Ionis Widens Its Modality Horizons

Over the weekend, blue chip antisense oligonucleotide company Ionis and genome editing competitor Intellia presented data on targeting prekallikrein (PKK) for treating hereditary hemeangioedema (Ionis donidalorsen here, Intellia NTLA-2002 here).

Using CRISPR Cas9 endonucleolytic disruption of the KLKB1 gene coding for PKK following LNP delivery, Intellia came out as the apparent winner in this showdown.  Not only did they demonstrate more pronounced PKK inhibition, but also more consistent elimination of debilitating attacks characteristic of the disease.  Moreover, by exploring less frequent antisense oligonucleotide administrations despite suboptimal low -60% knockdown, Ionis indicates that it is worried about the safety and tolerability profile of donidalorsen. 

Whether reversible approaches like antisense and RNAi or irreversible approaches like CRISPR gene disruption will ultimately prevail in the HAE race remains to be seen and will likely be decided by the safety of suppressing PKK expression over the long-term. If there is an overshoot of CRISPR-mediated gene disruption that would e.g. result in blood clotting abnormalities, even for a subset of patients, the field would be wide open for reversible methods.  

Ionis invests in genome editing

But whether that will be antisense remains to be seen.  Especially for targets in the liver, RNAi currently clearly rules the land for gene knockdown: highly potent, titratable and reversible knockdown with 5 years counting without a notable setback, especially related to off-target toxicity.  By contrast, Ionis is being held back by persistent safety issues as it has been beating a dead horse with its phosphorothioate-based backbone chemistry although it appears to be finally weaning itself off with chemistries such as the Mspa backbone.

So it is probably the hope of leap-frogging the RNAi competition by adopting genome editing as Ionis today announced that it was partnering with CRISPR genome editing company Metagenomi.  The HAE data comparison could not have come at a more opportune time.  

One declared aim of the investment in genome editing is life-cycle management of existing franchises.  In the liver, these franchises (TTR amyloidosis, ApoC3, PCSK9 etc) are currently and in the foreseeable future being dominated by RNAi despite Ionis’ heavy investments, so it clearly makes sense to amortize its investments in disease-specific market research, commercial infrastructure and clinical trial experience to accelerate the success of a more promising approach. 

TTR amyloidosis is a great example where even GalNAc-conjugated follow-on antisense compounds are unlikely to challenge Alnylam’s suite of RNAi triggers.  Also due to this dominance, it makes less sense for Ionis to develop an RNAi competitor drug despite its access and now actual adoption of this modality for targets in the muscle.  But as TTR shows, other genome editing companies are already competing for some of these targets so it won’t be all that simple trying to leap-frog RNAi and Alnylam like that.

 

The rise of the multi-modality oligonucleotide therapeutics companies

After straight-forward antisense for gene knockdown and then splice modulation, with the recent adoption of RNAi and genome editing, Ionis is rapidly expanding its oligonucleotide modality toolbox.

In fact, it is becoming a little bit like smaller competitor Wave Life Sciences which has been practicing all types of antisense (knockdown, splice modulation, more recently RNA editing) and RNAi using a bewildering mix of chemistries.  Not only are they burning through cash as if there was no recession and inflation problem, I never liked that because clinical failure after failure (esp. minute target engagements at best) suggest that the company is stretching itself too thin.

By comparison, Ionis, with $2 billion in cash and a more experienced and bigger operation is a different beast altogether and may be able to pull it off, at least on a technical level.  However, instead of spending $80M in upfront alone on a modality that is somewhat further removed from its traditional chemistries (longer mRNAs, LNP delivery for CRISPR), it could have much more synergistically leveraged its investments in chemistry and delivery by investing that same amount in the ripe-for-the-picking RNA editing.  Accordingly, $80M is more than the market cap of my currently favourite RNA editing investment, ProQR.

I’m sure the opportunity to expand druggable targets and indications by applying existing delivery technologies and chemistry know-how by adopting RNA editing is not lost on RNAi players such as Alnylam and especially Arrowhead Pharmaceuticals.  Arrowhead in particular, having scooped up the RNAi assets of Novartis and Roche for peanuts has demonstrated an ability to recognize and act on similar opportunities.

Antisense Technology Produces Huntington’s Disease Breakthrough

Huntington’s Disease (HD) is one of the most prevalent severe, ultimately fatal genetic neurological diseases of our times for which impactful treatments are desperately lacking.  Based on results from a clinical study aiming to knockdown the disease-causing gene using RNaseH antisense technology, this is about to change over the next 3 years.

IONIS-HTTRx study results presented at CHDI

At the major annual HD gathering this week, lead investigator Tabrizi from the University College London presented data from a phase I-II study of antisense drug candidate IONIS-HTTRx in patients with early symptoms of the disease.

IONIS-HTTRx (aka RG6042), developed by Ionis and partnered with Roche, targets both wild-type and mutant huntingtin protein to provide all patients with the most potent and safest sequence possible.  While mutant allele-specific approaches have been discussed and are being pursued by antisense rival Wave Life Sciences, the concern that suppressing wildtype huntingtin in adults seems to me a far-fetched theoretical discussion, if not wishful thinking given that antisense is unlikely to completely eliminate wildtype huntingtin and that the drug is being given to adults.  

To wit, the concern is based on genetic mouse models where complete huntingtin knockout causes embryonic lethality. Importantly, this is not recapitulated in mouse model where knockout is delayed until adulthood.

Call me cynical, but the choice of an allele-specific approach by Wave Life Sciences has likely been primarily motivated by allowing for some follower differentiation.  Ironically, by limiting themselves to a much reduced sequence space around co-segregating single nucleotide polymorphisms, the sequences may not only lack the potency, but also the apparent safety of IONS-HTTRx.

Good safety profile maintained until highest dose tested

In this study, IONIS-HTTRx was given monthly over 3 months.  Speaking to the safety of the approach, apart from transient side effects due to the intrathecal mode of administration, no adverse events of note were reported and all 46 subjects have subsequently rolled over into the open-label extension study.

Importantly, doses as high as 120mg monthly were well tolerated at which dose the knockdown effect seems to have plateaued.  This not only bodes well for IONIS-HTTRx, but for the entire neurological disease franchise of Ionis Pharmaceuticals. 

The human safety and tolerability results are consistent with a range of small and large animal models (including dogs, pigs, non-human primates), some of which were a year or longer in duration.

Robust huntingtin lowering

It is widely thought that mutant huntingtin protein is harmful to the cell expressing it, ultimately resulting in neuronal death throughout the brain.  This can be visualized by considerable, 30%-level atrophy of the brain over the course of the disease.

Although it is not possible to assess huntingtin levels directly in brain tissue in humans from biopsies, the experience in the preclinical animal models and human data from spinal muscular atrophy drug SPINRAZA allows one to model the correlation between ASO-dependent protein changes in the cerebrospinal fluid (CSF) and the various areas of the CNS quite well.

Since SPINRAZA is based on chemistry that is highly similar to that of IONIS-HTTRx, they behave identically in terms of biodistribution for all intents and purposes.

In the two highest cohorts, 90mg and 120mg, mean 40% mutant huntingtin reductions in the CSF were observed shortly after the last dose with further reductions (50%+) seen thereafter.  Such levels are predicted to reflect 55 to up to 85% reductions in the cortex and 20-50% in the caudate.

This degree of knockdown exceeds that necessary to see changes in HD animal models, some of which with manifest disease symptoms at treatment onset.

The future is bright

The study results therefore raise hopes that  IONIS-HTTRx may halt or even reverse disease in manifest HD patients.  As exciting and medically likely with the biggest bang for the buck is the prospect that the drug candidate may prevent disease symptoms to emerge in the first place when given early to those at risk of developing the disease (~1 in 2 with a parent having HD, 200,000 in the US alone). 

The companies and clinical investigators are now busy designing a pivotal outcomes study which I would guess will ultimately involve 150-250 early-stage patients over a period of 16-24 months.  The size and extent of the trial will depend on which endpoints are chosen.  A pre-planned interim look at ~12 months may also make sense to look at brain size as a potential way to obtain accelerated approval.  Accelerated approval under the new FDA may also be possible based on functional analyses the investigators are currently conducting using data from the present study and the open-label extension with dosing well beyond 3 months..

In drug development, sometimes you can tell early on whether a drug candidate is destined to breeze through to approval. In this regard, IONIS-HTTRx certainly feels like Spinraza.

Roche Buys Santaris (Because It Could Not Buy ISIS)

Minutes ago, Roche announced the acquisition of Danish antisense company Santaris for $250M in upfront cash and $200M in contingent payment.  Given that Santaris only has a microRNA inhibitor for HCV in the clinical pipeline which many in the industry believe is outdated given recent successes in the treatment of HCV, this purchase is a big vote of confidence by Roche in the RNA Therapeutics platform.

This latest development comes after Roche struck a Huntington’s-based deal with ISIS Pharmaceuticals in April 2013 resulting in a development candidate as revealed today by ISIS in its earnings report.  Roche was fortunate to get that deal, because ISIS was about to strike a muchbroader neurology deal with BiogenIdec, leaving it hungry for all those other targets.  Apparently, Roche was happy with what it saw, also following a smaller deal with Santaris just in January this year on undisclosed targets.

This deal flow is reminiscent of what Roche did in RNAi Therapeutics in 2006/7.  After Merck took out Sirna Therapeutics, Roche saw a need to partner with Alnylam instead, at least this is how the narrative goes. 

You can bet your house that Roche at one point or another wanted to buy ISIS, but given its varied partnerships and general unwillingness to sell itself had to settle with archrival Santaris (--> ongoing litigation which perhaps Roche is more able to settle on ISIS' terms).  But, hey, maybe history is repeating itself and ISIS already signed itself away to AstraZeneca and we will hear about it soon.

PS: for the stock junkies (I know there are many here), this news also bodes well for Marina Biotech given its highly similar chemistry to that of Santaris: LNA and CRN.

Don’t Bank on OncoGeneX Phase III Data

As much as RNAi Therapeutics is contributing to and benefiting from the fact that RNA Therapeutics as a whole is rapidly materializing as the 3^rd major drug discovery platform, this relationship could also backfire to some extent.  I am particularly thinking here about the upcoming OncoGeneX 2nd gen RNaseH-antisense (ASO) phase III results in prostate cancer (partnered with TEVA).

Obviously, a successful outcome of the SYNERGY trial which is analyzing the ASO compound custirsen (aka OGX-011) in combination with chemotherapy would be a big win for the field and represent the first more significant commercial success.  In February, the company announced that the pre-specified number of ‘events’ (i.e. deaths) had been reached in this ~1000-patient study, and depending on the robustness of the results (good or bad) we could be hearing any day now of the top-line results.

Reasons to be skeptical

In evaluating the prospects of an Oligonucleotide Therapeutics compound, I first ask whether the delivery has been firmly established and how good the target is. 

Usually, since Oligonucleotide Therapeutics enjoy the benefit of being able to pursue virtually any target, target risk should be minimal.

Unfortunately, in this case, there are considerable risks in both the delivery and the target.

Custirsen targets clusterin expression.  Clusterin has chaperone activity and is thought to protect cancer cells from stress, including stress arising from cancer therapies such as radiation and chemo.  Accordingly, limiting clusterin activity by knocking it down would be predicted to improve the effectiveness of conventional cancer therapies.

The most important validating preclinical study in that regard comes from a publication by Zellweger and colleagues…from the year 2001 (which got me wondering about the patent clock).  While some of the predicted effects were seen such as increased sensitivity to chemotherapy agents, the effect sizes were rather modest and their correlation with ASO potencies poor. 

In general, in reviewing even more recent ASO chemistries such as cET2.5 for cancer (e.g. STAT3 by ISIS/AstraZeneca), I was struck by the poor correlation often seen between knockdown potency and therapeutic effect (e.g. much more potent anti-cancer effects with an ASO that had 20% knockdown activity compared to one that had 60% knockdown activity) and sometimes also between mRNA and protein levels (mRNA e.g. reduced by 50%, protein essentially disappearing; patent application WO2012/135736).

This raises the prospect that some of the apparent anti-cancer activities were not due to on-target efficacy, but due to some unintended side effects such as immune stimulation.

Promising phase I PK-PD data at very high dose of 640mg

Consistent with the concern of unintended/undesirable immune stimulation, the apparent increase in overall survival observed in the phase II prostate cancer study of custirsen was seen at the dose of 640mg, a dose at the very upper limit of phosphorothioate chemistry (Chi et al. 2010).  This study in ~80 prostate cancer patients provided the rationale for investing in the ongoing phase III trial.

Unsurprisingly, immunostimulatory side effects in the form of chills and fevers were seen in the majority of subjects taking custirsen in combination with chemo, but not those on chemo alone (plus the liver and kidney tox you’d expect from such a high PS-ASO dose).  Moreover, the apparent clusterin knockdown, as assessed on the basis of the protein found in serum (not in cancer tissue directly), was a very modest ~30%.

This unsatisfactory degree of knockdown stands in stark contrast to a promising phase I PK-PD study which showed >90% knockdowns in prostate/tumor biopsies (Chi et al. 2005).  This was accompanied by mid- to high single-digit microgram of oligonucleotide concentrations per gram tumor tissue.  Such concentrations do not necessarily predict (normal) mRNA knockdown efficacy, but do not exclude such activity either. 

One explanation for the apparent discrepancy could be that the phosphorothioate oligo preferentially localizes to solid tumors and that solid tumors are not a significant source of serum clusterin. Possible, but...  

Under the radar for a reason

A sub $200M market cap for a company with imminent pivotal phase III data in prostate cancer and low trading volumes indicate that the market has not been paying all that much attention to Oncogenex. 

In this case, I would like to think for a good reason, and while I do not exclude ‘positive’ results (at the cost of considerable side effects and an uncertain mechanism of action), I hope that the public won’t read too much into them regarding the ongoing RNA Therapeutics revolution.  Every mechanism and chemistry has its place. PS-ASOs knockdown of (cytoplasmic) mRNA for cancer is not it. 

For cancer, RNAi and microRNA Therapeutics have the edge.

Ebola, Advantage Tekmira

These are critical weeks for Tekmira.  Not only is Tekmira fighting to get back control over its LNP delivery technology, the full-fledged termination of the Ebola biodefense program following the Stop-Work Order would have given the company serious headaches.  As much of the current cash-flow derives from the $140M biodefense contract from the Department of Defense (DoD), the company may have been forced to decide between laying off or furloughing staff until the decision in re Alnylam would provide more funding clarity (November-December 2012).

Luckily, with the DoD deciding to continue Tekmira’s Ebola program, the advantage is again with Tekmira.  My main concern with a negative biodefense decision was that it might have particularly affected the long-term ability of the company to continue to innovate on its LNP technology.  The benefits of the biodefense contract were particularly visible in manufacturing (such as scale-up and lyophilization), thereby allowing the company to build on a key competitive strength.

Of course, it is fair to say that I feel vindicated by the simultaneous decision by the DoD to discontinue Sarepta’s competing Ebola development program: Tekmira’s RNAi Therapeutics approach, now in phase I studies, had the stronger scientific data compared to Sarepta’s antisense approach which had once enjoyed a multi-year head-start (here my 2010 comparison of the two programs).  

Liposomally formulated RNAi Therapeutics also has the advantage of facilitating multi-targeting.  This opens up the prospect for targeting multiple viral, but also host factors, some of which may actually be shared by a number of hemorrhagic fever viruses (HFV). I start getting the impression that setting the stage for the development of such broad-acting HFV therapeutics appears to be an increasingly important aspect of Tekmira’s Ebola program.

I should add that Sarepta will continue to be funded under an analogous $140M contract for Marburg (also a HFV).  Here, the company seemed to have achieved important post-infectionmilestones.  Tekmira does not compete here.  Continuing to fund both an RNAi Therapeutics and an antisense program may also be a strategic risk-mitigating decision by the DoD in case one approach encounters terminal platform issues.

Nevertheless, comments by David Hough from the DoD indicate that the decision was mostly based on the scientific merits:

"Our job is to ensure we're making the most out of every dollar we spend," he noted. "We evaluated each contractor's efforts independently.  The evaluation was conducted using all current data and in accordance with the criteria set forth in the awarded contracts and as stated in the initial solicitation.  This was certainly a tough decision but the final decision was made to move forward on continuing the development of the drug candidate that represents the best value to the Government based on what we know today."

Simply SNALP: RNAi TTR Knockdown Data Impress

Clearly, RNAi Therapeutics is back in fashion.  Well, almost.

Alnylam’s announcement of profound gene knockdown in the ALN-TTR02 phase I study for the treatment of TTR amyloidosis was greeted with an over 50% jump in the stock price on very high volume.  Various observers pointed out that this might have been an over-reaction given the early nature of the data.  On the other hand, considering that the over 80% peak knockdowns achieved with only a single dose of 0.15mg/kg are representative of a widely applicable delivery strategy, namely Tekmira’s SNALP technology, and considering that an accelerated approval path  should be possible for this orphan disease, determining the fair value of a SNALP-based company like Alnylam becomes difficult.

Yes, this was a study that enrolled just 17 subjects, healthy volunteers at that.  But it may be this that makes the tight variability in the pharmacodynamic response (=TTR knockdown) even more remarkable so: less than 5% relative standard deviations in the 0.15mg/kg and 0.30 mg/kg 3-person dose cohorts, the relevant groups for this analysis.  

When I had mentally listed the technical risks of SNALP-mediated delivery several years ago when I did my fundamental research on SNALP delivery, intersubject knockdown variability was one of the major ones.  However, given the present results and those from the recent hypercholesterolemia study (ALN-PCS02), I am much less concerned about this now, also in patient groups where liver health may be affected.

Moreover, the dose-related TTR knockdown in humans was essentially what has been seen in non-human primates.  Although a number of analysts were apparently puzzled by the importance of this observation, its value to a scientist is obvious: you can essentially eliminate the risk that an SNALP program fails in phase I due to lack of knockdown efficacy (and if you are one of those scientists that also dabble in the stock markets, you can start investing in the stock based on the monkey data that you’ll see presented at an informal presentation).   

The safety profile with ALN-TTR02 in this study was good.  There was one infusion reaction seen in other SNALP studies before and that was managed by slowing the infusion rate.  This was said to be possibly related to the pre-treatment with corticosteroids, and not the liposomal formulation itself.  In any case, while corticosteroids and other transient immune suppressions will not be an issue for the initial patient populations in diseases like TTR amyloidosis, Tekmira and Alnylam should start to try and wean themselves of it.  The improved, more predictive immune stimulation assays may form a sound scientific basis for this.

The reason for why the study was limited to 17 healthy volunteers was mostly due to competitive concerns.  In particular, the ISIS/GSK TTR antisense compound is said to skip phase II studies altogether and go straight into a pivotal phase III.  In data presented in May of 2011, this antisense compound was reported to effect a 44% mean TTR reduction at the 200mg/week dose level when given for four weeks, and 81% knockdown at 400mg which was the next higher dose tested.  400mg/week, however, is a dose that I believe is too high for chronic phosphorothioate oligonucleotide administration.  Although ALN-TTR02 should thus be 100- to 200-fold more potent on a weight basis than the antisense drug candidate, being the first-to-market is seen by some as an important factor for the commercial success in this orphan market, especially since these compounds work by the same mechanism of action.

The value of SNALP delivery

Some readers of this blog may be tiring by now of reading me sing the praises of SNALP delivery technology and Tekmira.  Yes, I’m invested in the technology via my Tekmira shares.  In all fairness, however, it is the clinical data from the SNALP compounds, really starting last November with the ALN-TTR01 data (SNALP Works!) that are reviving the RNAi Therapeutics field: ALN-VSP, ALN-PCS, and ALN-TTR.  We have heard a lot about exciting new, revolutionary RNAi delivery technologies, often claiming to solve the putative problems with  SNALP technology.  However, in the end, it is SNALP that is set to unlock the first wave of RNAi Therapeutics value creation.

Even Arrowhead Research and Benitec which develop competing delivery and RNAi trigger technologies, respectively, admitted so much in congratulating Alnylam to the results.  Sadly, they missed the opportunity to also congratulate the real innovator behind SNALP technology, Tekmira.  It seems that since Alnylam is getting all the media attention, it was probably not a good PR strategy to do so.

RNAi delivery technologies are tough to develop and rare to find, but when they work, they open up a range of therapeutic opportunities.   

Preview: For my next post, I am planning to write something about last week’s MC3 paper and how it reveals that covering all the tracks is hopeless when a scheme is that elaborate (RE Tekmira-Alnylam litigation), and why Alnylam the stock may be a great short here. 

ISIS Claims Scalp of Santaris CEO

After noticing that the former President of the US operations of Danish LNA antisense company Santaris, Art Levin, had just joined muscle microRNA Therapeutics company miRagen, I was alerted that this has been in the wake of a major shake-up at Santaris. Most notably, it appears that the CEO of Santaris, Soren Tulstrup, either handed in his resignation, or, more likely, was let go.


Privately-held Santaris, of course, has been a fierce rival of ISIS for supremacy in the antisense space.  Having former ISIS insider Art Levin join Santaris 2 1/2 years ago was in fact a bit of a coup for that company.  Its LNA technology is probably the most potent/high-affinity in the space for many applications.  ISIS, however, has had the benefit of increased financial might and having built a large patent estate that it is (ab-)using to sue, preferably smaller competitors with the obvious intention of making it difficult, if not impossible for those to raise capital and pursue business development opportunities.  It is rumored that ‘partner’/satellite company Alnylam has probably paid the price in the tens of millions for the privilege of going public.   


Not long ago, ISIS sued Santaris for unduly hiding under the Research Exemption in its various relationships including Pfizer, Enzon, GSK, and Shire.  In parallel, ISIS and microRNA spin-off Regulus have been haunting Santaris by questioning Santaris’ ability to develop its exciting lead candidate, miravirsen, a miR-122 antagonist for the treatment of HCV.  As a result, Santaris was dumped by GSK in favor of Regulus’ miR-122 program despite having the superior data package, which is also born out by the clinical data so far (here for the latest update at EASL).


Regulus announced this week that it was awarded another HCV miR-122 patent, in Japan, concluding with the remark that it was continuing to pay 'attention to hepatitis C'- somewhat ironical given that its clinical entry seems to be delayed forever.  Seeing that announcement, I had thought that this was one of the unnecessary PRs that I often take as a sign of weakness. In hindsight, it was probably just a case of adding insult to injury.


I myself have had reservations about the wisdom of building the company’s financial future on a program for which it may be lacking access to some of the key patents in the space.  Although one may assume that in the wake of its scientifically leading efforts in miR-122/HCV, Santaris will have in turn built IP to which Regulus would need access if it were to develop its own miR-122 antagonist,  such a strategy has been a high-stakes gamble for sure.  Along with the ISIS lawsuit questioning Santaris’ fundamental business development strategy, I would therefore not be surprised if the CEO had faced some tough questions by Santaris' investors about his decisions and consequently was ousted. 


Clearly, an exit in the form of an IPO or takeover had long been overdue for that company.  The company’s technology, pipeline, and relationships, also in light of what’s out there already (i.e. ISIS and AVI Biopharma), should have easily supported this, if not for the lawsuit and threats thereof.

Merging Antisense with RNAi Therapeutics to Create Fitter Companies

Yesterday’s long awaited data presentation on phase III results from ISIS Pharmaceutical’s lead antisense gene knockdown program was met by disappointment in the investor community: ISIS down over 15% on the day. While the exciting news is that the drug, mipomersen, looks like it is very close for approval for the severe, but very rare condition of homozygous familial hypercholesterolemia (hoFH) and importantly also possibly other forms of highly elevated LDL-cholesterol, part of the disappointment may be related to the long-term outlook on antisense for gene/mRNA knockdown: a 27% reduction in ApoB levels following 6 months of 200mg weekly injections with a safety profile (a number of cases of elevated liver enzymes, injection side reactions) that may be adequate for the severe cases of hypercholesterolemia, but not necessarily for less severe diseases. And this is for an organ, the liver, which has one of the best pharmacologies for antisense. Also, while a 27% knockdown may be therapeutic for a few targets, especially in metabolic disease, for most other targets it is insufficient.

Lack of high-quality gene knockdown opportunities would lead to an inefficient use of its capital that includes an enviable $600M+ cash pile and an equally remarkable cash-flow from their ‘satellite businesses’. Maybe partly because of this realization, but also of course because it believes that its IP is transferable to all areas it considers antisense, ISIS is not standing still and continues to innovate in areas such as RNAi Therapeutics and the promising gain-of-function antisense technologies of splice modulation and microRNA inhibition.

On the other side of the fence, RNAi Therapeutics is also facing a challenging investment environment. Capital is particularly difficult to raise for early-stage platform technologies. While some companies such as Alnylam, Tekmira, Sirna Therapeutics (historical example), and mdRNA are making good strides in establishing broad RNAi drug development platforms, this alone does not make up for lack of steady newsflow from mid- to late-stage clinical results that can support rich valuations in biotech. This is also not helped by some obvious mis-steps within the industry itself with the main sins being spending money on lawsuits and prematurely entering programs into the clinic, ironically not least in an attempt to keep investor interest levels high.

By merging with antisense, both the pipeline maturity profile could be enhanced and resources spent more efficiently by avoiding the temptation of entering programs into the clinic prematurely and otherwise weeding out programs that serve to artificially fill pipelines. The latter point, of course, would also apply to the antisense company, and the antisense company would further benefit from gaining access to the most potent and therapeutically promising gene knockdown technology known, RNAi Therapeutics. As the intricate relationship between Alnylam and ISIS Pharmaceuticals or the acquisition of Coley as a launch pad for Pfizer’s RNAi Therapeutics ambitions demonstrate, the scientific barriers for such mergers should be relatively minor. One risk, however, that cannot be ignored, particularly for those companies that have been built for sale, is that by combining various drug development platforms, the new entity may become a less attractive candidate for a Big Pharma acquisition. When it comes to mere survival, however, a combination should be the lesser evil.

I will now briefly discuss three fantasy combinations, at least one of which I would speculate to see within the next year.

1) Alnylam Pharmaceuticals and ISIS Pharmaceuticals (probably not within the next year). Without a doubt the most influential and potent leaders in RNAi Therapeutics and antisense, respectively. Already highly entangled through their IP cross-licensing agreements and microRNA therapeutics spin-off Regulus, a combination would create an almost cash flow-positive dream team with over $1B in cash, blocking IP, unmatched expertise in nucleic acid chemistry, clinical pharmacology, and RNAi Therapeutics. First revenues from the sales of mipomersen and continued IP licensing revenues would support a solid pipeline consisting of mipomersen label extensions, RNAi Therapeutics opportunities for liver and solid cancers, full ownership of the miR-122 program for the treatment of HCV, and transitioning antisense towards splice modulation, microRNA inhibition (with Regulus), and possibly other gain-of-function antisense applications. As part of the re-organization, some programs could easily be sacrificed without punishment by the markets. John Maraganore would be the CEO of the combined company, allowing Stanley Crooke to follow his passion in the science of oligonucleotide therapeutics.

2) mdRNA and AVI Biopharma (how soon?). The transition of antisense for gene knockdown to gain-of-function applications is most noticeable in the case of AVI Biopharma. After many years of attempting gene knockdown with their steric-block morpholinos, the company finds success in applying its technology for the modulation of gene splicing. It is considered the closest competitor to Prosensa’s Duchenne Muscular Dystrophy exon skipping program which has only recently entered an attractive $25M upfront plus multimillion bio$$$ milestone and royalty deal with GSK after a series of high-profile publications on DMD-related exon skipping. The partnership potential of AVI’s DMD program then as well as its recent capital raising should provide the needed capital cushion for the combined company to invest in RNAi Therapeutics for gene knockdown and some new splice modulation opportunities. mdRNA, of course, is scheduled to run out of cash early next year and it would be a miracle if the two companies had not contemplated such a merger, particularly after AVI Biopharma moved in as mdRNA’s neighbor not too long along up from Oregon. With AVI’s cash reserve, the combined company may then find it easier to attract platform partners for mdRNA’s technology further bolstering the financials.

3) Silence Therapeutics and Archemix (not really an antisense company, but close enough). It is almost two months now that Silence announced to be in reverse merger talks. Since then, however, not a word except for so-so news on the results of opposition proceedings at the EPO related to their core patent. What Silence needs is cash (who doesn’t?), and what it can offer is a phase I cancer program, RNAi Therapeutics drug development expertise, and an siRNA structure that is not without use, although facing very serious patent challenges. What Archemix needs with its growing pipeline is access to public markets as evidenced by a previously failed reverse takeover attempt with cash-rich NitroMed, as does by the way Quark Pharmaceuticals which should also be counted as a possible Silence Therapeutics merger candidate. The fact that Silence Therapeutics has some aptamer in its blood line and the increased investment by Archemix into aptamer-mediated delivery of RNAi (Dicerna) and microRNA (miRagen) Therapeutics should help a combined company find a common language. Until any such deal is announced, however, Archemix would have to be prepared though that yet another potential partner will walk away from it last minute as Silence Therapeutics should also be receptive to other offers.

Which one is the most likely combination? Add your vote on the right.

Antisense or Guide Strand? That’s the Billion Dollar Question

As somebody who has spent the better part of my last decade in academic science, which in its ideal form is guided by the self-less pursuit of the ‘truth’, I must admit that I am somewhat irked when I read a paper on Argonaute 2 enzymology and find science being used also (though not exclusively) as a tool of corporate propaganda. You may think that this is nothing new at all. However, I’d like to use the latest publication by ISIS Pharmaceuticals on RNAi as an excuse to highlight a few of the issues that come up when ISIS Pharmaceuticals, by sheer repetition, claims that RNAi Therapeutics is an antisense technology.

In the paper by Lima and colleagues in the Journal of Biological Chemistry (‘The Binding and Cleavage Specificities of Human Argonaute 2’), the investigators study the effect of sequence length and modifications of what they call the ‘antisense’ strand on Argonaute 2 binding. What the authors call here ‘antisense’ is widely referred to by the rest of the field as the ‘guide strand’, that is the small RNA of the double-stranded RNA trigger that gets loaded onto Argonaute 2, the enzyme in RNAi that carries out the target mRNA cleavage. Admittedly, the ‘antisense’ is sometimes used in the RNAi literature to operationally describe the relative polarities of the RNAs involved (guide strand, passenger strand, target mRNA). However, when ‘antisense’ comes up 243 times within a manuscript, but ‘guide’ not at all, even as a verb, I believe it is a valid hypothesis that this cannot be a coincidence, but is calculated corporate speak.

I have explained before why RNAi by its very nature is a double-strand RNA technology, most tellingly illustrated by the fact that the discovery that what had been thought was mediated by antisense was actually mediated by the double-strand RNA contaminants of antisense preparations was awarded the Nobel Prize, so I will not repeat here myself. This is also not meant to criticize the scientific merits of the paper which furthers our understanding of the chemistries that could be used for the less efficient form of RNAi, namely single-strand RNAi and must be seen in the context of the recently announced ssRNAi initiative by ISIS and Alnylam, except maybe for the observation that the finding that single-strand RNA binds purified Argonaute 2 apparently better than double-strand RNA does not reflect Argonaute 2 loading in a living cell.

The point is that whether RNAi is labeled as an antisense technology or not can have significant consequences for the interpretation of the scope of patents, some of which date from before the discovery of RNAi in worms. It is all good for Alnylam for example to implicitly subscribe to ISIS notion as long as they have exclusive access to that IP as it helps constrain the freedom-to-operate of competitors such as Merck and Silence Therapeutics. However, when these early patents are ultimately not deemed as critical, can this leave Alnylam with patents that were pursued less rigorously so as not to conflict with the ISIS’ IP? One of the Tuschl patents comes to mind, I believe dubbed ‘Tuschl IV’, which for the first time explicitly described the use of single-strand RNAs for the induction of RNAi, but, to my initial surprise, had received very little attention among all the ssRNAi talk.

Aside from this political minefield, labeling RNAi Therapeutics as ‘antisense’ just does not do justice to the many ground-breaking discoveries this field has seen, and the people and institutions that made them and that you would think deserved their rightful place in RNAi history and a larger piece of the royalty pie. Specific modifications when used within an siRNA yes, but not the fundamental mechanism of action.

I leave it up to the reader whether this makes ISIS Pharmaceuticals an even better investment, and I must say they clearly deserve credit for their pioneering role in oligonucleotide chemistry and clinical application, and are now harvesting the financial fruits not only from their own drug development pipeline, but also from licenses for all types of therapeutic RNA approaches, including microRNAs (progress so far have exceeded my expectations, and according to a just published comments by Regulus’ CEO on HCV-miR122 this looks set to continue for the foreseeable future) and aptamers. Regardless, 243 times antisense does not make RNAi such.

RNAi is not Antisense

From the abstract of an important paper in RNAi:

"Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC.
Carnegie Institution of Washington, Department of Embryology, Baltimore, Maryland 21210, USA. fire@mail1.ciwemb.edu

Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts...Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference..."


RNAi is not antisense- This very finding formed the basis for awarding the 2006 Nobel Prize committee to Andy Fire and Craig Mello. Despite this authoritative decision, as the promise of the RNAi Therapeutics platform is getting closer to reality by the day, the promise also of financial fortunes has led to efforts to re-brand RNAi as yet another antisense technology. Although this in a way could be considered as some kind of validation of the competitive nature of RNAi, it has important implications for RNAi intellectual property.

In the past, ISIS Pharmaceuticals, undoubtably a leader in RNA therapeutics and with nucleic acid modification IP very relevant for RNAi Therapeutics, has made a number of statements I cannot agree with. Essentially, the company claims having discovered RNAi and has long regarded Alnylam as one of their many “satellite companies”. This, however, does not reflect reality in as much as repetitively claiming that RNAi is an antisense technology does not make it a true statement.

The latest example can be found in ISIS Pharmaceutical’s press release on the issuance of an ApoB-related antisense patent summarizing the claims as covering “the use of both single-stranded and double-stranded (siRNA) antisense drugs complementary to any site of the mRNA of human apoB regardless of their chemistry or antisense mechanism of action.” “Double-stranded antisense drug”- quite an oxymoron. But note that nowhere in the claims of the patent are duplex RNAs mentioned, instead “12 to 30
nucleobases in length targeted to a nucleic acid molecule encoding apolipoprotein B wherein said compound is 100% complementary to the nucleic acid sequence set forth in SEQ ID NO: 3” are claimed.

RNAi being antisense, having discovered RNAi first, and Alnylam being a satellite company, just does not agree with the fact that the very reason why Fire and Mello’s discovery is considered a major breakthrough, is because they found that RNAi is induced by double-stranded RNAs and not by antisense molecules as had been widely believed, and this indeed was the very working hypothesis that Fire and Mello famously disproved and for which they were awarded with the Nobel Prize. Come to think of it, RNAi as an “anti-antisense” technology may be a more apt description.

The claim why RNAi is supposed to be an antisense technology is largely based on the observation that during RNAi, the double-stranded siRNA is unwound, and the single-stranded guide RNA strand incorporated into RiSC which it then guides to target mRNA partly, but not exclusively, based on base-pair complementarity. Just because base-pairing interaction is involved, however, does not make it antisense. What is important for a biotechnology that harnesses an endogenous biological pathway are the features that make it being recognized as a bona fide substrate. In the case of RNAi, this clearly is double-stranded RNA with a couple of additional characteristics, and not single-stranded antisense RNA. This is the result of RNAi having evolved as a way to recognize and fight RNA viruses which are characterized by double-stranded RNA intermediates. Re-branding RNAi after one of the components of its inducers would be like saying the wheels of a car are the car.

While single-stranded antisense RNAs may also induce RNAi, research by ISIS itself and others has shown that they do so much efficiently, about 20- to 50-fold less efficient than double-stranded RNAs, that is about as efficient as a conventional RNaseH antisense molecule (probably another reason for ISIS’ growing interest in RNAi).

With so much “new fundamental” trigger IP being claimed, RNAi now becoming an antisense technology etc., are we about to see a different approach by the RNAi Therapeutics companies in possession of the real fundamental IP? I would not necessarily expect legal proceedings as long as it does not start to affect their business dealings. However, such rhetoric has the potential to lead to market inefficiencies, hurt smaller companies with a less active PR department (see Tekmira and their ApoB program), and lead to a situation in which as the first RNAi Therapeutics are approved and patent disputes to be resolved, the debate will have been re-framed to the disadvantage of RNAi Therapeutics companies.

PS: The RNAi-is-Antisense claims also get to the important issue of target IP. Target-specific IP should be encouraged as it incentivizes the identification of suitable RNAi Therapeutic gene targets, as long as certain minimum validation standards are satisfied. I would therefore agree that if the ISIS antisense patent had been a milestone in demonstrating the suitability of apo-B inhibition for the treatment of hypercholesterolemia (note: ribozymes targeting Apo-B were specifically exempted from the claims which already suggests that the present patent is not the seminal ApoB-hypercholesterolemia patent), this should also have consequences for drugs based on other mechanisms of actions such as RNAi, monoclonal antibodies etc. In cases where there is a proven benefit in targeting at the RNA level, the royalty rate should climb commensurately, although I do not believe that in practice this means that an antisense therapeutic will be able to stop an RNAi Therapeutics from reaching the market and vice versa.

Note: The abstract of the Fire and Mello paper has been added after the first publication of the blog entry.

RNAi Therapeutics versus Antisense- Where Delivery Makes a Difference

Regulus, the microRNA joint venture between Alnylam and ISIS Pharmaceuticals, is the most visible manifestation of the scientific overlap that exists between antisense and RNAi. The overlap, however, is not just limited to the science, but also extends into the capital markets. To better help the investor differentiate between the two technologies, I’d like to use this blog to provide an overview of some of the fundamental differences underlying the development of RNAi and antisense into therapeutics and their long-term prospects, with an emphasis on delivery. Needless to say, beware this discussion will be heavily biased in favor RNAi, but then again these are the reasons why I’ve been attracted to RNAi in the first place.

Certainly, progress in both areas in the last 3-5 years has mutually benefitted the investment climate for both technologies as it has heightened interest and increased confidence in RNA therapeutics in general. However, the two technologies also compete for investment dollars with many of the same investors, which are typically upbeat about the future of gene-based medicines but unsure where to place their bets, allocating their investments based on where they see most promise. One issue that often comes up in making this decision is the observation that while for systemic applications antisense, as practiced in the most advanced programs today, is typically administered without a particular delivery formulation, the development of specialized delivery technology is frequently cited as the key challenge for RNAi to realize its ultimate therapeutic potential.

Antisense for gene knockdown works largely by two mechanisms: interfering with translational initiation (e.g. AVI Biopharma's morpholinos) or through an RNase H-type mechanism (e.g. Santaris and ISIS Pharmaceuticals). For this, the key factor is to achieve efficient hybridization of a single-stranded oligonucleotide antisense with its target mRNA which either prevents productive ribosome association to the mRNA (inhibition of translation initiation) or may be recognized as a substrate for the RNase H enzyme which may degrade the RNA portion of the mRNA-DNA duplex, but normally functions in the degradation of the RNA primer during DNA replication.

Various oligonucleotide chemistries have been developed to optimize these processes for in vivo applications. Essentially all of these are single-stranded oligos of which the sugar phosphate backbone is heavily modified to a) increase their in vivo stability; b) improve their pharmacokinetics and avoid rapid renal excretion by promoting their association with components of the blood; c) similarly allows them to be retained in tissues; d) facilitate crossing of cell membranes; and finally e) increase their target mRNA binding. By contrast siRNAs, because of their charge and more rigid double-stranded nature and with apparently some exceptions that include mucosal epithelia, do not cross cell membranes efficiently on their own and therefore need to be specially formulated for most applications.

The use of unformulated antisense is consistent with their mechanism of action. Since antisense does not harness a naturally existing endogenous gene silencing pathway, it relies on achieving concentrations of oligonucleotides in the target tissue over a prolonged period of time that are high enough such that, as a result of the rules of thermodynamics, a sufficient fraction of target mRNA will be recognized. Similarly, unlike RNAi, the specificity of antisense is largely governed by biophysics and benefits only relatively little from biological proof-reading.

In practice, to achieve the necessary tissue concentrations, patients are typically dosed frequently at the initiation of therapy so that the tissue concentrations reach steady-state therapeutic levels. Targeted delivery of antisense into cells of interest may allow one to achieve a knockdown earlier, but any benefit would only be short-lived as antisense is not retained in specific gene silencing complexes but will soon redistribute according to their partition coefficient throughout the entire tissue so that ultimately similar amounts have to be administered and a formulation would only be a nuisance with little benefit.

By contrast, RNAi harnesses an endogenous and catalytic gene silencing mechanism, which means that once it has been delivered, either by conjugation or in nanoparticles into the cytosol, they are efficiently recognized and stably incorporated into the RiSC silencing complex to achieve prolonged gene silencing. In fact, measurable RNAi-mediated gene silencing can be observed at siRNA concentrations so low that it becomes difficult to detect them (e.g. fluorescently-tagged siRNAs by microscopy). This means that as the majority of siRNAs that do not reach the cytoplasm may disappear quite rapidly, the total exposure of the body to the nucleic acid can be much lower compared to antisense which should be beneficial both in terms of safety and pharmacodynamics (activity profile of drug over time).

This is not to say that chemical modification is not practiced in RNAi. However, unlike in antisense, the purpose of modification in RNAi is mainly to avoid triggering innate immune responses, making the siRNA sufficiently stable so that they survive their journey into their target cells, and also to stabilize them as part of RiSC (Merck has been talking about that concept on several occasions); and as we learn more about the biochemistry of endogenous RNA silencing pathways, modification is also increasingly used to increase the inherent biological specificity of RNAi. Unfortunately, it is surprising to me that compared to RNAi only very little, if at all, is reported about the specificity of antisense and I would be grateful if somebody here could point out pertinent studies that I should be aware of.

Targeted delivery may also avoid unnecessary drug exposure of non-target tissues. For unformulated antisense, no matter what the indication and target tissue, the biodistribution is essentially the same, and toxicities of the liver and kidney due to extended exposure to large amounts of the heavily modified antisense compounds is well known.

Certainly, improving the therapeutic index is an important issue for RNAi Therapeutics, too, but as the many transgenic mouse models which express ample and highly efficient RNAi throughout their life without causing overt toxicity attest, ultimately the improvement in the therapeutic index of RNAi is not limited by its very mechanism of action.

While it is a certainty that antisense companies will come out with 4th and 5th generation antisense technology, advances after decades of antisense research aiming to improve target mRNA recognition will only be marginal and based on trying out yet more nucleic acid modifications, although it appears to be a challenge to improve upon the efficacy of probably the most potent antisense modification that have now been known for a while, namely LNAs and their derivatives.

While RNAi efficacy in animals has already surpassed that of antisense for applications of the liver and lung as well as other tissues, I am confident that future advancements in RNAi will be more than marginal. For example, even as recent liposomal formulations achieve 90% gene knockdown in the liver at 1mg/kg, this still means that only about 1 in 10,000 siRNAs that have reached the liver makes it into the cytoplasm (assuming it takes about 1000 cytosolic siRNAs to achieve that level of knockdown according to a recent presentation by Phil Sharp). Alone a better understanding of the endosomal uptake of these nanoparticles, which is only in its infancy and starting to be explored, should allow for more than incremental improvements in the therapeutic index of RNAi Therapeutics.

And if you are still undecided on where the future is heading, numerous transfection studies in vitro where it can be assumed that equal amounts of antisense and siRNAs are present in cells, have shown that RNAi is quite a bit more potent on a mole-by-mole basis comnpared to antisense.

I am aware that some in the antisense community, including investors, may take offense with this blog, but since I am often asked about this issue, I think a more straightforward approach is better than to keep beating about the bush. And, of course, there is always the comment section.

RNAi by single-stranded RNAs

Before RNAi, there was antisense. Antisense strategies typically aim to suppress gene expression by either preventing translation initiation or degrading the messenger mRNA through an RNase H mechanism. As more mechanistic insight into the RNAi pathway was gained, it became clear that the final active, so called “guide” RNA that recognises and causes cleavage of the target RNA by RNAi is a single-stranded 19-23nt RNA bound to the catalytic RiSC endonuclease Argonaute 2 (in humans).

Sure enough, the provision of single-stranded RNAs was then shown by the Tuschl lab to be sufficient for inducing RNAi (Martinez et. al., 2002), although considerably higher concentrations were needed compared to the conventional double-stranded siRNAs in vitro. This work and potential applications thereof are the subject of a USPTO patent application by the Max Planck Society.

Nevertheless, ISIS Pharmaceuticals would claim that its antisense patents cover any therapy making use of a single-strand antisense mechanism, and in their interpretation this includes RNAi with single-stranded RNAs. ISIS’ leadership in nucleic acid technology such as nucleic acid modification and manufacturing technology is without doubt. This is also the reason why Alnylam, the leading RNAi company (of which Tuschl is a co-founder), has taken an exclusive license to a number of ISIS’ patents for the development of RNAi therapeutics, particularly for the use of modified siRNAs.

Although in tissue culture it is clear that double-stranded siRNAs are much more potent inducers of RNAi than single-stranded small RNAs, single-stranded RNAi molecules may have some utility for in vivo applications. In addition to the potential cost of goods advantage of a single-stranded versus a double-stranded approach, the delivery of single-strand RNA-like molecules to organs such as the liver may not even require as much as a formulation into a special delivery composition, as is suggested by the ability of single-stranded antisense compounds such as ISIS’ ApoB100-targeting 301012 to enter liver cells following subcutaneous injection. It is also possible, although not proven, that some of ISIS’ antisense compounds, particularly those with limited 5’ modifications, actually work through an RNAi mechanism.

The issue therefore relates to the value of a claim to a mechanism that was not even known to exist at the time when ISIS applied for patents relating to RNase-mediated gene suppression by an antisense mechanism involving ANY RNAse activity. While I think it is almost impossible that these claims will supersede the fundamental Fire-Mello and Tuschl II RNAi patents, they will probably carry some weight for the use of single-stranded RNAi molecules and ISIS is indeed pursuing such single-strand RNAi molecules for therapeutic purposes. Importantly, however, their utility in an in vivo setting still remains to be demonstrated.

ISIS Copies Alnylam’s Heart Disease Strategy

Today, ISIS Pharmaceuticals, a company developing antisense therapeutics, announced a collaborative deal with Bristol-Myers Squibb to target PCSK9 for the treatment of hypercholesterolemia. As dicussed in my May 6 post, this is the same gene targeted by the RNAi Therapeutics company Alnylam. Since both antisense and RNAi are nucleic acid-based methods for down-regulating gene expression, I will use today’s news to contrast the two technologies.

Both technologies use short nucleic acids designed to seek out their complementary RNA targets and thereby down-regulate corresponding gene expression. Antisense typically comes in two flavours. One is to target a certain region in the complementary mRNA that should inhibit its translation into protein without destroying the RNA target, the other is to use DNA-based antisense molecules that when paired to their target RNA will trigger RNaseH in the cell to degrade the RNA. Although the latter method takes advantage of an enzyme, the natural function of this enzyme is thought to be mainly involved in facilitating DNA synthesis, and therefore neither of the two strategies takes advantage of an existing gene repression mechanism. By contrast, RNAi makes use of such a pre-existing gene repression mechanism that is catalytic and therefore theoretically more efficient. Accordingly, antibodies and recombinant proteins have demonstrated the power of harnessing biological pathways for therapeutic purposes. In the case of RNAi, this is borne out by the finding that in tissue culture and in vivo, the amount of nucleic acid needed to efficiently knock down a gene is significantly less and gene knockdown generally more specific and predictable for siRNAs compared to antisense molecules. This is important as lower doses and higher specificity should reduce side-effect risk. Furthermore, for certain applications it may be desirable to use DNA directed RNAi induction methods, which is not a viable option for antisense.

But one of the main reasons why I am so excited about RNAi Therapeutics is because of the immense interest in the scientific community to understand and leverage the endogenous functions of RNAi. This not only attracts some of the scientific minds to solve potential bottlenecks, but also public recognition and consequently financial support. The Nobel Prize last year has certainly helped, and it is therefore gratifying to see the governor of Massachussetts announce yesterday a plan to support the biotechnologies in that state, specifically mentioning RNAi Therapeutics as one of the focus areas. Antisense, however, has had a long history of failures which has affected investor confidence and hurt its development. Interestingly, however, a number of issues that led to these failures like medicinal chemistry, delivery, and target selection are quite similar for the development of RNAi Therapeutics, and it is fortunate that RNAi can take advantage of lessons learned in the antisense field. It is therefore maybe not surprising that Alnylam and ISIS have extensively cross-licensed their IP, and given the size of the heart disease market, both drugs should ultimately be able to co-exist on the market. My bet, however, is on RNAi.