Checkpoints Drugs Promise to Unlock RNA Therapeutics Cancer Potential

If you are involved in cancer drug development, you are probably thinking of ways to exploit checkpoint inhibitors for your purposes.  Checkpoints are the mechanisms whereby cancer cells avoid being recognized by the immune system as foreign and the initial clinical results of inhibiting them, e.g. by targeting CTLA4 and PD-1/PD-L1, has caused immune-oncology as a whole to take the cancer drug development space by storm.

Suddenly, every scientist and their technician believe that by taking checkpoints out of the equation, their immune approach to cancer, which in many cases was marked by failure after failure in the past, will work. 

Take e.g. cancer vaccines and the idea of grinding up tumors and use the cell mash to train dendritic cells.  Sounds compelling to me, but I also know that I lost a bunch much money on the very same idea 10 or so years ago with nothing, but some early promising data from the clinical Wild East to support it. 

Others meanwhile believe that their technologies may enable immune-oncology by e.g. being able to modulate the tumor microenvironment so as to permit better access of the tumor killing cells.

RNA Therapeutics are no exception.  RNA Therapeutics, due to its endless targeting opportunities, has always been considered promising for oncology, but has struggled to show clear-cut success either due to drug delivery issues or due to the difficulties of predicting immune activities based on rodent studies.

Emblematic of this reversal of fortunes is the toll-like receptor (TLR) field of activating the innate immune system with oligonucleotide stimulants.  The idea here is to provide an environment that is more conducive to tumor cell killing and/or to directly impact tumor cell survival (e.g. interferon stimulation).  Art Krieg, of TLR9 (CpG) fame, but who has been erring as its lost son in the (blooming) deserts of RNAi, RNaseH antisense, splice modulation, and RNA activation, is apparently re-energized enough to go back to TLR Therapeutics and has started Checkmate Pharmaceuticals which will likely try and harness TLR agonists for cancer immunotherapy.

Nanoparticle delivery may also see a revival.  This is because they have a propensity, usually undesired, to be taken up by phagocytic cells, some of which may be effective in antigen presentation (e.g. dendritic cells).  I have thus noticed that a number of mRNA cancer vaccine approaches involve nanoparticle delivery with the aim of expressing tumor antigens in the training camps of the immune system, the lymph nodes.  

Nanoparticles may also be a way to knock down the gate-keepers in the tumor microenvironment which inhibit tumor infiltration by cytotoxic T (incl. CAR T-cells) and other helpful immune cells.  Since gate-keepers should be most useful when positioned at the entrance, this might actually take advantage of another limitation of many nanoparticle delivery technologies, namely getting stuck close to the vasculature instead of penetrating deep into the tumor.

Once deeper into the tumor, the struggle may not be over for cytotoxic T cells and tumor eating cells due to potentially immunosuppressive activities in the tumor microenvironment.  This is the new positioning for the phosphorothioate-based antisense molecule ISIS-STAT3 by Isis Pharmaceuticals and AstraZeneca for which, like for other phosphorothioate-based approaches, the demonstration of robust uptake and gene knockdown activity in tumor cells themselves is lacking, but functional uptake in cells of the tumor microenvironment has been reported.

Finally, RNA Therapeutics such as CRISPR genome editing or straightforward self-delivering RNAi (see recent license of MirImmune from RXi Pharmaceuticals) can be tools for the ex vivo preparation of T- and dendritic cells.  A self-delivering approach may be advantageous here as it may function in normal cell culture media and thus not confound cell signaling pathways in the maturation of these cells.

Investor, tread carefully

Overall, I’m convinced that there are synergistic potentials to be exploited and checkpoint inhibition may open the door to certain that have failed in the past.  Nevertheless, one should be mindful that most immune oncology drugs only work in a fraction (maybe 25%) of patients and the current hype around immune oncology guarantees that there will be many bad apples for investors to avoid.  Trust me, I've seen it when RNAi was indiscrimately hyped and abused for short-term financial gain in 2006-8.  Just because you can contrive a link to immune oncology or because a desperate Big Pharma does a deal, does not mean that all the biological problems will dissolve.  The bubble will burst after which mostly only well-financed quality plays will recover.

Disclosure: I am fishing for short opportunities in the immune oncology space as a hedge against a (hopefully temporary) correction to what seems to be an in a number of areas (e.g. gene therapies, immune oncology, one-drug orphan wonders such as Alexion etc) overheated biotech space.

Dicerna Trying to Succeed Where Novartis Has Admitted Defeat

This week, newly public RNAi Therapeutics company Dicerna initiated its first phase I study of a Dicer-substrate-based RNAi Therapeutic.  DCR-MYC targets the well-known Myc oncogene utilizing a liposomal delivery formulation (EnCore) for targeting a variety of cancers, solid and hematological (à Myc and lymphoma) malignancies alike, but with a planned focus on primary liver cancer in future studies.

The cancer trial start coincides with Novartis’ bitter, brake-slamming exit from internal RNAi Therapeutics development, largely blaming lack of suitable delivery technologies.  In particular, in classic Big Pharma style, Novartis seems to have selected its 31 RNAi trigger picks under the 2005 Alnylam license not based on where delivery is most advanced, but based on where it wished to strengthen its disease franchises.  It is this putting the cart-in-front-the-horse attitude that is at the root of Big Pharma’s miserable failure with an emerging platform technology that has its own mind of where it wants to go first.

According to commentary by Alnylam, cancer appears to have been a focus of Novartis’ target selection.  With regard to delivery to cancers, I would agree with Novartis to the extent that it is not as far developed as for example for the liver.  A problem with it is the inter- and intra-cancer heterogeneity of the EPR effect that most current cancer delivery approaches rely on.  You therefore have to be quite careful as to which cancers you select.  The same heterogeneity applies to target receptor expression (e.g. LDL-receptor, folate receptor) and Tekmira will have its good, not necessarily publicized reasons for why it chose neuroendocrine (NET) and adrenocortical carcinoma (ACC) for its ongoing phase II trial with TKM-PLK1, preliminary results from which are expected this year.

I’ve had the pleasure of attending the European Symposium of Controlled Drug Delivery in the Netherlands this week and presentation after presentation showed that for most liposomal formulations, tumor penetration is a major issue.  The good news is that EPR is very real, but the field has come to a point where it needs to establish the rules for which cancers are amenable and which strategies (size, lipophilicity) can be employed to aid in tumor penetration.

Imaging studies presented at the conference and the recent (conditional) European marketing approval of the companion-diagnostic/folate receptor-targeted cancer drug pair by Endocyte (Vintafolide) strongly suggest that patients should be pre-selected based on whether they have cancers amenable to EPR.  For example, pre-treatment with a small dose of the drug co-formulated with a diagnostic contrast reagent would both visualize amenable tumors as well as have the side benefit of de-sensitizing the patient to hypersensitivity reactions that are typically observed for infused drugs during the first administration.

So while I remain uncertain about the specific prospects of DCR-MYC partly due to concerns around the target and partly due to the relative inexperience of Dicerna in liposomal delivery, RNAi Therapeutics will become a reality in the treatment of cancers.  Just don’t expect clumsy Big Pharma R&D to rise to the challenge.

MicroRNA Therapeutics Company InteRNA Technologies Selects Clinically De-risked AtuPLEX Delivery Technology

Silence Therapeutics and InteRNA Technologies announced today the signing of a collaboration agreement following which the companies will use the endothelial cell-directed AtuPLEX gene knockdown delivery technology for the development of microRNA cancer therapeutics.

The news comes as Atu027, Silence’s RNAi Therapeutics candidate in oncology which also uses AtuPLEX delivery technology, is about to wrap up phase I studies (enrolment to complete by year-end at current rate of recruitment, with follow-up putting data announcement 3 or 4 months after that). Importantly, this study suggests a quite promising safety profile for AtuPLEX with apparently no dose-limiting toxicities so far as doses reach levels where meaningful gene knockdown in endothelial cells can be expected based on the pre-clinical rodent and non-human primate studies.

The deal makes sense to both Silence Therapeutics and InteRNA Therapeutics.For Silence Therapeutics, it is a quite obvious and facile monetization opportunity of its already established AtuPLEX technology with a nucleic acid payload (microRNA mimics discovered by InteRNA) where it does not have a core interest. For InteRNA, it promises to speed up its clinical development efforts as the safety of AtuPLEX has been considerably de-risked by now in the clinic and a lot of the learnings of AtuPLEX, also with regard to regulatory issues, should be applicable to the envisaged microRNA Therapeutics. In this regard, InteRNA may be different from some other microRNA Therapeutics companies that intend to do the heavy lifting of bringing a new small silencing RNA systemic delivery technology into the clinic.

Consequently, I consider this collaboration a win-win for both companies, even more so in financially constraint times. There is considerable synergies to be gained if companies would think more about how they could use their respective strengths to help each other develop promising therapeutic candidates instead of trying to do everything themselves and replicate the mistakes of others.

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 Grants Takeda RNAi Therapeutics License for Cancer and Metabolic Disease and Anoints Asian Partner of Choice

As if executing from a script, Alnylam today announced its first major Asian licensing deal giving Japanese pharmaceutical company Takeda access to the US biotech's RNAi Therapeutics IP for cancer and metabolic disease. The deal also provides Takeda with the preferred option of co-developing Alnylam product candidates for the Asian market. In return, Alnylam gets a sizeable $100M in immediate upfront, and another $50M in "near-term technology transfer payments". Adding the typical development and commercialisation milestones, field area expansion payments, and royalties built into these deals, the eventual value may well exceed one billion US dollar.

The agreement is also another validation as to how important SNALP RNAi has become for the current valuation of RNAi Therapeutics, as both metabolic disease and cancer are the two major near-term opportunities for SNALP RNAi. Remember, Roche’s therapeutic areas also included metabolic disease and cancer, as well as certain liver diseases and respiratory disease.

That this deal was announced one day before the close of the Tekmira-Protiva reunion should also be no coincidence and the exact timing may relate to the interests of various Tekmira and Protiva shareholders. Furthermore, my expectation is that the announced $50M in near-term technology transfer payments refers to the final closing of Tekmira-Protiva as well as finalization of an R&D collaboration agreement for SNALP similar to the ones that Tekmira currently has with Alnylam and probably soon with Roche as well. It also reminds us that there are at least another 3 Big Pharma and biotechs that are currently evaluating SNALP RNAi with Protiva-Tekmira. So plenty of more news to come and auction dynamics to set in.

Takeda’s move once more emphasizes the thirst of Big Pharma for innovation. Takeda itself has just propped up its sales numbers and cancer franchise with a headline-grabbing $8.8billion purchase of Millennium Pharmaceuticals and adding to that another $150M for planning more long-term may not be bad for balance. And on the same business trip at that. This is because Millennium is also Alnylam’s Cambridge, MA, neighbor and former employer of Alnylam’s CEO John Maraganore. In fact, it is rather curious that John Maraganore was chosen by the Boston Globe to voice his views on the Millennium deal, to which he responded that “the deal could potentially free Millennium from worries about making every quarter's profit figures, allowing it to invest more money in drug development.” Well, with the $150M in added cash, and ~$400 already stashed in the bank, he has just gotten one step closer to that dream.

Takeda has just had a high-profile failure for an cholesterol-lowering drug candidate, and may now be keen more than ever on exercising its right to partner Alnylam’s PCSK9 program for Asia. ALN-RSV01 interestingly is the only program that Takeda does not have first right of refusal, and may indicate yet another deal in the Alnylam pipeline.

Takeda is also strong in the diabetes area, and it will be interesting as to whether Takeda will pursue such programs with RNAi soon and for which liver-expressed genes may be targeted. This may particularly benefit Alnylam since, unlike in the Roche deal, it now has also the opportunity to opt into Takeda’s RNAi Therapeutics programs in a 50:50 relationship.

Based on statements such as "We believe this alliance will accelerate our initiatives to establish the foundation for RNAi drug discovery" in the presss release by president of Takeda, Yasuchika Hasegawa, it also appears that Takeda has been quite a bit active in RNAi Therapeutics already. Moreover, the deal provides for a cross-licensing of delivery technologies, and it will be exciting to learn what these are.

Novartis, Roche, and now the Takeda deal by yet another major foreign company may illustrate two points. One is that the US currency has fallen so low that foreign companies cannot resist the opportunity to secure their rights to US innovation, in this case to RNAi Therapeutics at bargain exchange rates. The other is that Alnylam may want to keep US rights to itself for the time being.

Finally, the deal is yet another validation that if you have money and are serious about developing RNAi Therapeutics, Alnylam is the company to partner with for access to fundamental IP and, thus far, enablement, too.

Impressions from the 3rd Annual Meeting of the Oligonucleotide Therapeutics Society: Day 1

The 3rd Annual Meeting of the Oligonucleotide Society (OTS) got under way in the stylish Langenbeck-Virchow Auditorium situated in the centre of a culturally vibrant Berlin. The OTS brings together the many disciplines in the rapidly expanding field of oligonucleotide-based drug development, ranging from aptamers, ribozymes, antisense, immunostimulatory nucleic acids to RNAi. Reports from clinical studies of these treatment modalities and oligonucleotide chemistries were at the centre of today’s lecture sessions.

Brett Monia from ISIS Pharmaceuticals chaired the first session and set the stage by reporting that while there are only four approved oligo-based therapies, more than 50 are currently in clinical development. Many of these are designed for cancer applications, but infectious, metabolic, and inflammatory disease applications are emerging as major new focus areas. This may not only be due to considerable unmet medical needs, but also because these may offer better success rates for clinical development. A few talks on cancer therapies with ASOs (antisense oligos) documented the challenges of designing smooth clinical trials for cancer therapies. One problem is the heterogeneity of cancers and matching the gene target with the right cancer. Early trials on a given compound typically involve small patient numbers with a wide spectrum of cancer manifestations. Although safety trials, almost anecdotal evidence of biological activity in a few patients then often guide the design of later phases of clinical development. Clearly, for cancer, there is a need for the development of more reliable biomarkers as early indicators of therapeutic efficacy and for patient stratification, which may be something that Rosetta Genomics’ microRNA diagnostics may be able to facilitate.

Focusing on diseases like viral infection and metabolic disease where efficacy can be routinely evaluated in early stages of clinical development (viral titers, LDL-cholesterol, glucose) should therefore be lower hanging fruits for the next wave of oligonucleotide-based therapeutics. This transformation in the industry is illustrated by ISIS’ new focus on metabolic disease and its out-licensing of the more challenging ASO applications. Rosanne Crooke recounted the clinical experience with 301012, ISIS’ lead compound for the treatment of hypercholesterolemia. As I wrote earlier, one major concern with targeting ApoB100 is the risk of steatosis of the liver due to inhibition of a protein required for the export of triglycerides and which has indeed been observed by a number of groups targeting ApoB100 by RNAi. Possibly motivated by the existence of individuals with null mutations for PCSK9, the new target of choice for hypercholesterolemia, with no evidence for adverse effects due to lack of the LDL-receptor processing protein, a conference participants asked about the existence of ApoB100 knockout mice. Interestingly, no such mice exist, because ApoB100 would have additional roles during development.

Despite reports of steatosis due to knockdown of ApoB100 with RNAi, Sirna Therapeutics is yet another biotech company interested in targeting it for lowering cholesterol. In a talk about siRNA delivery to the liver, Barry Polisky, CSO of Sirna Therapeutics/Merck also noted that in Sirna’s experience, there are genes that can be knocked down extremely well (e.g. ApoB100) and some that are relatively refractory to knockdown by RNAi. Although anecdotal, given the extensive experience of Sirna Therapeutics with screening for effective siRNAs by tiling them across entire genes, and similar findings by Qiagen and others, it will be interesting to determine what makes a gene a good target for RNAi, particularly whether it involves its normal regulation by microRNAs.

Polisky further focused on the need for siRNA modifications to make RNAi therapeutics a reality. Currently, Sirna’s standard siRNA design seems to involve si(R)NAs that lack essentially all 2’OH groups, except for the three 5’ nucleotides of the guide strand, while the passenger strand has 5’ and 3’ modifications that avoid it to be loaded into RiSC to reduce off-targeting. Other speakers and posters also noted that since most nucleotide modifications had been developed with antisense therapies in mind, new modifications may exist for optimal RNAi efficacy, particularly in vivo. Indeed, new synthesis strategies are being developed that will allow this to be tested, such as the 6-membered carbohydrate substitutions of ribose reported by Piet Herdewijn from the University of Leuven, Belgium.

Polisky further characterized siRNA delivery to the liver as efficient, albeit not optimal. Progress is clearly illustrated by the fact that >30% of siRNA formulated into liposomal nanoparticles now end up in the liver with first-pass kinetics with no evidence for toxicity in the 1-9mg/kg range (mostly rodent studies), whereas >99% of unformulated siRNAs are rapidly filtered out by the kidney.

The liver is also the organ where the first microRNA-based therapies are likely to be targeted. Like Regulus and Rosetta, I was amazed, or maybe not, at how many groups are currently working at abrogating miR-122 function for treating either hypercholesterolemia or HCV infection. LNAs, owned by the privately held Danish company Santaris, appear to be a particularly promising oligonucleotide class for achieving potent microRNA down-regulation in vivo. Similarly, antisense strategies based on LNA appear to rival siRNA potency in a number of settings, and it will be interesting to learn more about the toxicologies of these compounds, which would certainly be facilitated if Santaris decided to enable to field by allowing better access to their technology. It also highlights the need for specialized chemistries for therapeutic targeting of microRNAs which in my mind is something that Regulus ought to consider.

With regard to delivery outside the liver following systemic administration, PEI-formulated siRNAs (see recent PolyPlus-Alnylam deal) are now being used successfully by a number of groups for lung delivery. Other interesting delivery data was presented by a Portuguese group in a poster on RNAi in the brain. By non-covalently adding transferrin protein to cationic liposome-siRNA particles, they were able to efficiently target neuronal cells both in vitro and in vivo which, similar to cancer cells (see Calando’s cancer cell targeting with transferrin), are studded with transferrin receptors.

PS: A nuisance to most scientists in the field, immunostimulation by oligonucleotides is exploited by Dynavax as an adjuvant for vaccines. Poor adjuvants activity lead to insufficient immune responses and the need for multiple booster injections affecting compliance. HBV vaccination is a case in point, and as a consequence around 50% of patients in the US fail to successfully complete vaccination. Very high HBsAg antibody titers after only two injections of recombinant HBsAg with an immunostimulatory oligo from Dynavax as adjuvant, instead of alum, suggests that they may be able to greatly improve on those numbers.

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.

Promising Cancer RNAi Therapeutics Study Published in Cancer Research

Due to the relative ease of delivering RNAi effector molecules to the eye, lung, skin, and liver, it is expected that RNAi-based therapies for these organs will be the first to successfully navigate their way through the clinical trial maze. Nevertheless, a considerable amount of the early development efforts of RNAi Therapeutics is spent on cancer. I do not think that I need to remind anybody here about the need for more and better cancer therapeutics, but RNAi in particular offers the potential to greatly benefit cancer care. Delivery, however, represents a major obstacle for the development of cancer RNAi Therapeutics. This is even more so when faced with metastases which emphasises the need to find systemic RNAi delivery solutions to reach disseminated cancer cells throughout the body.

I was therefore very encouraged on reading a study published by Pirollo and colleagues from the Lombardi Comprehensive Cancer Center at Georgetown University Medical Center. In a Priority Report in the reputable journal Cancer Research (Cancer Res. 2007: 67 (7)), they report the development of a novel siRNA delivery system made up of nano-sized liposomes that harbour effector siRNAs and antibodies directed against the transferrin receptor. Transferrin receptors are known to be overexpressed on tumour cells and targeting them emerges as a promising strategy for cancer RNAi Therapeutics (Note: Calando, a subsidiary of Arrowhead Research, uses a similar approach in their first clinical RNAi cancer program). Consequently, the authors could show through imaging and nucleic acid detection techniques that accordingly formulated siRNAs were specifically enriched in the tumours of mouse models. The siRNA itself targets HER-2, a gene known to play a role in the survival of cancer cells. Although the siRNA in isolation already exhibited antiproliferative effects in tissue culture and the living animal, the most impressive data was that it could greatly sensitise cancer cells to the commonly used chemotherapeutic agent gemcitabine at low mg/kg doses. In addition to a dramatic reduction in tumour growth, the mice continued to gain weight and generally looked healthy. This study therefore shows the promise of using siRNAs in combination with chemotherapeutic agents, allowing for treatment regimens that are both more efficacious in addition to reducing debilitating side-effects.

Behind the success of these studies were 2 small tricks applied to the basic concept of nano-immunoliposomally formulated siRNAs. One was the addition of a pH-sensitive
peptide. This significantly enhanced cellular siRNA delivery, possibly by facilitating endosomal release of the siRNA cargo. The other trick was the modification of the siRNA, in which the non-targeting strand of the blunt-ended “siRNA” was greatly modified. This suggests that the systematic modification of basic siRNAs can be considerably advantageous and may yield even better siRNAs in the future.

Let us hope that other laboratories will find this immunoliposome technology equally promising so that it will eventually be tested in clinical trials.