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)

Mirus Scientists Publish Elegant Paper on Targeted siRNA Delivery to Hepatocytes

The ability to systemically administer siRNAs and functionally modulate gene expression in tissues of interest is considered by many an important step to opening up the therapeutic potential of RNAi to a wide range of diseases. The liver is arguably the best example where intravenously administered siRNAs have already been shown to potently knockdown target genes from mice to non-human primates.

The most promising delivery technologies so far (next to gene therapy vectors such as AAV which I will not discuss here) involve liposomes such as the SNALP particles which were first pioneered by Protiva scientists and subsequently became the subject of intense legal battles involving Protiva, Tekmira, Sirna Therapeutics, and Merck. While facilitating highly efficacious gene knockdown, toxicities such as liver enzyme elevations and non-linear relationships between knockdown and siRNA dosage have been repeatedly reported with these chemistries. While the toxicities were observed at relatively high dose levels, non-linear dose responses complicate the choice of the right dose for entering clinical trials.

One step in the right direction was taken when Daniel Anderson from the MIT reported earlier this year at the Keystone meeting the identification of a slightly different class of compounds which they termed “lipidoids”. Importantly, this class of chemistries appear to efficiently promote RNAi gene knockdown with little if any apparent toxicities and linear dose responses.

The just released paper by Rozema and colleagues from Mirus Bio Corporation (Rozema et al. PNAS Early Edition 24 July 2007: Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes) sheds light on some of the issues above and holds out a new paradigm for achieving safe and efficacious therapeutic RNAi knockdown in select cell populations of the liver. Rather than regarding drug delivery to the liver as a passive process given that the bulk of intravenously injected drugs will pass through it with a relatively high chance of entering resident cells there, the present approach involves attaching a simple galactose-derived ligand thereby actively targeting asialoglycoprotein receptors (ASGPr) displayed on hepatocytes.

Knockdown of ApoB100 and microscopic analysis of fluorescently tagged small double-stranded nucleic acids confirmed that hepatocytes were indeed efficiently transfected. Equally important, however, was their observation that Kupffer cells, a major population of macrophages in the liver that are implicated in many cases of drug-related liver toxicities, did not take up the siRNA mimicks, whereas non-ASGPr targeted particles were able to transfect surrounding cells, including Kupffer cells.

Taken together with the SNALP and lipidoid data, this study supports the hypothesis that Kupffer cells may act as a sink for certain siRNA formulations such as SNALPs, thereby not only causing non-linear dose responses, but also an immunogenic response and related toxicities. It also shows that it should be possible to design simple and small siRNA nanoparticles for RNAi delivery from relatively cheap materials. Although lipidoids appear to be a clinically viable technology already, it is comforting to know that alternative routes exist and gratifying to see almost daily improvements being made in the RNAi delivery field.

PS: An interesting aspect of this publication was data the authors obtained on ApoB knockdown. In addition to serving as a target for early proof-of-concept studies for systemic RNAi delivery, ApoB has been a favourite for treating hypercholesterolemia using both antisense and RNAi. ISIS Pharmaceuticals in particular has an anti-ApoB antisense compound in late phase II clinical trials. However, only a month ago, Alnylam reported that in their hands ApoB knockdown led to unacceptably high levels of fat accumulation in the liver (fatty liver phenotype), a finding that was confirmed in the present study. Moreover, both reports indicate that this was a siRNA sequence specific effect and was achieved using two different delivery methods. This physiologic response makes a lot of sense, since ApoB’s main role is in the export of cholesterol and triglycerides from the liver. Failure to export them should accumulate them in the liver.

This is a good example where RNAi can serve both as a target validation tool AND a platform for developing innovative drugs. Consequently, I fully support Alnylam’s decision to target PCSK9 in their hypercholesterolemia program instead, which is a genetically well validated target for reducing LDL-cholesterol and heart disease (see also Blogs from 6 May, 2007: “Preventing Heart Disease with RNAi Therapeutics”, and 9 May, 2007: “ISIS Copies Alnylam’s Heart Disease Strategy“). It is curious then that ISIS maintains and has published the absence of such a phenotype. For the sake of patients and stakeholders in ISIS, I can only hope they are right. The explanation for the apparent discrepancy? I cannot really offer a good one except to speculate on a fortuitous ISIS 301012 antisense off-target effect.