Alnylam and The Medicines Company to Position RNAi PCSK9 Drug Candidate as Fast Follower

Yesterday, Alnylam made the somewhat surprising announcement that it partnered with the ~$1.6B market cap The Medicines Company to develop and commercialize its RNAi Therapeutic program targeting PCSK9 for hypercholesterolemia.   This program includes ALN-PCS02 which had completed a phase I study last year and contemplates subcutaneous formulations, too.  

This means that as a number of PCSK9 monoclonal antibodies are moving forward in clinical development at lightning speed, including phase III trials involving more than 20k patients, the ambition here is not to have a Big Pharma try and catch up (maybe they did try, but nobody was willing to partner), but to carefully study the experiences of the monoclonals and exploit the biological differentiation that an RNAi approach offers over antibody.  With The Medicines Company on board, some of that potential at least is seen in the hospital setting.   

Mechanism of Action of RNAi vs Antibody 

There are various points of differentiation which might translate into a clinical benefit for RNAi. Expect the companies to look hard for such evidence and, if found, beat the drum about it. These differences include: 1) reduction of both intra- and extracellular PCSK9 thus replicating human genetics from which PCSK9 emerged in the first place; antibodies merely bind existing extracellular PCSK9; 2) because antibodies form complexes with their targets and do not act catalytically, the percent target inhibition efficiency of antibodies depends on target abundance; therefore, in patients that have many more PCSK9 molecules than the number of antibodies you can fit in a subQ syringe, PCSK9 antibodies will not work well; RNAi, however, works with similar percent knockdown efficiency more or less regardless of target gene expression levels.

Efficacy

In general, the LDL cholesterol reductions with monoclonal antibodies have been between 40-70% in multi-dosing regimens.  In Alnylam’s single-dose phase I trial, the liposomal ALN-PCS02 achieved a ~30% reduction (area under the curve). 

Frankly, given the number of clinical trials involving PCSK9 monoclonals, I have given up tracking the results of each and every study.  Having said that, in reviewing the phase II trials of the candidate that may be viewed as the most advanced/exciting one, AMG145 by Amgen, it seems that the higher end of LDLc reduction was only achieved when given on top of statins.  Althought to me this seems a bit counterintuitive since statins are thought to act mechanistically essentially the same as PCSK9 inhibitors, namely via increasing LDL-receptors on hepatocytes, that's the way it looks right now, and the ALN-PCS02 trial may have been disadvantaged as it was mono-therapy.

Finally, with continued improvements in the potency of RNAi Therapeutics technologies, it should be possible to achieve similar LDLc reductions with RNAi as with PCSK9 antibodies. 

  

Acceptance

I like the fact that PCSK9 has become a small battleground between RNAi and monoclonal antibodies as this may be the best way for RNAi Therapeutics to work on its wider acceptance by the medical and investor community.  Notably, the often glorified monoclonals frequently suffer from injection reactions (some notable serious ones were observed in Regeneron’s PCSK9 trials), other immune-related issues and manufacturing challenges to name a few issues.  RNAi Therapeutics, of course, are facing some of the same challenges, but it irks me that when it comes to this technology, they suddenly are supposed to be show-stoppers.

Financials

The financials (including a $25M upfront, up to $180M in sales and commercialization milestones and double-digit royalties) were not all that exciting for Alnylam and reflect the fact that only one single-dose phase I trial had been conducted.  Also, as Alnylam cannot claim a blocking IP any more and has licensed hepatic targeting rights to other companies, including Roche/Arrowhead, the value of ALN-PCS as the only RNAi candidate for PCSK9 has been lost.

Still, $25M is serious money for a company the size of The Medicines Company and you do not turn this over just to help out an old friend.  

Merck Double-Knockdown Strategy to Ameliorate Toxicity from Mtp and ApoB Inhibition

The signs are that the next RNAi Therapeutics metabolic/cardiovascular disease candidate will be a dual-targeting one.  While the initial attempts in this area were directed at specifically reducing the well-known cardiovascular risk factor LDL-cholesterol (preferred targets: ApoB and PCSK9), it has become quite tempting to exploit the rare opportunity offered by RNAi Therapeutics to target multiple gene targets with just one formulation to both broaden the therapeutic benefits in patients that typically suffer from a plethora of metabolic dysfunctions (obesity, insulin resistance, and hypercholesterolemia to name a few) and to balance the adverse effects that may result from inhibiting certain targets.

Most notable among the latter is the liver fat accumulation following ApoB knockdown.  Clinical studies with ISIS Pharmaceuticals’ antisense compound mipomersen/KYNAMRO have clearly evidenced such liver fat accumulations which were often accompanied by increases in liver enzymes, general indicators of liver toxicity.  These results further are corroborated by similar clinical observations with the small molecule lomitapide by Aegerion targeting microsomal triglyceride transfer protein (Mtp) which acts essentially at the same stage as ApoB in packaging triglycerides and cholesterol for transport out of liver cells into the circulation.  Aegerion obtained this drug candidate from BMS via UPenn as BMS did not want to further develop this compound due to these safety risks.

Both mipomersen and lomipatide have completed phase III studies and new drug applications for approval in the rare genetic disease homozygous familial hypercholesterolemia (hoFH), and in the case of mipo also for severe heterozygous FH have been submitted to the FDA and EMA.   In terms of therapeutic profile, mipomersen seems to have the edge as, being a phosphorothioate antisense compound, it preferentially accumulates in the liver.  Consequently, it does not cause the side effects resulting from the intestinal inhibition of this pathway that  have been observed with small molecule lomatipe (note: SNALP-delivered RNAi Therapeutics should have similar benefits over small molecules).  Moreover, mipomersen not only lowers LDLc, but also moderately reduces the independent cardiovascular risk factor Lp(a).   Although not a prospective primary goal of mipomersen clinical development, incidental positive findings like this one can go a long way in having regulators take a benevolent look at drug candidates.  This can be seen in the related obesity space where one of the attractive benefits of Arena Pharmaceutical's lorcaserin is that it lowers blood glucose levels.

Obviously, there should be plenty of potential gene targets involved in triglyceride synthesis and utilization/oxidation that could be exploited to concomitantly lower triglyceride content in ApoB/Mtp-inhibited livers while maintaining LDLc-lowering.

Merck Tests ApoB and Mtp Knockdown, Finds Mtp-DGAT Co-Knockdown Promising

Tep and colleagues from Merck published a paper on a study that tested whether an RNAi co-knockdown strategy could be implemented to alleviate the liver fat accumulations due to Mtp and ApoB inhibition.  To be clear, Merck did not state that they have firm intentions of developing such a co-knockdown strategy, but nevertheless noted that such a strategy would have the advantage of  not having to ‘add[ ] a novel compound on top of an approved drug’ and that dual-targeting RNAi Therapeutics candidates are already in clinical development, therefore paving the regulatory path (see ALN-VSP02, and TKM-EBOLA).

In a first step the scientists confirmed the liver fat accumulation following Mtp and ApoB siRNA knockdown.  Not only were they of similar magnitude, the effects of the two knockdowns where essentially the same in almost every other investigated regard.  Notably, there was no reduction in liver fat accumulation following prolonged siRNA treatment as one might have expected based on claims by ISIS Pharmaceuticals of liver fat normalizations with time, but widespread changes in the expression of lipid-related genes were nevertheless observed- this time consistent with claims by ISIS Pharmaceuticals.

Among the genes that were downregulated following Mtp siRNA treatment, presumably as a result of negative feedback, was DGAT2, a key enzyme in triglyceride synthesis that is also thought to represent an important regulatory node in lipid metabolism (e.g. by promoting fatty acid oxidation).  Reasoning that further reducing DGAT2 with liposomally formulated siRNAs may lead to a measurable reduction in liver fat, they then co-formulated the Mtp siRNA with a DGAT2 siRNA and injected them into mice.  Indeed, this resulted in not only the expected LDL-cholesterol reduction, but liver triglyceride increases were mitigated.  According to data not shown, it was claimed that the same beneficial effect could not be observed with an ApoB-DGAT2 siRNA combination, suggesting that Mtp may be the better target for co-knockdown strategies.

It should be added, however, that the day 14 time-point data these conclusions were based on were somewhat of an outlier as at this time the co-formulation with DGAT2 siRNA reduced the LDLc-lowering potency of Mtp knockdown.  On the other hand, the scientists report (also in data not shown) that they tested the co-inhibition strategy using DNA-directed RNAi and thus validated this conclusion.    Moreover, given the multitude of genes involved in lipid metabolism, the Merck scientists stated that the Mtp-DGAT2 co-knockdown is a proof-of-concept and that other targets besides DGAT2 are also being considered (especially Gpat1).

Given Tekmira’s interest in ApoB as a target and recent LNP work by Alnylam and their PCSK9 collaborators from UT Southwestern (Horton, Goldstein) on SCAP knockdown to alleviate hepatic steatosis and Alnylam's general interest in co-knockdown for metabolic applications, Merck will not be alone in their endeavor of finding an RNAi Therapeutic candidate that can do it all, LDLc lowering, triglyceride lowering, and more.  Given that SNALP technology would likely be used in such a clinical program, this could particularly benefit Tekmira.  

New Clinical Results Put ALN-PCS Firmly in Business in PCSK9 Race

Alnylam reported on Friday updated results from the phase I single-dose, dose-escalation trial of its hypercholesterolemia candidate ALN-PCS02.  Importantly, the data demonstrate that ALN-PCS can produce clinically meaningful 30-40% reductions in LDLc (the ‘bad’ cholesterol) for much of the 28 study days of the study after only a single infusion of the drug.  This bodes well that ALN-PCS may be useful for the millions of patients that cannot achieve their LDL cholesterol goals despite the availability of statins.

After presenting solid PCSK9 knockdown results in January from subjects receiving up to 0.25mg/kg of the PCSK9 siRNAs which are critically enabled by Tekmira's SNALP delivery technology, the latest data include the results from subjects receiving 0.4mg/kg PCSK9 siRNAs after the decision had been made to increase the top dose based on the favorable safety-efficacy profile thus far.  This was important because despite the solid plasma PCSK9 reductions seen up to 0.25mg/kg (roughly 50% mean reductions throughout the 28 days), both the peak and average mean LDLc reductions until then had been less than 30% and 20%, respectively.  Probably not enough to effectively compete in the hot PCSK9 field.  

Based on the latest data, which included impressive >80% target plasma PCSK9 reductions, it is reasonable that ALN-PCS will be able to achieve average (=sustained) ~45-50% LDLc reductions in a once-every-4-week dosing regime.  This would put it just slightly behind Regeneron’s PCSK9-targeting monoclonal antibody REGN727 in terms of LDLc lowering.  It may be more difficult for ALN-PCS to achieve the LDLc lowering efficacy of Amgen’s PCSK9-targeting antibody AMG145 which has been hailed at the recent ACCconference to produce LDLc lowering of ‘up to’ ~60-80% at apparently pristine safety and tolerability.  I should add, however, that the warts-and-all data on AMG 145 have yet to be made publicly available.

In light of the intense competition in the PCSK9 space and a possible potency disadvantage for this surrogate marker, it will be interesting whether, firstly, the pharmaceutical industry (note, a larger partner will have to be found to financially shoulder the unavoidable outcomes trial), and eventually the market-place will adopt this new class primarily for their absolute LDLc reducing ability or in order for patients to achieve their lipid goals.  Some LDLc proponents, of course, stress that the lower the LDLc, the better.  On the other hand, whether a drug can lower LDLc by 50% or 70% won’t matter for many patients when it comes to simply achieving standard LDLc goals of 100mg/dL or even 70mg/dL.  In that environment, the focus in making the choice between competing drugs would shift to factors beyond mere LDLc lowering potential, particularly safety.

While ALN-PCS is mainly suffering from the fact that it is given following pre-treatment with corticosteroids, something that also had important impacts on the PCSK9 and LDLc efficacy data, the once much-touted safety profile of the PCSK9 antibodies have taken a significant hit with the publication of phase II data from REGN727 (McKenney et al. 2012).  In addition to one serious adverse event, a case of potentially life-threatening inflammation of blood vessels (leukocytoclastic vasculitis) that, however, readily resolved after giving corticosteroids, there was an apparently dose-dependent, adverse event-related increase in the rate of discontinuations, with ~15-20% discontinuing treatment in the biweekly dose cohorts in study 11565.  Next to the case of vessel inflammation, the AE-related discontinuations involved cases of neutropenia, fatigue, injection site rashes, chest pain, and headache/nausea. 

Amgen has yet to publicize more detailed data for AMG145.

Next to multi-dosing and the safety and efficacy impacts of omitting corticosteroids in future studies, another important question for ALN-PCS that remains to be answered in the upcoming trials will be testing the candidate in patients on statins.  REGN727 encountered problems here as patients concomitantly taking statins experienced an LDLc rebound effect after around 2 weeks.  Whether this is a class effect of agents merely blocking extracellular PCSK9 (such as antibodies), instead of inhibiting the synthesis of both intra- and extracellular PCSK9 such as ALN-PCS is an interesting question.  

This mechanistic difference could also be the big wildcard when it comes to the ultimate arbiter of clinical utility, the outcome trials: despite the clear evidence that LDLc levels are inversely related with cardiovascular events, with all the complicated feedback loops of lipid biology and related diseases, mechanistic differences could easily erase 20% differences in LDLc lowering.  An indication that these theoretical considerations are translating into the clinic may be deduced from the observation that (in Alnylam's words) ‘importantly, ALN-PCS demonstrated consistent clinical activity toward both PCSK9 and LDL-C independent of baseline levels of PCSK9, highlighting the unique mechanism of action for a PCSK9 synthesis inhibitor.’

For Alnylam, the near-term challenge will be thus to find a large partner, probably without a stake in PCSK9 yet, willing to take the risk with RNAi Therapeutics mainly for the differentiation it offers over the other PCSK9 approaches.  A stumble of the monoclonal antibody class could easily mean that ALN-PCS becomes the lead candidate in the PCSK9 market, estimated by many to be a multi-billion dollar one. 

Metabolic Disease Drug Development Anxieties Highlight Benefit of Diversified RNAi Therapeutics Pipeline

Like the commodity bubble, it seems that the regulatory hurdles to drug development are mounting just when we thought we had reached the top. This is the impression one gets from the vote taken by an FDA advisory panel this week that can be essentially boiled down to the presumption that lowering blood sugar increases the risk of heart attack. The prospect of longer and more expensive clinical trials has certainly caused a few mini heart attacks to a number of drug companies developing medicines for diabetes and other metabolic diseases thinking that the obesity epidemic in the prosperous half of the world was bad enough such that regulatory agencies, politicians, and certain clinicians involved in drug development would have a keen interest in new treatment options. Don’t get me wrong here, I see very well the problem of millions of real patients and maybe not-so-needy folks taking drugs from which they do not derive any benefit, but there certainly are other ways of ensuring drug safety and efficacy without rendering drug development, and eventually the cost of drugs just unaffordable to anybody, and often without good reason.

Now that I got this off my chest, what does the broad attack on metabolic disease drug development mean for RNAi Therapeutics?

Metabolic disease, because of the ability of current systemic delivery methods to effectively knock down genes in the liver which is critically involved in regulating blood sugar and lipid levels, is one of the main therapeutic focus areas of current RNAi Therapeutics development. It shares this position with cancer, followed by respiratory, ocular and CNS-related disease. It may therefore be tempting, if you have the ability to target a range of hot metabolic disease targets, to create a development pipeline based on metabolic disease and little else. ISIS Pharmaceuticals from the related field of antisense therapeutics is probably the best example here as it has very much limited their internal drug development activities on metabolic disease, while handing off some of the more challenging targets such as for cancer to one of their many satellite companies.

Of course, if you don’t have the capability to diversify, concentrating on building a franchise around a single, but potentially very lucrative market may be the most efficient way of maximizing shareholder value given the many synergies derived from largely having only to exchange the siRNA inside and comparatively few other changes. However, if you can afford it and you are interested in establishing RNAi Therapeutics as a broadly applicable drug development platform, you probably would want to spread your risk more widely even if all the programs added up individually would yield a higher valuation. The fact that the regulatory environment may change overnight while drug development is a 12-15 year effort being one reason.

Next to insuring from regulatory risk, diversification also means that failure of one drug candidate such as for a given organ does not necessarily have to impact the perceived probability of success for candidates aimed at other organs. In the same vein, it may also be wise not to be too dogmatic about delivery technologies, siRNA modification or DNA-directed RNAi methods and structures. For example, if your entire IP claim depends on just one siRNA modification pattern even without so much as functional validation in a non-human primate, you may have ended up totally reliant on clinically non-viable chemistries. Related technologies have shown that developing chemistries in generations rather than multiple chemistries in parallel may mean that if your chosen chemistry generation turns out to be either inefficient or unsafe, it may take another 7 years to get a shot with the next generation.
Meanwhile, all bets are off what steps the FDA will take next.

The Race to Knocking Down Cardiovascular Disease

Given the burden of cardiovascular disease in the Western world representing a multibillion dollar drug market, finding a drug to complement statins in reducing complications due to high levels of bad cholesterol is naturally high on the priority list of many drug developers. The recent OTS Meeting and a Press Release by Alnylam emphasising their leadership by having obtained first-ever data on safely and effectively knocking down PCSK9 with RNAi in non-human primates, illustrate home the promise of RNA-based therapies for CVD. The interest is largely rooted in the fact that targets such as PCSK9, ApoB100, and potentially microRNA-122, well known determinants of blood cholesterol levels, but which have proven impossible to target by traditional small molecule approaches. Moreover, these targets are expressed in the liver, and it is clear by now that current systemic oligo delivery technologies allow them to be knocked down in vivo. Hence, the race is on to who will be first to develop a safe and efficacious oligonucleotide-based therapy for hypercholesterolemia and stand to reap the benefits of a potential blockbuster in the first phase of RNA-based drugs.

Assuming that it is a safe bet that cholesterol levels can be reduced with oligo-based strategies, what will determine regulatory success? Given that low cholesterol is a life-long effort, any drug taken over a long period of time, even before disease onset, will have to be safe first of all. Risk can be largely grouped into four categories: target risk, risks inherent to the therapeutic platform, sequence risk, and risks associated with route of delivery and drug formulation. Arguably the target best validated on the grounds of human genetics is PCSK9, a protease that degrades LDL-receptors and therefore inhibits clearance of bad cholesterol from circulation. Research mostly from the University of Texas Southwestern has shown that mutations that increase the activity of PCSK9 increase cholesterol levels, whereas individuals with nonsense mutations in PCSK9 that reduce PCSK9 activity have lower cholesterol levels and, importantly, a much reduced risk for cardiovascular events. Moreover, the absence of any functional PCSK9 throughout life has no obvious adverse side-effect while retaining the health benefits of low cholesterol.

Before PCSK9 came to the fore, ApoB100, a protein required for the assembly of LDL-cholesterol, used to be the target of choice. Indeed, the development of PCSK9-based treatment strategies have extensively made use of ApoB100 as a marker protein for evaluating RNAi delivery and knockdown in the liver. Pioneering research mostly by ISIS Pharmaceuticals has shown that indeed ApoB100 knockdown has the ability to lower LDL-cholesterol. Although ISIS has not seen fatty liver in clinical trials and preclinical research of their lead antisense compound ISIS 301012 (currently in late phase II) to be a problem, various other groups have observed this side-effect following ApoB100 knockdown, which would not be that surprising given the role of ApoB100 in fat metabolism. However, even if fatty liver will be observed in larger phase III trials and post-approval, ISIS has made the right decision to test 301012 first for patient populations most at risk for CVD.

Similar to ApoB100 and PCSK9, inhibition of microRNA-122 by antisense technologies has been now shown numerous times to also have LDL-cholesterol lowering effects. Strangely, despite the fact that this is by far the most abundant microRNA in the liver, no obvious toxicities have been associated with miR-122 inhibition. Consequently, a number of groups such as Regulus and Santaris hope to develop this into a treatment for hypercholesterolemia.

Taken together, my bet is on PCSK9 knockdown to lead the way in oligo-based therapies for the long-term treatment of hypercholesterolemia. New targets, however, should emerge, partly as a result of now being able to apply RNAi itself for target identification, for example by transiently targeting essentially any gene of interest in the liver in vivo and the use of transgenic RNAi mice (Artemis), a combination of the two latest Nobel prize-winning technologies.

Next to target choice, the nature of the knockdown technology, antisense versus RNAi, itself will also have important safety implications. As I am quite fascinated about the prospect of RNAi for various reasons, please keep in mind that my natural inclination is to favour RNAi any time. In terms of potency, once equal amounts of oligos get delivered into the cell, RNAi has been shown frequently to be generally superior to antisense oligos (ASO), although antisense technologies can be quite diverse. Lower dosages will not only reduce cost of a treatment that has to be taken long-term, but, more importantly, allow for dosages that fall well within therapeutic windows. Moreover, in the case of RNAi, I feel quite comfortable with a technology where the risks such as immuno-stimulation, off-targeting, and potential interference with the endogenous microRNA pathway are reasonably well understood, intensely studied, bioinformatics- and chemistry-based solutions devised, and well taken into account in current RNAi-based drug development efforts. This in fact reflects a new awareness in RNA-targeted therapies, largely driven by the renewed interest generated by the discovery of RNAi. Accordingly, the therapeutic utility of any two RNAi compounds, or antisense compounds for that matter, may differ dramatically due to sequence-dependent toxities.
These toxicities may also be linked to route of delivery and related oligo formulation. A technically quite uncomplicated approach, as taken by 301012, is to simply administer relatively large amounts of unformulated oligos (200mg/week in the case of 301012) to make sure that enough of it ends up in the liver. By contrast, liver uptake of siRNAs is thought to require additional formulation. Indeed, liposomal formulations that are set to enter the clinic within the next year increase liver uptake of siRNAs from less than 1% of injected material to over 30%, allowing for lower dosages to be used. Some toxicities, however, were observed at relatively high dose levels with some of the cationic liposomes, and it remains to be seen whether lipidoids and other “not-so-cationic” liposomes will come to dominate the liver delivery field. Also, while most of the disclosed liposomal delivery vehicles efficiently enhance liver uptake, they are often not specific for uptake into the hepatocyte population in the liver, the cell type of interest. Particularly uptake into Kupffer cells, a type of immune cell in the liver, can lead to dosing and safety complications, and ultimately the path taken recently by scientists from Mirus, which by the way has an RNAi delivery collaboration with Pfizer, to specifically target formulated siRNAs to hepatocytes, but not other liver cell types, may substitute non-specific liposomes in the second wave of RNAi-based therapies for hypercholesterolemia. While delivery is often described as the Achilles Heel for RNAi therapeutics, the charge (ironically) and chemical similarity of siRNAs as a class makes them ideally suited to devise drug targeting strategies that can be broadly applied and should lead to safer therapies, something that is nearly impossible for say small molecules.

ISIS’ ApoB100-targeting antisense 301012 has good chances of becoming the first oligo-based therapy for CVD, at least for people with familial hypercholesterolemia and for whom statins don’t work. Although only a fraction of the overall market, the sheer size of the cholesterol market makes this a lucrative goal nonetheless. I am somewhat surprised that, to my knowledge and despite potential target risk, there is little talk of other ApoB100-targeting therapies. It will be interesting to see what companies like Merck, which has clearly stated their admiration for 301012 at the last OTS Meeting, are willing to pay for rights to 301012. PCSK9-targeting therapies are in late preclinical development and therefore about 3 years behind 301012, but I believe these to be the safest bet for a widely applied oligo-based drug for hypercholesterolemia with a number of organisations ramping up their PCSK9 programs.

Alnylam appears to be leading this race with the recent announcement of first-ever non-human primate data of an RNAi compound that safely and effectively knocked down PCSK9 with concomitant reductions in total and LDL-cholesterol. An IND is planned for the end of this year, or early next year, and probably will depend on finding the delivery solution that most importantly is safe for long-term administration. Importantly, Alnylam enjoys a particularly strong IP position and know-how in targeting PCSK9 by RNAi, due to their own position in fundamental RNAi technology, and important collaborations on the biology of PCSK9 with UT Southwestern, which has been leading in the genetics of PCSK9, as well as in delivery with the Anderson/Langer lab at the MIT and exclusive access to Tekmira’s cationic liposomal delivery IP for RNAi. Sirna-Merck may want to dispute this with an patent on targeting the same PCSK9 by RNAi that issued recently and was filed in July 2006 as part of their brute-force approach to patenting genes for RNAi. Alnylam, however, presented their first PCSK9 RNAi data in mice at last year’s 2nd Annual OTS Meeting, and it is anybody’s guess when their or rather UT Southwestern’s first lab-book entry on PCSK9 RNAi occurred. Probably at a similar stage to Alnylam is the PCSK9-antisense collaboration of ISIS with Bristol-Myers Squibbs for which mouse data have been published earlier this year. Santaris’ antisense compounds for PCSK9/ApoB100 and miR-122 should also be heading soon towards the clinic.

New delivery technologies, including oral formulations, and targets should ensure that the oligo-CVD field will remain lively in the years to come. Also, since there have been a number of recent data demonstrating efficient targeting of RNAi to the endothelia of blood vessels, new RNAi strategies aimed directly at the atherosclerotic plaques may emerge.

It would not be the first time that several similar compounds, small molecule, antibody or recombinant protein, with essentially the same molecular targets, would co-exist in a market, a concept also very familiar to the hypercholesterolemia field. IP, careful clinical development involving the best scientists in both oligonucleotide technology, delivery and the biology of the drug targets, together with a bit of luck, will decide who will reap the largest benefits from the potentially first knockdown blockbuster.

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.

Preventing Heart Disease with RNAi Therapeutics

High blood cholesterol, a major risk factor for heart disease, already is an enormous public health problem that is likely to worsen given current lifestyle and demographic trends. Fittingly, statins, a class of small molecules that inhibit cholesterol synthesis and therefore have a wide range of effects that together lower the risk of developing heart disease, are probably the best-selling drugs on the market today. However, many patients still do not achieve their cholesterol goals and there is significant demand for new innovative treatment options.

An RNAi Therapeutic alternative, due to the ability to target almost any gene, is particularly interesting because of well validated targets, but which have proven refractory to targeting by the existing drug classes. These genes are not directly involved in cholesterol synthesis and targeting them should be synergistic with statins. Currently the most interesting gene targets are ApoB100 and PCSK9. ApoB100 is the sole protein component of “bad” LDL cholesterol and is produced in the liver. Notably, targeting ApoB100 by RNAi in the using cholesterol-conjugated siRNAs in 2004 by Alnylam scientists was also the first demonstration of gene silencing following systemic administration of siRNAs. This study not only showed considerable reductions of ApoB mRNA and protein, but also the hoped for decrease in blood LDL cholesterol. I should add that clinical data from phase I and II trials conducted by ISIS Pharmaceuticals using antisense oligo technology further document the promise of ApoB100 as a target for hypercholesterolemia. It will be interesting to follow their further clinical progress, but I expect siRNAs to do even better, because of increased specificity and potency thus allowing for lower amounts of nucleic acids to be administered.

Two years later after the demonstration of systemic RNAi in mice, Alnylam scientists then reported even enhanced ApoB-100 silencing and improved lipid profiles in monkeys, this time using liposomal formulations originally developed by Protiva Biotherapeutics. These and similar liposomal formulations have proven to be very efficient for liver gene knockdown in general and are now being pursued by a number of companies in preparations for the first systemic RNAi clinical trials. Unfortunately, however, their promise has also led to legal haggling as to who owns the IP behind this delivery technology. Companies involved in this dispute involve Protiva, Inex Pharmaceuticals, and Sirna Therapeutics/Merck and I hope that legal issues will not do further damage to the development of this promising delivery technology. No matter who owns the commercial rights to the technology, Protiva scientists have to be credited with this major achievement.

Interestingly, despite their publication record on ApoB100, Alnylam decided to target PCSK9 for the treatment of hypercholesterolemia. Although I cannot exclude that this move is partly due to a deal with one of their collaborators in siRNA delivery, PCSK9 has a lot riding for it. In fact, they are pursuing this program in collaboration with scientists from UT Southwestern Medical Center that arguably are world-leading in the genetics of hypercholesterolemia. PCSK9 itself is a protease that degrades LDL-receptors (LDL-R). LDL uptake by the liver is important for clearing LDL in circulation and it is expected that increasing LDL-R levels by suppressing their inhibitors should lower LDL cholesterol. Indeed, data presented at this year’s Keystone Meeting support this thesis. However, it should be kept in mind that many drug development projects fail, not because the drug fails to reach its target, but because of side-effects. Side-effects are a particularly important consideration for drugs that have to be taken chronically as is often the case for hypercholesterolemia. So one of the major questions here is whether long-term downregulation of PCSK9 can have adverse consequences. Here, the genetics of PCSK9 are compelling: Naturally occurring mutations in the human population that increase PCSK9 activity have been shown to increase LDL and lead to hypercholesterolemia, while those that inactivate it lower LDL dramatically- without any obvious detrimental consequences! Of course, compensation mechanisms cannot be excluded, but this is probably as good as you can get with choosing a target based on human genetics.

In summary, due to the availability of excellent “non-druggable” targets and the ability to knockdown genes in the liver with current delivery technologies, RNAi Therapeutics are a promising strategy for treating hypercholesterolemia. Alnylam is expected to initiate phase I studies in the second half of this year, and I would not be surprised to see further studies being initiated in the near future by Protiva or Inex (mere speculation here though). The major obstacle for these trials that I see are side-effects due to the liposomal formulations, and my advice would be to carefully characterise them in animal models before committing to phase I instead of simply bowing to investor expectations.