastructure to realize that goal.

There were two pieces of information that may give some quite interesting insights into Novartis’ RNAi Therapeutics plans. One was an overview of the RNAi libraries being used by GNF which essentially all somewhat disappointingly were focused on the “druggable genome”, disappointing because a lot of genes may be missed that could be addressed by RNAi Therapeutics. There was one notable exception, however, and that was a library devoted to genes involved in cancer. As the time approaches for Novartis to make their move on Alnylam, this confirms my belief that cancer will be an important part of the new license agreement. With screening efforts such as these, there should be more than enough targets to keep all the Alnylam licensees busy without having them compete too much with each other.

The second insight was Miraglia’s presentation of a new “gene X” that when mutated had been found to lead to an increase in LDL-R, the new star in the hypercholesterolemia field. When tested whether RNAi knockdown would also elevate LDL-R, this was indeed the case with a nice correlation between degree of target knockdown and LDL-R elevation and subsequent cholesterol lowering, just what you would like to see in an RNAi Therapeutic. So, like for Takeda, cancer may be well joined by metabolic disease as one of the main initial therapeutic fields for Novartis’ RNAi Therapeutics efforts and driven by current delivery capabilities.

Systems biologist Sumit Chanda (Burnham Insititute, La Jolla) praised RNAi screening as an important tool to learn more about the vast part of the genome that is essentially left unexplored. He bemoaned the fact that it is paradoxically genes of which so much is already known about, think of p53 and TNF-alpha, that get the bulk of the research funding.

Chanda’s laboratory used RNAi screening to discover host factors involved in HIV replication, which could therefore also be potential targets for drug intervention. As a virologist/molecular biologist myself, I also see targeting host factors as a very promising approach for treating viral infection. Unfortunately for Chanda’s group, another group from Harvard just published the results of a similar RNAi library screen, meaning that they were “scooped”. Nevertheless, it appeared that there was only limited overlap between the results and one could explain this by the slightly different assay conditions employed or the quality of each screen. In any case, the point was well made that one screen will never answer all the questions and should be complemented by additional ones. Moreover, the importance for multiple negative controls and multiple redundant siRNAs towards a single gene (to confirm the sequence-specificity of the hit) was emphasized.

Natasha Caplen from the National Cancer Institute (NCI; Rockville, MD) picked up the personalized medicine theme for cancer therapy. As noted before, while monoclonal antibodies have changed the way cancer is treated today, it is often only few patients that respond to them. Moreover, many of the drugs’ mechanism of action converge on the few same signaling pathways, meaning that the mechanisms of actions are still limited and prone to mutational escape. RNAi Therapeutics could then either play a role in exploiting new mechanisms of actions or for sensitizing towards existing drugs. Caplen’s group chose the latter approach and focused on devising new strategies for the use of the bacterial L-Asparaginase enzyme (L-ASP) that has been used to selectively starve acute lymphoblastic leukemia (ALL) cells. One problem associated with L-ASP, however, has been the quite variable response to this treatment.

Caplen’s group suspected that cancers that do not respond well to L-ASP treatment may overproduce the human asparagine synthetase (ASNS) to compensate for asparagine deprivation by L-ASP. To test their hypothesis they first screened a library of cancer cell lines for L-ASP responsiveness and, indeed, L-ASP negatively correlated with ASNS expression levels. To demonstrate a causal relationship, they then knocked down ASNS which greatly increased the sensitivity of the cells to L-ASP. The sensitization was so unbelievable, up to 500-fold, that Natasha Caplen made her co-worker repeat the experiment again and again to convince her of the veracity of the results.

ASNS expression levels could now be used to select patients for L-ASP treatment. Alternatively, ASNS knockdown may be a viable way to increase L-ASP responsiveness. It more and more appears therefore that cancer drug sensitization, and not just targeting novel oncogenes appears to be a very promising avenue for future cancer RNAi Therapeutics. Similar patient selection and drug sensitization strategies were subsequently discussed for the topoisomerase inhibitor camptothecin (Caplen) and the Wnt signaling pathway by Paul Kassner (Amgen).

Finally, I’d like to summarize the talk given by Xiao-Dong Yang from Intradigm (Palo Alto, CA) on the delivery of RNAi Therapeutics to cancer. Using siRNAs provided by Qiagen, it appears that Intradigm screens for siRNAs essentially based on potency. I am somewhat skeptical whether this is sufficient and consequently raises some questions as to their findings that were largely centered on targeting the VEGF pathway in mouse cancer models.

Intradigm’s RNAi Nanoplexes consist of a cationic polymer to which the siRNA is complexed, a hydrophilic steric polymer such as PEG, and optionally a cell targeting ligand such as an RGD peptide. The Polytran polymer-based system is particularly interesting as it is a polypeptide composed of branched histidine and lysine residues. While the positively charged lysine condenses the siRNA, histidine only becomes positively charged once internalized into the endosome as they suck up the incoming protons. This supposedly leads to an increase in osmotic pressure causing the rupture of the endosomes to release the siRNA into the cytoplasm.

The Polytran system consists of fairly uniform 100nm particles that are slightly positively charged. Like many RNAi nanoparticle systems, of the Nanoplexes that make it into a tissue following administration, many are either found in the liver, the spleen, or tumor tissue. Many of them are also taken up by phagocytic cells such as macrophages which may pose challenges with dose prediction and requires that siRNAs are carefully characterized as to their potential to activate the innate immune system. Unfortunately, this aspect of their work was little discussed although unmodified siRNAs, prone to elicit innate immune responses and could therefore cause non-specific anti-tumorigenic effects, were used. With this in mind and the fact that only one control siRNA sequence was used, quite potent mouse antitumor effects were found at low mg/kg dosages with apparently knockdown efficiencies of up to over 90%.

While these approaches are valid for the development of an RNAi delivery system, the presentation reminded me of the fact that for companies like Intradigm it would really make sense to collaborate on the siRNA chemistry side with one of the established RNAi operations rather than committing insufficiently characterized siRNAs into the clinic.

On the way home, I was once again amazed by the fact that RNAi has emerged as the only gene knockdown technology that can be applied in high-throughput to explore the depths of our genomes. This both speaks to the potency and specificity of RNAi as well as the ability to translate our understanding of how RNAi silencing works on a molecular level into bioinformatic tools to separate the wheat from the chaff, as Sumit Chanda put it.

Thanks a lot to Qiagen for organizing a day full of excellent scientific talks in a pleasant Mission Bay setting, and I’d recommend everybody running qPCRs Qiagen’s robust and reliable SYBR Green QuantiTect reagents.

Alnylam Progresses RSV RNAi Program into Phase II Clinical Studies

Alnylam Pharmaceuticals, arguably the leading company in RNAi Therapeutics, today announced the start of phase II clinical studies for ALN-RSV01, an siRNA for the treatment of Respiratory Syncytial Virus (RSV) infection. RSV infection is the leading cause of infant hospitalization in the US and a significant risk factor for the immune-compromised and elderly. Although a neutralising antibody exist for the prevention of RSV infection, no drug has been shown so far to be effective in the treatment of RSV infection.

Today marks another milestone in the rapid, but at the same time circumspect development of ALN-RSV01 from the test tube to the clinic. It demonstrates just how quickly it is possible to develop RNAi as antivirals. This is because the viral sequence alone allows us to start designing and testing siRNAs for their antiviral activity. It is not surprising therefore that the NIH and Department of Defense is interested in fostering this technology for the fight against bioterrorism and pandemic flu. Indeed, Alnylam may leverage their experience in RSV and translate it into their pandemic flu program for which an IND is planned by the end of the year. Another company, Nastech, of Bothell, Washington, is also in the discovery phase of an RNAi therapeutic for pandemic flu, as was Sirna Therapeutics before their acquisition by Merck.

Alnylam’s first phase II study is an experimental challenge study in which volunteers are nasally infected with an attenuated strain of RSV and ALN-RSV01 given either before or after infection. The goal of this randomised 90 patient double-blind, placebo controlled study is to first establish safety, but more importantly antiviral efficacy as measured by incidence of infection, viral titer, and symptomatic differences. If successful, this would constitute human proof-of-concept of a human RNAi Therapeutic and represent another major de-risking event on the path to establishing RNAi as a whole new platform for innovative drugs.

PS: In another RNAi pipeline event, Intradigm, an RNAi delivery company based in Palo Alto, California, announced today that they have officially selected ICS-283 as a development program for cancer. ICS-283 is designed to target cancer angiogenesis and phase I studies are expected to start in 2008.