RNAi Therapeutics Portfolio Update: RXi Pharmaceuticals (Sell)

I have decided to sell all of RXi Pharmaceuticals in the RNAi Therapeutics model portfolio (for $4.49). This is partly an opportunistic move that comes after a remarkable meteoric ~200% run-up from its all-time low of $1.51 in the absence of a change in fundamentals (a recent overview can be found here), and a new CEO that needs to be able to learn as fast as the stock price has risen. I will keep the proceeds as cash for now, as I continue to research one particular company as a potential candidate for re-investing the proceeds. Stay tuned.

Disclaimer: Investments in RNAi Therapeutics are highly risky and not suited for most people. The purpose of the blog and model portfolio is to convey a sense of the dynamics in the field and is NOT an endorsement for making related investments. I also have financial interests in some of the companies included in the portfolio. I do not, however, have short positions in any of those.

Tekmira Reports First Knockdown in Man Following Systemic RNAi Delivery

Tekmira today reported very encouraging results from their first clinical study with SNALP siRNA delivery targeting apolipoprotein B for the treatment of hypercholesterolemia (PRO-040201, aka SNALP-ApoB). While the ~20% ApoB/LDL-c knockdown following single dose administration and evidence for flu-like toxicities at the highest tested dose level indicate that the present formulation is not suitable as a therapeutic, the company is rightly optimistic that based on these results, follow-up formulations that it intends to enter into the clinic later this year will be able to provide significant improvements in the absolute dose and therapeutic index. This is because the new knowledge can now be applied to the rapid progress that has been made in finding much more potent SNALP/LNP formulations (see recent PNAS paper and ILS2009 presentation) since the present formulation was locked down 2 years ago. In addition, much has been learned since on how to predict and mitigate SNALP siRNA-triggered immunostimulation in humans (extensive review). I therefore agree that it was probably the scientifically and financially best decision by the company to stop this trial at this stage and focus their resources on the next formulation.

When I first adopted SNALP/liposomal delivery as my pet systemic siRNA delivery approach 2-3 years ago, my main outstanding nightmare stemmed from an unexpected early-stage clinical disaster during a phase I trial by the same company (Protiva branch) almost a decade ago employing related stabilized cationic liposomes for the delivery of plasmids (SPLPs). In that trial, severe flu-like symptoms were observed at the very early and low dosages and the was trial terminated and the drug never heard of again. Instead of giving up, this has stimulated them to undertake almost heroic and industry-leading work aimed at mitigating immunostimulatory effects of liposomal nucleic acid delivery. It is thus quite remarkable that after just seven years after stumbling on siRNAs as more promising payloads for the technology, Tekmira has now demonstrated unambiguously functional liposomal siRNA delivery following systemic (intravenous) administration, with first, transient flu-like symptoms observed at what should be much higher dosages compared to the SPLP trial.

It is worth bearing in mind that the decision to stop the trial was not because the one case of flu-like adverse event was deemed a serious one, but because they felt that at this point they had already learnt what they wanted to learn and given the superior performance of the next-generation formulations it would therefore not have been ethical to further expose patients to a drug that will not be developed for commercialization (all my interpretation). It also might have risked tainting the asset with little to gain. To put the toxicity into context: in the most recent published clinical results on ISIS’ mipomersen targeting the same gene, 70% of patients receiving drug exhibited flu-like symptoms at similar 20% ApoB/LDLc knockdowns.

Equally encouraging is the fact that the knockdown, which based on the natural intra-person lipid variability and the pharmacokinetics of the reported knockdown (fast onset as expected for an RNAi drug) was almost certainly due to drug, occurred at dosages where the company expected to start seeing efficacy based on pre-clinical animal studies. One cannot underestimate the importance of being able to predict efficacious dose (and toxicity) based on these models, particularly at the early stages of evaluating such a novel technology where very little human pharmacological experience has been obtained.

The trial design involved cohorts of 4 patients for each dose level: one treated with placebo and three with study drug. While 8 cohorts had originally been planned, the trial was stopped in approximately the 6th or 7th cohort. According to the company, at this point the dose had been escalated to somewhere in the 0.6mg/kg ballpark and is thus consistent with IC50s for SNALP-siRNAs in non-human primates of ~1mg/kg at that time. Considering that according to a presentation at the International Liposome Society a month ago by Pieter Cullis, scientific founder of Tekmira, SNALP-like formulations have now reached IC50 potencies in non-human primates of ~0.03mg/kg, i.e. around 1.5 magnitudes better, there is every reason to believe that significant improvements in absolute dose and therapeutic index can be achieved with the follow-on SNALP-ApoB candidate. Strengthening this claim, similarly potent formulations have just recently surfaced from a study supposedly (Alnylam IR: I still cannot find the paper) published in PNAS by Alnylam and collaborators at the MIT on lipidoid-containing SNALP-like particles (allow a delay of at least a year from experimental finding to publication).

While all this sounds good, a critical question to ask is what gives them also the confidence that the new formulations (= more potent liposomes + less immunologically active siRNAs through modification) will not perform worse immunologically than the first one, thereby eliminating some of the benefit from the improvements in SNALP potency. The ability to answer this question would hinge on the ability to sensitively detect immunostimulation and translate such findings pre-clinical models into humans. The JCI paper last year by Tekmira scientists on the application of SNALP for distant tumors in mice showed that Tekmira indeed understands how to detect such responses even in the absence of cytokines in the serum (tissue-based PCR assay for IFIT1 mRNA). And from all the presentations I have seen, another important piece of the puzzle in being able to translate from animals into humans is the good correlation between immune responses seen in in vitro human PBMC immuno-assays and responses in animals. In combination, these elements allow for efficiently modifying siRNAs until predicted immune-related tox in humans have been minimized. Through such knowledge, Tekmira was able to provide a good explanation for why some non-human primates exhibited liver toxicity in the 2006 Nature study (the first demonstration of systemic RNAi in non-human primates) even in the absence of outward immune activation and remedy it through siRNA modification. It is interesting, however, that in the present clinical trial the presumed immune stimulation appeared to have (thankfully) been uncoupled from liver enzyme elevations as an absence of liver toxicities was noted. Although the reasons behind these are unclear, this uncoupling lends further credence to the notion that also efficacy and liver toxicity can be uncoupled: the mechanism that allows for functional entry of the liposome itself does not cause hepato-cytotoxicity.

According to a conversation with the company that I had following yesterday’s release of top-line results, it also appears that they have developed what appears to be a separate assay that they have not disclosed yet, and that should be even better in predicting immune responses in humans. Although, in the absence of seeing the data, I will have to take the company by its word here, as a proven leader in liposomal siRNA delivery, I’d like to give them the benefit of the doubt.

Looking ahead

The company indicated that the new formulation will go into the clinic later this year, although discussions with the FDA have yet to determine the exact regulatory requirements for this (e.g. an abbreviated IND package would make sense IMO). Like the first trial, the second one is likely to be a single-dose one, and a multi-dose trial in humans would similarly require additional pre-clinical data. All this makes it clear that the main value of this program to the company is that ApoB is an exceptionally good target to most rapidly develop insights into the entire SNALP-siRNA delivery platform. The market reaction tomorrow, however, will show whether this is shared by investors who may be disappointed by the delay on ApoB and may have also hoped that SNALP-ApoB could find a development partner soon. However, I would think that most invested in Tekmira as a leader in systemic RNAi delivery and are equally encouraged that the company is now able to build real data which have for the first time demonstrated unambiguous systemic RNAi gene silencing in Man. In addition to investor perceptions, the financial repercussions of these results also depend on what value such pioneering work will fetch from potential partners that are interested in the broad development of the RNAi Therapeutics platform.

Whether Tekmira’s enthusiasm is shared by current partners will be seen by their clinical actions. As such, Alnylam until recently reiterated that it was still on track to file an IND for the liver-directed SNALP-TTR. Since Alnylam apparently was aware of the status of SNALP-ApoB in November already (see RNAi Clinical Experience chart in this slide presentation), Alnylam also seems to be comfortable that the more potent SNALP formulations will have clinical applicability given Tekmira's results. I look forward to learning about the TTR trial design, including whether multi-dose will be attempted, and there should be results by the end of this year. In addition, the fact that we have just learned that Roche entered IND-enabling studies, most likely with a SNALP product, further validates Tekmira’s decision. The fact, however, that the IND submission of SNALP-PLK1 has been moved into the second half of this year represents a slight delay, and it remains unclear whether this might be related to today’s news.

In any case, today makes me feel like we have witnessed possibly the beginning of the most important line of RNAi Therapeutics clinical research thus far. Many more data-points on clinical SNALP-siRNA delivery should accumulate over the next two years.

Disclosure: I have financial interests in the company, largely by holding TKM shares.

Note added in proof: Scientific papers by Tekmira and Alnylam on the development of significantly improved SNALP formulations:

1) By combinatorial chemistry screen (December 2009 PNAS paper)

2) By rational design of lipids (January 2010 Nature Biotech paper)

2010 in RNAi Therapeutics Starts Off with Big Pharma Validations

If the first week is any guide, 2010 could develop into one of the most interesting years in RNAi Therapeutics history and comes after a somewhat lackluster 2009. Both the Dicerna-Kyowa Hakko platform collaboration announced earlier this week and the pre-IND milestone payment Alnylam received today from Big Pharma partner Roche are important proof-points that Big Pharma/Biotech (BBP) continues to invest in RNAi Therapeutics.

The Roche payment due to the initiation of IND-enabling studies marks the first time that a BBP is close to entering the clinic with its in-house developed RNAi Therapeutics candidate. The strength and clinical value of Alnylam’s strategy to further monetize on its mainly RNAi trigger IP has been increasingly questioned by skeptical investors as around half a billion dollars in realized IP funding has apparently not been enough thus far for one of its BPP partner to enter the clinic. This event should also be a boost to Tekmira Pharmaceuticals both in terms of platform validation and financially, as Roche has stated before that its first 2 RNAi Therapeutics INDs will involve SNALP delivery and Tekmira stands to collect ~$9M for each of the 2 candidates it helps Roche to get to the IND stage. For speculations about the indication of Roche’s first RNAi Therapeutics candidate, read the recent blog entry here.

While the Roche news is a definite plus for Alnylam, the Dicerna news has somewhat negative implications as it could be interpreted that BPP is becoming more hesitant to pay the Alnylam premium for what had been considered a toll-gate into RNAi Therapeutics. This is particularly so because Kyowa Hakko once paid Alnylam $15M upfront for Asian rights for Alnylam’s lead RNAi Therapeutics program for the treatment of RSV infection, and very little has been heard about the Kyowa relationship after ALN-RSV01 has been dropped as a candidate for development in pediatric populations. [update] However, as a reader rightly points out in the comments section, Kyowa's 'slight' may also simply reflect the fact that Takeda is Alnylam's exclusive Asian platform partner until 2013 and that Kyowa Hakko was sufficiently eager to speed up the development of its internal RNAi Therapeutics efforts by partnering with a pure-play RNAi Therapeutics company such as Dicerna.

Listening to some of Alnylam’s recent comments, especially its more frequent comparisons of RiSC vs Dicer-substrates, it also appears that Alnylam is not claiming to control the IP around Dicer-substrates. dsRNAs of 25bp and longer may therefore be a tempting, cheaper alternative into RNAi Therapeutics. I still believe that while Dicer-substrates are of comparable potency to Tuschl-type siRNAs, they are more complicated and therefore more costly to develop (especially innate immunity and more restricted chemical space due to requirement for Dicer-cleavage), although they these should not be considered show-stoppers and Dicer-substrates may actually allow for unique delivery strategies and in that regard may be considered complementary. The relatively early stage of Dicer-substrates, however, may explain why the immediate financial benefit to Dicerna was limited ($4M upfront, the rest biotech bucks), but should help the VC-stage company to raise further funds.

Price competition in RNAi trigger IP in the face of financially struggling competitors such as Dicerna, recent IP uncertainties, and the relatively increasing value attached to delivery vs RNAi trigger IP may indeed be responsible for Alnylam’s missed guidance of two major deals in 2009. While SNALP/LNP delivery is making great progress, Alnylam may have to offer more high-quality delivery options to further command terms similar to the Roche and Takeda collaborations. The decision of Novartis, which is about to spend $50B on eye-care provider Alcon, to pay Alnylam ~$100M this year for the broad adoption of Alnylam’s full RNAi Therapeutics IP, will be a good indication of the perceived strength of Alnylam’s IP if not the maturity of the technology.

Both the Alnylam-Roche and Dicerna-Kyowa news further underline the strong momentum of liposomal delivery and RNAi Therapeutics for cancer as also indicated by the Silence-Intradigm merger. Liposomal and lipid-based deliveries were highlighted in the delivery capabilities of Kyowa Hakko and Dicerna, respectively, and the first target picked by Kyowa was for oncology. Antibody-directed targeted delivery, also seems to gather pace as Kyowa Hakko further high-lighted such capabilities in the press release ('POTELLIGENT').

The two news items thus should be a good prelude to two other events this quarter that could decisively shift sentiment on the RNA Therapeutics sector into positive territory, namely the clinical trial results from Tekmira and ISIS Pharmaceuticals on their respective ApoB-lowering drug candidates.

Some RNAi Therapeutics Trivia: Dicerna almost appropriately means 'digested' in the Malay language.

RNAi Therapeutics Highlighted in Roche Media Day Presentation

Roche can be considered the bellwether of how Big Pharma/Biotech is looking at RNAi Therapeutics for rejuvenating their pipeline, and their moves will be closely watched by the investor community. I therefore found it extremely interesting what a Media Day presentation in 2009 revealed about their RNAi Therapeutics intentions. This presentation was given by Lee Babiss (President & Global Head of Roche Pharma Research) who has been a champion of RNAi Therapeutics at Roche. It is worth noting though that according to musings on the cafepharma discussion board, he has unfortunately left the company.

Roche sees RNAi Therapeutics as an integral part of their personalized medicine efforts conducted in concert with their diagnostics unit and which offers fast development timelines. Notably, doing the math, there were about 5 RNAi Therapeutics research programs in 2008 (0 in 2007) at what appear to be Lead Identification and Optimization stages. Relevant to RNAi Therapeutics, this portfolio of about 100 programs is jammed full with solid cancer programs which is THE poster child of personalized medicine (almost 20% of programs). As has been made public in a previous RNAiNews interview with, Roche intends to work with Tekmira to bring two RNAi Therapeutics programs into the clinic near-term, including one IND in 2010. According to slide 26 this is likely to be a program (‘MVD’) in metabolic disease as this has been the most advanced program (‘phase 0’) at the time of the presentation. ‘MVD’ is categorized under ‘metabolic disease’ and therefore could mean ‘macrovascular disease’. Type II diabetes, respiratory, and solid cancer RNAi therapeutics programs follow not far behind. It would not surprise me that the second program would be one for solid cancer, paralleling efforts at Tekmira (phase I SNALP-ApoB ongoing; solid cancer 2010 IND).

In delivery, it is, however, not all about liposomal delivery. Roche’s dynamic polyconjugates were highlighted in rhesus monkey studies which showed quite significant (>90%) and very prolonged (almost maximal at 3 weeks although this could be repeat administration data) knockdown, albeit at the relatively high doses of 20mg/kg. siRNAs coupled to antibodies were also highlighted in cancer applications which together with SNALP and DPCs may form Roche’s 3-legged stool of siRNA delivery.

Alright, this is it for me in 2009. I would like to thank everybody for reading some of the highly speculative stuff and who have made writing the blog particularly interesting to me either through your comments or by personal contact. I look forward to what promises to be a very exciting year in RNAi Therapeutics.

Rosetta Genomics Business Strategy Back on Track

Following the recent hiring of Kenneth Berlin as CEO, Rosetta Genomics is re-focusing their microRNA diagnostics efforts to leverage their extensive bioinformatic know-how and associated IP in the field. Having emerged as the leader in microRNA diagnostics churning out 3 microRNA tests in short order, initiatives such as betting the future on an early-stage blood-based colon cancer screening test and an one in plant biology ('Rosetta Green') first left investors perplexed and eventually disappointed. According to today’s press release and conference call, however, the company has decided to revisit its proven strategy of going back to their treasure trove of bioinformatic information and select new development candidates.

What is also remarkable is that, for the first time, the company is providing guidance about market size and clarity on pricing. This is a welcome change from previous years that had investors in the dark about expected revenues. In hindsight, it would appear that this was actually was due to lack of strategic planning. It is therefore expected that with the strong commercialization background of Mr. Berlin as former worldwide General Manager for the J&J molecular cancer diagnostics outfit Veridex, this essential aspect of Rosetta Genomics' business can only get better. Predicted revenues of only 2010 $2-4M based on 1,200-2,400 samples processed from their first 3 tests, however, represent business development challenges considering current cash levels of about $10M and a predicted $900k monthly burn-rate in 2010.

Mr. Berlin will therefore largely be measured for his ability to

a) penetrate the following predicted markets (annual numbers of potential tests US/ex-US):

1) miRview Squamous: >60k/250k; 2) miRview Meso: >60k/200k; 3) miRview Mets: >60k/300k;

b) develop 5 tests for which they already have identified the microRNAs, among which were disclosed the following tests and time-line:

1) an extended miRview Mets test (H2 2010 launch); 2) a test predicting superficial bladder cancer to become invasive (late 2011 launch and 350k annual market); 3) a test further differentiating lung cancers from fine-needle aspirates (2011 1M annual market)

c) achieve all this all the while minimizing shareholder diluation

As to the last point, I am curious whether, considering the first 3 tests have obviously been partnered already, Rosetta can further monetize their IP and know-how in the microRNA therapeutics area, and find a Roche-like partner for developing companion diagnostics. Because of trends making it more and more difficult to get reimbursement for home-brew tests, it is surprising that unlike Rosetta’s competitor Asuragen (companion Dx deals with Biogen and Merck), we have heard nothing about their plans in the high-value area of molecular companion diagnostics. Ongoing post-marketing studies for their present tests, however, should partly address the reimbursement challenge in addition to convincing patients/physicians to adopt the tests.

With the proposed strategic review, Rosetta Genomics should become a company worth considering again given their strong scientific foundation. It will take some time, however, to re-build confidence in management. After all, the misguided colon cancer ambitions did not seem to have been based on scientific progress from their exploratory published data, and it would not seem fair to blame the former CEO alone for all these blunders. Rosetta Green also needs more explanation. However, I do want to credit former management for having established the scientific engine behind Rosetta and having brought to market 3 out of the 4 currently marketed microRNA Dx tests. Privately-held Asuragen should also receive credit in this regard. This could also mean that people like Victor Ambros, Gary Ruvkun, and maybe 'even' (because plant biologist) my D.Phil. examiner David Baulcombe are even stronger contenders for a Nobel Prize for their discovery of micro- and other silencing small RNAs which in turn could give companies like Rosetta Genomics a significant boost in the near- to midterm.

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 Trends from the International Liposome Society Meeting 2009

Christmas to me means going to London to learn about what liposomes have in store for RNAi Therapeutics. As you know, liposomes have emerged as the leading platform for the systemic delivery of RNAi Therapeutics and offers considerable promise for diseases of the liver, solid cancers, and after that potentially enhanced vaccines, infectious disease and immune cell-related disorders. The fact that liposomal drugs are on the market already should further speed up its development, not least from a regulatory point of view.

The liposome field appears to be split between between cancer and vaccines about 2:1. Especially the success story of doxil (liposomal doxorubicin) has shaped the field and should partly explain why it is attracted so much towards solid cancers for RNAi Therapeutics. The EPR effect, targeted delivery, tumor vascular biology have been recurring topics and research using various advanced imaging modalities nicely informs the application of liposomal siRNAs in oncology. For example, liposomes (not necessarily unique in that regard) are not uniformly distributed within the tumor, but form a concentration gradient around the blood vessels. Strategies to achieve more uniform drug delivery may involve metronomic dosing in which the drug is administered more gradually at lower dosages such that the simultaneous onset of cell killing in proximity to the vessels will relieve intratumoral pressure so that subsequently arriving liposomes are freer to move within the tissue. Hyperthermia or low-dose TNF-alpha were two suggested strategies for increasing the leakiness of tumor vasculature, also to improve liposomal tumor distribution.

Since the highest concentration is achieved around the tumor vasculature and because there are differences between normal and tumor vasculature, targeting genes within the endothelial and supporting cells has become an attractive anti-tumor strategy. This for example is also the strategy that Silence Therapeutics is pursuing and the lipoplex formulation (siRNAs associated with liposome on the outside, not inside as in a SNALP) for some reason might have its application here.

But once within the tumor, mostly the result of the overwhelming EPR effect, the question becomes how to get the liposome into cells and then release the drug cargo into the cytoplasm in the case of siRNAs. Ligand-targeted liposomes are one answer to increase the cellular uptake of the liposomes. Antibodies are an increasingly popular means (immunoliposomes), although they present certain manufacturing challenges. It is still debated how PEG affects cellular attachment. Some claim that you cannot take up a pegylated liposome, some say you can. The answer may be that it depends on the size of the PEG. This is important for example in the context of a targeted liposome and whether to add the ligand to the PEG or directly incorporate it into the lipid bilayer as a lipid-conjugate, and when and how the PEG should leave the liposome. For efficacy and safety, triggerability of membrane lysis is of course important. Too much charge for example can be cytolytic, so you would like to expose membrane lytic activities in a pH-dependent (endosomal acidification), redox or protease-dependent manner. For the liposome field, this typically means pH-sensitive lipids.

Combined chemo-siRNA therapeutics is another trend that will become important for liposomal cancer RNAi Therapeutics. Combining ever more chemotherapeutics appears to be a game of diminishing returns with triple chemo Rx often not any better than double chemo Rx. This is because of limited mechanism of actions of chemotherapeutics and associated resistance mechanisms. RNAi Therapeutics therefore can either target these resistance pathways directly or bring to the table an entirely new anti-tumor mechanism of action.

I have always found it attractive to apply siRNAs for developing more effective vaccines. While I am not familiar with the literature around virosomes, viral ghost shells, a presentation by Swiss company Pevion Biotech caught my attention in this context. Basically, they are using flu viruses, disassemble them to get rid of the viral RNA and associate protein, and then reassemble them. During the reassembly process, they can then variably add their lipid-anchored antigen of choice and use the re-formulated virus as a vaccine. The fact that our bodies have seen flu virus before apparently helps to obtain a more favorable immunological response. By this, they believe to have broken the potency-toxicity linear correlation that seems to plague adjuvant science. While promising and based on a credible scientific presentation, it still does not appear to be among the most potent adjuvants, so I wonder whether an siRNA targeting the right immunomodulatory gene loaded into the virosome, essentially replacing the viral RNA there, could improve this equation even further. A challenge for nanoparticle-formulated siRNAs for enhanced vaccines is that although they are easily taken up by phagocyotic antigen-presenting cells, the phagocytic pathway usually destroys the siRNA and little escapes into the cytoplasm. The virosome could offer a solution since the flu virus is THE paradigm of how membranes fuse with each other and also explains how an extracellular antigen, the vaccine, can produce a favorable cellular immune reponses in this system. What I noted at the conference is the surprising lack of cross-talk between people using liposomes for vaccines versus those using them for oligonucleotide delivery which is unfortunate in cases like virosomes.

Pevion also seems to be producing good-quality liposomes. For liposomal RNAi Therapeutics, it is probably making the jump from rodents to primates and scalable manufacturing that separates the boys from the men. There are many interesting early-stage strategies, but if the goal is translational medicine, it is of utmost importance to take manufacturing issues into account from the very beginning. Of course, if you have such know-how, this challenge also presents an opportunity/entry barrier that can be capitalized on and should also present a significant barrier for generic competition. A scientist from Gilead told the story of the attempt by a fairly large Argentinian generics manufacturer to copy Gilead’s AmbiSome formulation by just mixing the individual components together and that this went quite wrong.

In order to address that gap between basic and applied science, the opening talk advocated the creation of companies whose sole business model is to take promising academic research and transform it into industrially partnerable technologies. Certainly, this concept appears to be gaining traction and it will be important in how the compete with or complement small biotech companies. The Centre for Drug Research and Development in Vancouver is one such entity that can be put into this category, also with a liposome focus (Pieter Culles e.g.). Vancouver in general can be considered the world's capital for liposomal research, which brings us to the Tekmira, UBC/Alcana, Alnylam triangle.

Pieter Cullis, co-founder of a number of liposomal companies including Tekmira, but now apparently more closely associated with Alcana and Alnylam, gave a nice talk about the rational design of lipids for use within SNALPs. Only small changes in the lipids can have large biological and physico-chemical effects. For example, by just modulating the chemical strength of the linkage of the fatty acids with the lipid head-group, SNALPs of very different potencies are obtained. Yet another objective is the investigation of the pKa-potency relationship which again can greatly impact liposomal performance. Through this, Alcana-UBC, largely driven by two ex-Tekmira scientists have come up with around 100 new lipids which further help to refine the design rules in an interative process. Actually, the rational lipid design approach is the approach Tekmira itself has been taking- probably not surprising given this background and may explain why Tekmira is getting access to these lipids for free without having to fund their development. It also differentiates it from the ‘lipidoid’ approach by MIT-Alnylam. Maybe given the varied performances due to even only subtle changes, huge libraries of lipids are not that desperately needed as was thought just 3-5 years ago. It is also the animal model that would become the bottle neck, as the tissue culture system is of only limited utility in predictively characterizing SNALP performance and mice become the system for the screen. Still, ‘lipidoids’ may offer fundamentally new structures which can be further refined using similar rational design approaches. For the liver, at least in terms of potency, this may not be needed according to Cullis’ back-of-the-envelope calculation that predicts single digit microgram(siRNA)/kg potencies that the technology is approaching for this tissue, an area that is approaching certain theoretical limits.

In summary, the conference, has confirmed my view that liposomal delivery is a good place to be in RNAi Therapeutics at the moment. It may even be time to have a conference entirely devoted to liposomal strategies for RNAi Therapeutics. There is a need, however, to focus on those companies that operate on sound scientific principles and ideally have a track record in being able to produce high-quality/consistent liposomes in a manner that is scalable.