Dicerna Focuses Operations Following Settlement With Alnylam

Last Friday, Dicerna and Alnylam announced that they had settled all ongoing litigation between the parties just as the acrimonious fight was about to court. 

Settlement terms

As part of the settlement, Dicerna will pay Alnylam $25M in stock and cash and drop its anti-competitive practices counter-suit against the RNAi behemoth.  In addition, Dicerna has agreed to not pursue certain targets/indications (see discussion below).  In return, Alnylam will drop its trade secret lawsuit it had filed on grounds of suspected GalNAc-conjugate trade secret misappropriation in the wake of Alnylam’s acquisition of the Merck RNAi assets in early 2014.

Litigation background

Allegedly, ex-Merck employees who had been hired by Dicerna had taken along competitively advantageous information that Alnylam claimed that it paid for.

Personally, given the timing and fuzziness of the litigation, I had always believed that Alnylam primarily filed the lawsuit to drain a much smaller, but direct competitor of vital financial resources.  In particular, even if these ex-Merck employees took with them information important to Alnylam, Merck obviously didn’t protect such knowledge as expected for trade secrets.  Moreover, Dicerna’s application of GalNAc-RNAi conjugate looks extremely different from that of Alnylam.  Even a more negative outcome should therefore not have adversely affected Dicerna's ability to further develop its technology.  

Coming out from underneath the litigation cloak

Still, this strategy had almost worked out.  Ironically, it was Alnylam’s own success in the clinic (à primarily Patisiran phase III APOLLO data last fall, but also GalNAc-related data) that had provided Dicerna stock with sufficient strength for the company to be in a position to ward off the existential threat posed by the litigation: $25M just 15 months ago would have been a mortal blow to ~$50M market cap minion Dicerna.  Today, the $600M market cap makes the $25M almost immaterial.

Nevertheless, based on the extremely bullish market reaction to the deal (up close to 40% following announcement of the settlement before settling the day at +18%), I believe that an important facet of the deal has been widely underappreciated.  Specifically, the fact that Dicerna had to agree not to pursue certain targets could completely change the face of this company. 

From the Alnylam press release:

'Dicerna will be restricted in its development and other activities relating to oligonucleotide-based therapeutics directed toward a defined set of Alnylam targets, for periods ranging from 18 months up to four years.'

Instead of a broad pure-play GalNAc RNAi company, Dicerna now probably needs to be considered a 5-or-so development candidate company for which the end game will be its sale to a larger company.  Except for the long-promised deal on the mystery candidate (AAT?), this reduces expectations for future strategic blockbuster deals.

Disclosure: Especially in light of the target selection restrictions, I view the settlement as incrementally negative for Dicerna and expect the market to realize the same soon.  Following Friday's strong move to the upside, Dicerna is a short-term conviction short with a $10 near-term price target.  Also expect Alnylam to sell the 2M million Dicerna shares almost as soon as it receives them (please read comments section below for correction).  Long Alnylam.

Endocyte Success to Catalyze Interest in Folate-Targeted RNAi Therapeutics

One of the big stories on Wall Street this Friday was Endocyte and their folate-targeted drug vintafolide. All on the same day, Endocyte received a positive opinion by the CHMP in the EU, paving the way towards marketing authorizations of vintafolid in ovarian cancer in this important market.  Simultaneously, it announced encouraging phase II data in non-small cell lung cancer (NSCLC) in which the drug, in combination with chemotherapy agent docetaxel, met the primary endpoint in extending progression-free survival (PFS).

I hope I got all of that right.  What is more important in the context of RNAi Therapeutics and this blog is that folates have been considered by the field as a promising targeting agent in the past and the successes by Endocyte and progress in RNAi delivery could lead to a new push in that direction.

The idea behind vintafolide is simple.  Since non-targeted small molecule chemotherapeutics inhibiting basic cell division processes cause widespread toxicity in normally proliferating tissues (e.g. GI tract, bone marrow), often times preventing their use at therapeutic dosages, targeting such agents specifically to cancer cells should greatly increase their therapeutic window and utility.  In the case of vintafolide, the chemotherapeutic agent (DAVLBH) is targeted towards the folate receptor that is overexpressed in many tumors as tumors have an increased demand for folates to support their growth.

Following binding of the folate drug-conjugate to the folate receptor, the conjugate is taken up by the cancer cell through endocytosis.  Once in the endosome, the folate falls off the drug which in turn is then free to diffuse into the cytoplasm to inhibit cell division by binding to tubulin. Meanwhile, the folate receptor recycles back to the cell surface.  All analogous to ASGPR and galactose in the liver.

One challenge with folate-targeted RNAi Therapeutics that I have heard a number of times, is the concern that folate receptor expression is not sufficiently uniform to get at all/most the cancer cells.  This assumes that in order to be successful, a cancer RNAi Therapeutics would have to reach the majority of cancer cells.

Interestingly, vintafolide is used in combination with a companion diagnostic comprising folate with a molecule that can be imaged.  By first running the imaging test, Endocyte has been able to focus their studies on those patients which express folate receptor on all their tumor masses.  Importantly, apparent anti-tumor activity correlated with folate receptor expression.  Such enrichment strategies could obviously also be applied to folate-targeted RNAi Therapeutics.

Imaging tests, however, would not be able to address folate receptor expression heterogeneity within a tumor mass.  It would be interesting to know to what degree vintafolide activity was restricted to the cell that has taken it up or whether it could also act on by-stander cells.

RNAi Therapeutics companies that could take an interest

I can remember Alnylam-related OTS presentations before 2009 that looked at simple folate-siRNA conjugate delivery with promising localization data.  Knockdown efficacy, however, seemed limited. 

This is not surprising given the absence of endosomal release chemistries in those molecules.  Endosomal release of receptor-targeted RNAi Therapeutics could obviously be addressed by Arrowhead’s DPC technology.  A folate-targeted single molecule DPC would be my front-runner in realizing the potential of folate receptor-targeting for RNAi Therapeutics. 

Curiously, Merck has partnered with Endocyte on vintafolide and as we know had been busy copying delivery technologies by Arrowhead and Tekmira.   It would therefore greatly surprise me if Merck had not looked at folate-targeted RNAi Therapeutics, which might strengthen Alnylam’s effort in developing folate-targeted DPCs following their acquisition of Merck's RNAi assets in January.

In addition to the conjugate companies, liposomal players such as Tekmira and Dicerna could get in on the folate action as well.  This is because receptor-mediated endocytosis is believed to play an important role in the cellular uptake of liposomes that are not constitutively positively charged (àApoE-LDL receptor).  By adding folate to the mix, one could a) extend the utility of a liposomal cancer RNAi Therapeutic to cancers overexpressing either receptor, and/or b) enhance cellular uptake by interacting with 2 receptors simultaneously on cancer cells that express both receptors.

Alnylam Acquires Merck’s RNAi Assets and Gets a $700M Investment from Genzyme

I have expected RNAi Therapeutics business development activities this January, but I have to admit that when these deals are actually announced it gives me an adrenaline rush each time.  For $175M plus some milestones and royalties, it was announced a few hours ago that Alnylam will acquire the RNAi assets from Merck.  Merck had just undergone a corporate re-organization that I had speculated could spell the end of Merck’s RNAi Therapeutics platform development efforts, especially since protein guy Roger Perlmutter replaced RNAi supporter Peter Kim as Merck’s head of R&D (Merck’s RNA(i) Therapeutics Unit on the Chopping Block).  That's how it sometimes works, no complex science, just tastes and egos.

In addition to cultural issues, the transaction also solves the problems that the bean counters in Big Pharma face when justifying investments in emerging technologies.  How e.g. do you take into account that much of the value of a platform technology could be outside of your core areas of therapeutic interest?  Who e.g. would have predicted that TTR amyloidosis and HBV would materialize as the two commercially most attractive near-term opportunities when Merck acquired Sirna Therapeutics in 2006?  As an added sweetener for the bean counters, the write-off will allow Merck to exceed financial guidance in one of the coming quarters.

The $175M price tag has two important implications.  Firstly, for a biotech company the size of Alnylam, putting that much into the acquisition of largely IP is a vote of high confidence in RNAi Therapeutics.  This is in sharp contrast to the days when Alnylam made deals with Roche and Takeda and must have felt as if they made out like bandits by selling IP of uncertain value given the questions around Alnylam’s access to delivery at the time.

Secondly, the $175M indicates that there was competitive bidding going on for the assets, most likely by other Big Pharma companies.  In fact, I had suggested to some of the other RNAi companies to take a look at the assets in the hope of another Roche-Arrowhead-like 'steal of the century'.  The fact that Alnylam won out is likely explained by Merck being the best strategic fit for Alnylam whereas other Big Pharmas may be better off in getting unfettered access to RNAi Therapeutics via the likes of Tekmira and Arrowhead Research as Merck's RNAi assets, by largely copying the efforts of others, always seemed to overlap with others (except for modification chemistry) 

So what will the $175M get Alnylam?  The most valuable aspect to Alnylam should be Merck’s development efforts in polyconjugate technology where Merck has been copying Arrowhead’s efforts.  Thus, instead of spending something like $1B, likely the current acquisition value of Arrowhead Research, it was able to get access to a similar technology for much less.  Although Merck also pursued liposomal delivery, since Alnylam already has access to Tekmira’s technology, the add-on value of Merck’s liposomes to Alnylam is more limited.  Finally in terms of technology, Merck has conducted a deep screening of nucleic acid modifications which can now be incorporated into Alnylam’s RNAi trigger discovery and conjugate development efforts.

Another important aspect to Alnylam will be safe-guarding the value of the Tuschl II IP.  Merck got uncontested access, including sublicensing rights, as part of a legal settlement between the two companies in 2011.  Tuschl II is still an important asset to Alnylam and in a worst-case scenario, Merck’s disposition of their RNAi assets would have meant giving away access to Tuschl II to various competitors.

Genzyme Buys into RNAi Therapeutics Big Time

The Merck technology acquisition will be funded by the $700M investment of Genzyme into new Alnylam stock at a 25% premium to current trading (~12% of the company) plus additional associated biotech goodies to come (correction: only $25M of Merck's $175M was in cash, $150M in shares).

I know that I am starting to come across like a know-it-all, but this transaction did not come all that surprising to me, too.  Importantly, 3-4 years ago Alnylam started to lease out significant lab space to Genzyme in Cambridge, Mass.  This seemed odd to me since Alnylam was not in the business of dressing up its financials by moonlighting as a landlord.  Surely, something more strategic was going on here. 

When a little more than a year ago, Genzyme took the Asian rights to ALN-TTR02, it seemed a bit of a downer.  In retrospect, however, it is apparent that Genzyme took that license as a starter before more validation of the technology in the form of phase II results with ALN-TTR02 and phase I results with ALN-TTRsc would trigger a more expansive deal like the one today.  Specifically, today's deal gives Genzyme Sanofi-Aventis limited commercialization rights to at least 3 additional Alnylam drug candidates in the orphan drug space.

And finally, attention Big Pharma bean counters, a lesson of that transaction is that all you need to do to financially justify investments in RNAi Therapeutics is to create a line item called ‘orphan drugs’ and account for RNAi Therapeutics under that label. 

Welcome to a new pharmaceutical world.

Merck’s RNA(i) Therapeutics Unit on the Chopping Block

After more than 6 years of playing it safe and producing nothing tangible in terms of clinical development candidates, I fully expect that the time has come for Merck’s RNA(i) Therapeutics unit to be slashed.  This morning, Merck announced a company-wide cost-cutting re-org, as part of which it aims ‘to reduce its focus on platform technologies’.  Remember Roche 3 years ago? 

Similar to Roche, the immediate financial benefits of scrapping the unit not only come in the form of savings in R&D expenses, but also likely tax write-offs due to for example the $1.1B purchase of Sirna Therapeutics in 2006.  Expect the company to exceed analysts’ expectations for coming financial results.

However, the most important factor why I believe the unit's fate had been sealed was the departure of Peter Kim as the Head of Merck Research Laboratories.  It has been said that he had been a key backer of the unit, and with the biologics guy from Amgen, Roger Perlmutter, probably intent to erase Dr. Kim’s legacy…

It is hard for me to feel sorry about such an event.  Their RNAi science was probably the best among the Big Pharmas, but hiding behind the VIOXX experience forever while taking home monthly salaries was asking for trouble.  Meanwhile, the likelihood that Merck will partner with the real innovators in the space on specific product candidates has increased today. 

Merck Paper Reveals Interest in GalNAc-targeted RNAi Therapeutics

Merck’s efforts have to be considered to be the strongest in RNAi Therapeutics among Big Pharma.  Its RNAi Therapeutics strategy so far, however, has consisted largely of trying to replicate the most promising technologies in-house. 

The latest publication by Merck RNAi scientists on the expression pattern of the asialoglycoprotein receptor (ASGPR1; Shi et al. 2013) confirms this as ASGPR is the target receptor of the two most advanced SNALP alternatives for gene silencing in the liver: the trail-blazing DPCs by Arrowhead Research (first use of GalNAc-targeted RNAi Therapeutics in Rozema et al.2007) and more recently the GalNAc-targeted siRNA conjugates by Alnylam (what these really seem to be will be covered in an upcoming blog...so stay tuned!).

Surveying ASGPR expression levels

When developing ligand-targeted therapeutics, it is important that the corresponding receptor is present on the target cell population.  Especially when targeting cancer, it can be difficult to find receptors that are not only present in large quantities, but also throughout the cell population.  

In the case of liver cancer (hepatocellular carcinoma/HCC), this question has occupied the RNAi field ever since the finding that SNALPs work really well for knockdown in normal liver due to uptake mediated by the LDL-receptor.  But is this mechanism also present on cells in liver cancer, a cancer of high unmet need where RNAi Therapeutics could have the biggest impact in oncology near- to midterm?

 

The Merck scientists set out to answer essentially the same question, but instead of interrogating LDL-receptor expression, they wanted to know about ASGPR expression on HCC cells.  Using tissue microarrays, they were able to test an impressively large set of 100s of tissues, including healthy human livers, liver cancer biopsies, and biopsies for other hepatic diseases such as viral hepatitis, chronic active hepatitis and cirrhosis.

Despite some apparent limitations with the tissue microarrays (e.g. normal liver samples were often marked as false negatives despite the known very high ASGPR expression level), the results seem to confirm that liver cancers in general have a tendency towards lower ASGPR expression and that inter-sample heterogeneity is comparatively large (some liver cancer samples had much more ASGPR expression than normal liver).  

This suggests that just as in the case in breast cancer where treatment decisions are often based on receptor expression levels, ASGPR-targeted liver cancer RNAi Therapeutics should also be combined with a companion diagnostics for ASGPR. 

For those curious about the status of ASGPR expression in chronic HBV since Arrowhead’s exciting ARC520 program for this indication involves a GalNAc-targeted melittin-like peptide, relax: livers infected with HBV express ASGPR just as well as normal livers.

Home-brew versus licensing

Based on the literature and conference presentations, it is reasonable to assume that Merck is well behind Arrowhead, and even Alnylam in developing GalNAc-targeted RNAi Therapeutics.  This begs the question, as it has in the case of SNALP, why does Merck not take a license or even acquire the original?

Of course, it always takes two for a deal, but given the financial capabilities of a Merck, if it wanted access, it could get it.

The likely explanation is that Merck's strategy in replicating technologies in-house is to first identify target technologies by validating them and then to develop viable IP workaround solutions.  In fact, in the apparent absence of a broad gate-keeping patent estate around GalNAc-targeted therapeutics, ASGPR has been a recognized drug target receptors well back into the 90's, there should be relatively little restrictions on the use of GalNAc per se.

Instead, a competitive advantage is largely gained through specific know-how like how to best link the GalNAc ligand to the oligonucleotide payload and synthesize the molecules cost effectively. The latter issue came to my attention recently when access to 'proprietary process for manufacturing GalNAc conjugates' from Alnylam was mentioned as the top corporate highlight in the quarterly update provided by Regulus Therapeutics.

For the aficionados, the triantennary GalNAcs as practiced by Alnylam are much more costly to synthesize than single GalNAcs as in the case of the liver-targeted DPC versions (where high affinity through multivalency is achieved by having multiple single GalNAcs along the DPC).

This situation is not unlike other areas in the drug development industry.  Take for example antibodies where, despite the various patent battles, there have been a number of commercially viable platforms based on specific libraries or optimization methods.  Nevertheless, despite this apparent freedom-to-operate, the way by which Big Pharma ended up gaining access to monoclonal antibodies was not by way of successfully developing them in-house, but by acquiring them.

I don't expect this to be any different in RNAi Therapeutics.

A Sign That Big Pharma Could Recognize the Low-Hanging RNAi Therapeutics Fruits

It was with much amusement and head-shaking disbelief that I read the Li et al. paper oncancer RNAi Therapeutics  development from Abbott.   The amusement stemmed from the fact that in the paper, the authors had come to the obvious conclusion: current technologies should allow you to develop real-world therapeutics based on the RNAi platform if only you judiciously combine the delivery platform with the right target and indication.  Duh…

As such, Abbott is representative of the various Big Pharma companies that guttered in-house RNAi Therapeutics development as they chose to only see the challenges instead of realizing the obvious opportunities.  Even those still in the game like Merck have long liked to go around and teach everyone how super-diligent, but super-slow their RNAi Therapeutics game strategy was. 

As RNAi Therapeutics have created market values at lightning speed (Alnylam now at a market cap of $3.5 billion), not based on just hype, but based on paths well-trodden by orphan drug companies, I expect more and more Big Pharmas to re-think their strategies.  Maybe even listen to this blogger who has always advocated a pipeline strategy that is based on where your delivery technologies can go to, instead of the traditional cart-before-the-ox-I-want-the-next-blockbuster-pill wishful thinking by people who may have risen to the corporate tops in companies like Coca Cola.
  

C’mon scientists from Merck, Novartis, and Takeda.  Inside yourself is an innovator that finally wants to see how your technology performs in humans.  Instead of just focusing on what could go wrong, don’t you also have an obligation to address diseases of high unmet medical need? And if not you, then who is going to transform your organization into a dynamic science-driven one that you can identify with as a scientist?  On a more practical level, I cannot see how many of you will be with your present employer in 2-3 years if you don’t have the goods to show.  Look at what happened to your peers from AstraZeneca whose last job it was to look for partnership in the more innovative pure-play RNA Therapeutics space before they were given the boot. 

Merck Hot on the Trails of Dynamic PolyConjugates by Arrowhead Research

Not only was I impressed by the Dynamic PolyConjugate delivery data presented at the late 2012 OTS meeting, many others in this peer group were, too.  This included favorable comments and keen questioning by a Merck scientist in the following Q&A session.  Not entirely surprising therefore, the presentation at OTS by Merck at the meeting revealed that it has recently shifted its delivery focus onto DPCs.  This week then, published evidence of that activity emerged in the form of a publication in Bioconjugate Chemistry (Parmar etal. 2013).  The DPC work described therein is consistent with a strategy in which the company aims at internally replicating the industry’s most promising/advanced delivery technologies, possibly hoping that not only can they overcome the scientific and manufacturing challenges, but also will find holes in the originator company’s intellectual property.  

Learning the DPC ABCs

The paper is essentially a replication of the basic DPC studies published in 2007 by Rozema et al. in PNAS.  Accordingly, they encountered much of the same challenges that the scientists at Arrowhead Research (formerly Mirus Bio, then Roche) had encountered in building the DPC delivery platform and includes issues with manufacturing yield and solubility.  The major difference is that Merck utilized a polymer backbone incorporating  disulfide bonds that one might hypothesize to be degraded in the reductive cytoplasm of cells. The fact that the first published Arrowhead DPC was not biodegradable was cited as a key toxicity issue.  All this is consistent with what Arrowhead started to report last year in explaining which challenges had to be overcome before they were able to clinically mature the technology.  Overall, the consistency of the findings with those first reported by Arrowhead gives me a good feeling about the theoretical basis for and robustness of the technology.

Lavishly Screening for Improvements

In order to discover suitable disulfide-containing polymers, Merck generated a small library of amphiphilic polymers by reacting together three types of chemical groups: an amine, an imidazole, and hydrophobic tails.  Conjugation of the targeting ligand and other masking groups that define DPCs was via the same CDM chemistry pioneered and still utilized by Mirus/Arrowhead.

The library was winnowed down via a series of tests, starting with membrane lysis assays down to tissue culture and rodent knockdown activity tests.  

Curiously, the paper lacked an assessment of toxicity in animals which was stated to be the entire point of the library and the study.  Whatever the motivation for this early publication of DPC research, this illustrates the early stage of Merck’s DPC development efforts.  The absence of published toxicity results may also mask a basic flaw with the disulfide concept: a) in order for these DPCs to fully degrade in the body, they would all have to be taken up into the cytoplasm; or b) the source of the toxicity is exclusively due to the intracellular accumulation of polymers.  Neither a) nor b), however, are not plausible in my humble opinion.

In any case, Merck likes to perform screening when it comes to optimizing RNAi technologies.  The most amazing example of this is probably their RNAi trigger modification screen involving over a hundred different types of nucleic acid modifications.   Regardless of what one might think of the wisdom of such brute-force and logically possibly flawed efforts, the size of these investments illustrate that Merck likely outspends all other companies in RNAi Therapeutics development, including Alnylam.

Merck Strategy

I am struggling to understand Merck’s strategy with regard to RNAi delivery.  It is obvious that all they are doing is to try and replicate the industry’s most promising technologies.  First it was SNALP (which they are still pursuing according to the OTS 2012 presentation), now it is DPC that they have set their eyes on.  I can only come up with two mutually non-exclusive strategies behind the approach. 

According to the first strategy, Merck wants confirm the validity of the promising reports.  If the technologies perform well in their hands they go out and license in the IP.  Extensive internal technology validation would be insurance against spending millions on technologies that turn out to be duds in an industry in which most technologies have not lived up to promises made.  Importantly, the decision to License technologies from the inventors and therefore presumably real experts would accelerate their pipeline development by years.  Of note, Merck still has not brought an RNAi candidate into the clinic since their $1.1B acquisition of Sirna Therapeutics and the pressure to achieve that milestone should be building.  According to the second strategy, Merck hopes to recreate or even exceed the originators’ achievements, and with luck can use the technologies without concern of infringing IP.  

Given Merck's significant investments which would seem to exceed those necessary for simple due diligence, Merck seems to be pursuing strategy No. 2.  It seems to be a risky strategy though because of IP uncertainty in general.  I therefore personally favor a more flexible implementation of strategy No. 2, namely one that envisions licenses and R&D collaborations if the IP situation or insurmountable technical hurdles eventually demand them. 

In any case, Merck must be doing quite well given the generous time and money spent on such RNAi Therapeutics research.  Enviable.

Merck in Talks with Arrowhead?

There are some comments in the paper that leads me to believe that Merck is actually talking to Arrowhead Research directly.  Although there may be hints in the very recent patent literature and investor presentations that biodegradability had been an issue with first-generation DPCs (note: Arrowhead is now using biodegradable peptides as the polymer backbone), and/or some of that has emerged when Roche opened up their books during their RNAi sales process, I have not seen such comments in the still relatively sparse peer-reviewed DPC literature.  Yet strangely, Merck presents the rationale for the disulfide strategy as if it was common knowledge.

Merck is not entirely foreign to Arrowhead Research.  Just last year, they entered into a research alliance concerning Arrowhead’s new-fangled peptide drug conjugates (PDC).  So there are open lines of communication between the companies.  Whether it’s something to get excited about if you are an Arrowhead shareholder, I’m not sure: in this industry, lots of parties are talking to each other without it ever coming to a deal.  Having said this, the recent results with DPC technology have been tantalizing, and for RNAi Therapeutics at Merck to survive, I would think that they have to put something into the clinic over the next two years or so or face the cost-cutting axe of the new chief of Merck Research Labs, Roger Perlmutter.  DPCs would seem to be one of the few options they have to meet such timelines.

What to Expect from RNAi Therapeutics in 2013

2012 was the most exciting year in the ~12-year history of RNAi Therapeutics- both from a scientific and financial perspective.  Left for dead by most, unambiguous gene knockdown results in Man have allowed the technology to regain much-needed respectability.  With the start of 2013, the industry is looking to build on these successes with additional clinical trial results, interesting new therapeutic candidates and product-specific and platform-related deals, particularly in the area of delivery.  With appetite for innovation increasing in a low interest rate economy and with the orphan drug tsunami, 2013 could be a quite rewarding year for the discerning investor.

Clinical results to look out for

Clinical results in 2013 that will continue to shape perceptions of the technology include phase II study results for ALN-TTR02 in TTR-FAP by Alnylam, phase I results from its GalNAc conjugate version ALN-TTRsc, and phase I results from a number of other programs, foremost from oncology drug candidate TKM-PLK1 by Tekmira, ALN-AT3 for hemophilia by Alnylam, and finally RXI-109 for dermal scarring by RXi Pharmaceuticals.  

For ALN-TTR02, it will be important to confirm the impressive knockdown results from the phase I study, but over longer periods of time and with still acceptable safety.  ALN-TTRsc will be an important proof-of-concept for the subcutaneous delivery of RNAi Therapeutics and should provide a good idea of what to expect for ALN-AT3 which is based on the same GalNAc siRNA conjugate technology.  The success or lack thereof of Alnylam’s GalNAc technology will also affect the perception of Arrowhead’s DPC technology as either a competing or necessary subQ alternative to GalNAcs.

Tekmira’s TKM-PLK1 has not gotten much credit so far.  This, however, could change with the presentation of the full phase I results, possibly at this year’s ASCO.  I consider PLK1 as the single most attractive target for cancer RNAi and I am bullish that the molecular analyses will show molecular, if not clinical efficacy at this early stage.  And while TKM-PLK1 could overcome the safety-efficacy hurdle for some indications, the importance of PLK1 as a target demands that Tekmira will continuously work on improved follow-on versions.

Finally, RXi’s second phase into RNAi for skin applications.  I also consider dermal scarring as an interesting differentiated, because cosmeceutical RNAi product opportunity.  

Cool pipeline additions

As detailed in my last blog entry, there are two exciting infectious disease drug candidates for which clinical development will ramp up in 2013: Arrowhead’s ARC520 aiming to achieve for HBV what has recently been achieved in HCV (dramatically increased cure rates and less suffering from the side effects of interferons), and Calimmune’s ddRNAi-based HIV drug candidate aiming to keep the virus out of immune cells.

It looks like we will have to wait for clinical efficacy results from these programs for a while (2014-2015), either due to the nature of the cell competition approach involved in the HIV program or because of the use of healthy volunteers.  I believe the latter is what Arrowhead has guided for ARC520, but from an investor perspective this would be highly unforunate as this would delay the demonstration of gene knockdown with the DPC platform.  And from a medical perspective, I am struggling to see what the value or necessity of a volunteer trial would be.  

Deals and Big Pharma

In addition to clinical trial results, RNAi Therapeutics investors will be getting up each morning to check the internet for whether a deal has been announced.  Alnylam’s ALN-PCSK9 is the most imminent partnering candidate and will be an indicator of the mere differentiation value of RNAi Therapeutics.  While clinically more advanced monoclonal antibody-based programs for the industry’s most desired target, PCSK9, exist, should monoclonal antibody stumble as a class, RNAi Therapeutics and ALN-PCS could suddenly have the market for itself.  Considering the multi-billion $$$ potential of PCSK9, a gamble worth taking for a Big Pharma in my opinion.

Similarly to ALN-PCS in the hypercholesterolemia market, the size and complexity of the clinical program that would be required to turn ARC520 into a major HBV drug well exceeds Arrowhead capacities, and this could mean that we will see an early licensing deal around that asset, too.  While proof-of-concept clinical knockdown data would greatly increase the partnering value of ARC520, from a financial perspective (--> di-lu-tion!) early partnering may be prudent if no alternative non-dilutive capital alternative presented itself.

As delivery is gating for all of the above RNAi Therapeutics product opportunities, delivery naturally should be the subject of a few more platform-type relationships.  Tekmira’s SNALP technology for addressing diseases of the liver, lung, and cancer tops the list for a meaningful partnership (>$10M upfront), and also Arrowhead’s DPCs for liver-targeted gene knockdown ought to see some interest.  

Delivery-related deals should also reveal which Big Pharma company is still committed to the RNAi Therapeutics platform.  For the efforts at Takeda, Merck, and Novartis (the three most significant ones in terms of investment to-date), it could be a make-or-break year.  I cannot imagine that these groups are allowed to exist in their current forms for much longer before they get anything into the clinic.  For this, they probably need to swallow their own pride and accept that expert outside help is necessary for their delivery needs (rather than attempting home-brew versions).  Given the recent clinical and late preclinical results for SNALPs and DPCs, chances that they will finally do something have certainly increased.

A Happy New Year everybody.

ISIS ssRNAi Challenge to Gold Standard RNAi Delivery Comes up Short

The esteemed editors and reviewers of CELL got it wrong this time.  Usually, when a high-impact journal like CELL decides to publish back-to-back papers on a given topic, it believes that they mark a turning point of some sort that will be cited all over.  In this case (Lima et al.; Yu et al.), the turning point would be nanoparticle, or more precisely SNALP-formulated RNAi delivery ‘out’, and unformulated, single-stranded RNAi ‘in’.

For those new to my blog: it is the double-strand feature that is a defining property of the RNAi mechanism.  While a single-stranded intermediate is generated in the process, numerous studies, including indeed the one by Lima et al., show that these are contrived, and consequently about 100-fold less potent ‘inducers’ of RNAi gene silencing.

It is therefore surprising that CELL would publish a confirmation of this.  What is new though is that the in vivo ssRNAi data involved unformulated ssRNAi application, whereas previous in vivo ssRNAi work by e.g. Merck involved LNP-mediated delivery (Haringsma et al., 2012; also covered on this blog here).  However, as detailed below, the efficacy was not impressive. To me, the main point of interest related to chemistry and how this sheds light on the basic RNAi mechanism, which actually made this paper enjoyable to read.  For example, the metabolically stable 5'-(E)-vinylphosphonate modification and the positive effect of 2’F on Ago binding.  Nevertheless, such biochemical detail is not the groundbreaking stuff that lands you a paper in CELL, but more something for the dedicated aficionado.

Does ISIS feel threatened by SNALP delivery?

The complexity of SNALP delivery, by which actually the difficulty of re-engineering SNALP technology without access to Tekmira’s trade secrets and know-how is meant, is held against the technology also in a commercially competitive sense.  If patient outcomes is the main goal, as long as you master complexity, isn’t that a good thing, especially in terms of the all-important length of market exclusivity (note that the main cost of SNALP delivery is still the siRNA ingredient)?

A common criticism of my writings is that I connect all things to Tekmira’s SNALP technology.  But read the Lima et al. paper and see for yourself how ISIS equates formulated RNAi delivery with liposomal delivery (start of the abstract e.g.): 

‘The therapeutic utility of siRNAs is limited by the requirement for complex formulations to deliver them to tissues. If potent single-stranded RNAs could be identified, they would provide a simpler path to pharmacological agents. Here, we describe single-stranded siRNAs (ss-siRNAs) that silence gene expression in animals absent lipid formulation.’  

Or

'However, in their current state, the therapeutic utility of siRNA is limited by the requirement for complex lipid formulations to deliver siRNA to peripheral tissues (Vaishnaw et al., 2010).'

It looks  like 1-billion market cap ISIS feels threatened by $40M market cap Tekmira’s SNALP technology after all and is a very interested participant in the frivolous patent infringement lawsuit against Tekmira (frivolous for the reason alone that Alnylam expressly congratulated Tekmira on the BMS deal).

SNALP requires 1000-10.000-fold less oligonucleotides

The assessment that the ssRNAi work by ISIS does not mark a turning point in systemic RNAi delivery is based on simple math.  1000 to 10.000-fold higher amounts of oligonucleotides were required to achieve equivalent knockdowns in mice: conservative 50microgram/kg/month for SNALP vs 50mg/kg/twice a week for ISIS ssRNAi.    

As with any drug, large doses increase the risk of causing toxicities.  In this case, it is particularly the accumulations of large amounts of phosphorothioated oligonucleotides in the liver and kidney that causes such concern.  Of course, SNALP LNP delivery is not entirely without its safety issues.  For example, in the clinic it still involves the use of transient immune suppression which may e.g. be prohibitive to their use in millions of patients with less severe forms of hypercholesterolemia.

Although assuming for a moment that the amount of required oligonucleotides should be irrelevant as long as it was safe, antisense technologies still suffer from poor cost of goods.  Last week for example, the CEO of another antisense company, Sarepta Therapeutics (formerly known as AVI Biopharma), wrote in an unsettling Open Letter to the Duchenne Muscular Dystrophy community that the company essentially cannot afford the large, almost nutritional amounts of oligonucleotides that are required for attempting a therapeutic splice correction.  Closer to home, instead of acknowledging their current cost of goods, ISIS Pharmaceuticals is only providing estimates for their future oligo manufacturing cost goals.

Extrahepatic tissues, which ones please?

Another claim by ISIS related to their ssRNAi tech was that it would be applicable beyond the liver ('broadly distributed and active in multiple organs'), also following systemic administration.  However, once again, this was directly contradicted by their own data which showed a maximally 35% knockdown (the type of from 100 to 65, not from 100 to 35 mind you) for such a tissue (the kidney) when administering…100mg/kg of oligonucleotides in mice. 

The accompanying Huntington’s Disease paper achieved ssRNAi-mediated knockdown in the brain.  This, however, was observed following non-systemic, intraventricular infusion of large amounts of oligonucleotides in small mice brains.

Alnylam was right in  terminating ssRNAi collaboration

Taken together, the publications explain why Alnylam decided two years ago to terminate their ssRNAi collaboration with ISIS.  In addition to using Tekmira’s intravenously infused SNALP LNPs, Alnylam has been working on GalNAc-siRNA conjugates as a subcutaneously deliverable alternative for gene knockdown in the liver.  Although the gene knockdown achievable with that technology still pales in comparison to SNALP LNP, it is about 10-fold more potent than ISIS’ ssRNAi, which goes to show that despite the disadvantages in cellular delivery of unformulated, rigid dsRNAs, their dramatically increased potency more than compensates for it.

At least Lima and colleagues and I agree on this point (opening statement of the introduction): 'RNA interference (RNAi) is a mechanism by which double-stranded RNA triggers the loss of homologous sequence (Fire et al., 1998).' [Emphasis mine]

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.  

Technology Trends: MicroRNA Inhibitors and Single-Strand RNAi

There have been developments in the areas of microRNA inhibition and single-strand RNA-mediated RNAi that might have strategic implications for delivery technologies and RNAi Therapeutics, respectively.

MicroRNA inhibition: naked antisense no more?

Currently, all development-stage anti-miR programs to my knowledge envisage the use of unformulated phosphorothioated antisense molecules with various high-affinity modifications such as LNA/LNA-type conformationally restricted nucleotides or 2’F and 2’MOE. To some degree, antisense and certain microRNA companies are making a living out of advertising that, unlike (most) RNAi Therapeutics, no intravenous administration was required.

At the same time, it is becoming clear that more complex structures such as Dharmacon’s miRIDIAN hairpin microRNA inhibitors or the tough decoys (also the synthetic versions that were newly developed in collaboration with Japanese RNAi behemoth Kyowa Hakko: Haraguchi et al. 2012) are considerably more potent on a per molecule basis. Because of their structural complexity, however, they would require delivery formulations for therapeutic use. It remains to be seen how often such formulations would need to be applied, but the early research by Haraguchi in tissue culture shows that the anti-miR effect with these structures can be relatively long-lived. Nevertheless, the in vivo pharmacology of these structured anti-miRs remains to be better explored, but I could imagine that especially for antiviral or oncology applications, the more rapid onset of action and the potentially improved targeting due to the delivery technology could yield positive surprises.

Single-strand RNAi Therapeutics: Stable 5’ phosphate and 2'F

A little more than a year after ISIS and Alnylam ended their ssRNAi Therapeutics collaboration (for which I believe Alnylam had greatly overpaid), ISIS and Merck have made progress in the area.

It had been well known based on particularly protein structural work that the 5’ phosphate modification in the guide strand is important for incorporation in the RNAi effector complex RISC. There has also been corresponding early evidence in ssRNAi research (Martinez et al 2002) that ssRNAs with a 5’ phosphate are more efficient inducers of RNAi, albeit at much lower efficicay compared to dsRNAi triggers. Notably, in the case of double-strand RNA-induced RNAi, prior 5’ phosphorylation is not necessary as this is efficiently accomplished inside the cells.

Based on work by ISIS presented at the Keystone conference in January it now seems that lability of the 5’ phosphate is partly responsible for the reduced efficacy of ssRNAi. Using a new 5’ phosphate mimic (and some other lipophilic modification strategies) that is much more stable than the natural counterpart, it was now possible, following subcutaneous administration, to achieve solid knockdown in rodents. However, the cumulative dose required to get there is still very high, in the range of 2^nd generation RNaseH antisense.

ssRNAi work just published by Merck (Haringsma et al. 2012) confirmed that the 5’ phosphate modification was important, but more importantly worked out the beneficial role of the sticky 2’-fluoro (2’F) modification in ssRNAi. The impressive efficacy-enhancing activity of the 2’-F modification (for which, I believe, Alnylam holds rights to an important patent via a license from ISIS, but a modification that has also raised genotoxicity concerns) was found to apply to both tissue culture and animal settings [Note: an earlier version mistakenly stated that the IP belonged to Alnylam]. Unlike the ISIS work, however, Merck studied ssRNAi in mice using SNALP-like liposomal delivery. It therefore seems as if Merck would need to extra work on adding chemistries such as phosphorothioates or conjugates so that its ssRNAi technology can be used in the ‘naked’ form. This is because the prospect of using ssRNAi in the naked versus formulated form would really be the only motivation for pursuing ssRNAi.

Alnylam Squares Off with Dicerna

Dicer-substrate RNAi triggers are often seen as a (probably cheaper) alternative to Tuschl siRNAs. Initially, based on a small sample size, it was even claimed that Dicer-substrates had superior potencies and prolonged durations of knockdown. Of course, Alnylam, considering Tuschl siRNAs to be its property, has recognized this and regards Dicer-substrates along with its corporate champion, Dicerna, a competitive threat. Although Alnylam has long claimed that Tuschl siRNAs are preferable over Dicer-substrates for various reasons, until now it has largely been Dicerna’s word against Alnylam’s word.

This has changed with a publication by Alnylam in the journal RNA which provides a comprehensive, and I believe fair comparison between the two structures (Foster et al 2012). Comparing large numbers of RNAi triggers both in vitro and in KC2-SNALP animal studies, the study shows that Tuschl siRNAs and Dicer-substrates are essentially equivalent in terms of potency and the duration of knockdown. However, when these structures are compared in terms of innate immune stimulation, Tuschl siRNAs had a very slight edge when unmodified sequences were tested. Of course, it is well recognized that chemical modifications are required and very effective at abrogating these immune responses. When these were applied, the potencies of Dicer-substrates were more likely to suffer than those of Tuschl siRNAs, and in a few cases innate immune stimulation was not entirely abrogated. The former can be explained by the additional requirement for the Dicer processing step which can be affected by chemical modification.

Overall, this means that it may take a little bit more effort to identify a suitable Dicer-substrate clinical development candidates, and there could be an increased risk in encountering unforeseen innate immune stimulations in humans. On the other hand, the study also suggests that for some genes it may be possible to find more potent RNAi triggers with Dicer-substrates, so that in an ideal world one would keep an open mind. I should also add that, not discussed in this paper, there are also other considerations which may favor one structure over the other.

Unfortunately, we are not living in an ideal RNAi Therapeutics world, but one in which patent trolls and IP freeloaders abound. The timing of the comparison study is particularly ironic since the freedom-to-operate of US-based Alnylam is very much in doubt, thus increasing the attractiveness of Dicerna's offering. This is because of the recent issuance of the Baulcombe patent in the US which has put essentially all the Tuschl siRNA structures, bar one, in a straitjacket. Until Baulcombe is sorted out, all buy-out and partnering efforts will be on hold, and if Merck gets exclusive rights to that patent...then Alnylam may well be toast.

Merck Seeks to Optimize Value of ApoB as Target for Hypercholesterolemia with Combinatorial RNAi Therapeutics

A series of recent papers (e.g. Ason et al. 1; Tadin-Strapps et al.; Ason et al. 2) shows that Merck wishes to use RNAi Therapeutics for the treatment of hypercholesterolemia, a precursor of cardiovascular disease. Despite the success that widely prescribed drugs such as statins have had in lowering bad cholesterol, there are still many patients in need of additional treatment options, patients that either have very high cholesterol levels to start off with or those that do not respond to or tolerate these drugs. As a result, interest in the pharmaceutical industry remains high in developing new approaches to treat these underserved patient populations.

The Pros and Cons of ApoB as a Target

ApoB has emerged as a very attractive, hitherto undruggable target in this endeavor, and indeed ISIS in partnership with Sanofi-Aventis are currently knocking on regulators’ doors to get their ApoB-targeting RNaseH-antisense drug mipomersen (commercial name: Kynamro) approved for familial hypercholesterolemia. Meanwhile, Tekmira and apparently Merck are in the early stages of developing ApoB-based RNAi Therapeutics for hypercholesterolemia.

What makes ApoB such an attractive target is that it is the critical protein component of bad LDL cholesterol (LDLc) and knocking it down therefore very potently reduces LDLc levels in the blood. Not only that, it seems that through the wonders of sophisticated feedback control mechanisms of lipid metabolism, essentially all other atherogenic lipids are reduced, too, following ApoB knockdown (e.g. Tadin-Strapps). There remain, however, two concerns with ApoB as a target.

The first one relates to the observation that in most rodent models, not only all the atherogenic lipids are reduced, but also the ‘good’ HDL-cholesterol which is responsible for reverse cholesterol transport from the plaques (where they are dangerous) back to the liver for excretion in bile (which is where they belong to). Research by Merck, of course using LNP technology, shows that when ApoB is knocked down by ~95%, both HDLc and LDLc where reduced by more than 2/3 (Tadin-Strapps et al. 2011; 79-90% non-HDLc lowering and 67-78% HDLc lowering in Ason et al). This was highly unlikely due to an off-target effect as various ApoB-targeting siRNAs exhibited this phenotype while non-targeting LNP formulations did not.

Nevertheless, it is unclear whether these rodent and similar non-human primate findings translate into humans, and what ApoB knockdown levels would need to be achieved to start seeing an effect on HDL. Mipomersen e.g. reduces LDLc by about a third and does not seem to affect HDLc in humans. The Merck scientists also speculate that the HDLc reduction simply reflects that in the absence of LDLc, ApoE redistributes to HDLc leading to their more rapid uptake in the liver. Therefore, despite the mantra that it is all about the HDL:LDL ratio, HDLc reductions via this route would actually be positive.

The second, probably more pressing concern is that ApoB inhibition leads to a failure to export lipids from and their accumulation in the liver, a condition known as hepatosteatosis or ‘fatty liver’. This has not only been observed in pre-clinical studies of ApoB knockdown, but was also observed in the mipomersen clinical studies (Visser et al. 2010). ISIS Pharmaceuticals, the discoverers behind mipomersen, argue that this accumulation is likely to be temporary only as compensatory genetic circuits get switched on to reverse the phenotype, a mechanism that is supported by Merck's own gene expression analysis. Moreover, it has yet to be shown that ApoB-related fatty liver predisposes to the development of liver fibrosis and ultimately liver failure or cancer which is really why we care about fatty liver in the first place.

Enhancing the Therapeutic Profile of ApoB-targeting Drugs

Giving up on ApoB in hypercholesterolemia because of the fatty liver concerns would mean forfeiting the potential of one of the most if not the most potent target in the hypercholesterolemia space. I therefore fully agree with the strategy by Merck to exploit the combinatorial potential of RNAi Therapeutics to optimize the profile of an ApoB-targeting RNAi Therapeutic, a strategy that I would fully expect of Tekmira to be evaluating as well.

The combinatorial potential of RNAi Therapeutics is one of the major attractions of this technology. Because of the almost identical pharmacological behaviors of siRNAs, it is relatively simple to employ multiple instead of just a single siRNA payload in an RNAi Therapeutic. This is particularly useful for complex diseases such as metabolic syndromes and diseases that involve resistance/escape such as cancer and viral infections. ALN-VSP02 is a dual-targeting example in oncology that is already in the clinic, and Tekmira’s Ebola RNAi Therapeutic candidate slated to enter the clinic in early 2012 will also involve at least two different RNAi triggers.

You can thus imagine that knocking down a gene along ApoB that leads to increased lipid excretion via the bile, increased fat oxidation in the liver, or redu­ced hepatic fat synthesis or reduced uptake of dietary fats in the liver, would greatly enhance the therapeutic profile of an ApoB-based drug by countering the development of fatty liver. It is the latter approach that Ason and colleagues from Merck took in their recent paper by targeting fatty acid transport protein 5 (Fatp5) alongside ApoB as Fatp5 had been described, also through the elegant application of ddRNAi, to reverse diet-induced hepatosteatosis.­

To study the effect of Fatp5 co-knockdown on ApoB-induced fatty liver, the researchers formulated both siRNAs into LNPs and infused them into mice. Both genes were knocked down efficiently (89-95%) and as you can imagine, at these ApoB knockdown levels, the fatty liver phenotype was quite robust. Predicted Fatp5-dependent phenotypes, such as an almost 1000-fold increase in the ratio of unconjugated to conjugated bile acids in the bile, were also observed (Fatp5 plays a role in bile acid conjugation) confirming the functional knockdown of both ApoB and Fatp5.

Unfortunately, despite the potent knockdown of Fatp5, the ApoB-dependent fatty liver phenotype was not reversed in the mice which were fed a ‘Western low-fat diet’. It therefore appears that Fatp5 intervention is only useful for diet-induced fatty liver, and that approaches specific to fat excretion or fatty acid oxidation in the liver will be more promising. Nevertheless, the scientists seem to be on the right track, and with SNALP siRNA delivery, it should be relatively easy to characterize other candidate genes. Indeed, due to competition, the Mercks, Tekmiras, and Alnylams may not necessarily want to disclose their magic siRNA cocktail.

So as we are on the eve of seeing mipomersen being approved as the first ApoB-targeting compound for the treatment of hypercholesterolemia, a second generation of ApoB-targeting RNAi Therapeutics are being readied that not only aim at incremental improvements in potency and dosing frequency, but completely rehaul the therapeutic profile of ApoB-based therapeutics.