The Mipomersen Briefing Docs: Gymnotic Delivery Revealed

After Alnylam’s CEO John Maraganore famously failed to deliver on his Big Pharma platform partnership promise in 2009 (aka ‘SRTL’), and ISIS Pharmaceuticals announced a preferred partnership with GSK in March 2010, it seemed that Dr. Maraganore salesmanship was outdone by one of the best biotech salesmen ever: Dr. Stanley Crooke. 

In addition to likely having deprived Alnylam of a deal, what is this salesmanship claim based on?  It is the fact that not all that long ago, antisense technology had fallen out of favor in Big Pharma.  This was because it required large amounts of oligonucleotides and, partly as a result of that, was associated with non-specific immunostimulatory effects.  And this is where the more elegant RNAi Therapeutics was supposed to take over. Despite this poor track record, the technology managed a comeback, ironically partly on the back of RNAi Therapeutics which was seen stumbling at the ‘delivery’ stage.  In some cases out of the frustration of making RNAi delivery work, the 'naked' delivery approach of antisense would be embraced by the likes of GSK, BiogenIdec, Pfizer, Genzyme, and VCs making the application of Santaris' LNA technology the basis of start-up biotechs targeting emerging classes of non-coding RNAs such as microRNAs or long non-coding RNAs.

The reason, however, why I had always favored RNAi as the more ‘natural’ gene silencing modality over antisense is that antisense, to this day, relies on achieving extremely high steady-state tissue concentrations (>100-300mg/kg of liver and kidney tissue e.g., see ISIS TTR patent application US2011/0294868) either by using reactive/sticky chemistries such as the phosphorotioate backbone or more gentle backbones such as the morpholino chemistry that, however, require even larger amounts of oligonucleotides.  

Mipomersen Briefing Docs Surprises

Unfortunately, the publication last week of the FDA Briefing Document related to the new drug application (NDA) for Mipomersen (tradename: KYNAMRO) by ISIS and partner Genzyme, should serve as a reminder that giving an old technology new names such as ‘gymnotic’ or saying that the algorithms now allow for the discovery of much more potent antisense sequences without any new chemistry (despite the fact that narrow tiling had always been practiced) are telltale signs that the biggest change that has occurred was in the marketing department.

The selective disclosure strategy by ISIS Pharmaceuticals on mipomersen has certainly contributed to the impression that antisense technology had indeed made important progress.  Dr. Crooke claimed in a recent interview on Mad Money with Jim Cramer that ISIS had been very transparent with mipomersen.  I could agree that there has been a lot of scientific data released on mipomersen by ISIS and their clinical investigators, but in hindsight this was arguably not for the claimed sake of transparency, but for providing the impression that mipomersen was a safer drug than it probably is. 

Here are a few examples of the ‘surprises’ that were revealed in the Briefing Docs:  

1) Liver Fat: More and Persistent

While the significance of the non-alcohol-related accumulation of fats in the liver in predisposing towards liver fibrosis is still hotly debated among experts, ISIS tried to dispel the still understandable fears about ApoB-knockdown-related fat accumulation in the liver by claiming a) that it was only modest, and b) that feedback mechanisms allowed the liver to adjust and that liver fat would decrease over time.  Supporting the impression that ISIS was concerned about transparency was the publication (Visser et al. 2010) of liver fat data from a 13-week study in a small, 21-patient study population which only showed a trend of such accumulation (median control-adjusted increase from baseline to day 99 of < 1%).  

Compare that to the actual results (as revealed in the Briefing Docs): In study ISIS301012-CS7 and CS12, 61.8% (63/102) in the mipomersen group had a > 5% increase in hepatic fat content.  This already shows that the median increase is definitely higher than 5%, not < 1%. Moreover, in study CS6, 16% had average liver fat of > 20% measured at least once.  30-40% liver fat contents also were not uncommon.  Such values were practically not observed at baseline or in control subjects despite ISIS’ repeated claims that such values are of no concern since NAFLD is so common anyway.

Furthermore, the data such as capture in Figure 10 of the Briefing Document show that there is no basis for claiming that the liver adapts and liver fat contents come down over time.  They only come down after you discontinue treatment.

Suggesting that the fat accumulations could be of clinical significance, a fibrosis-related biomarker score (ELF) was elevated in the mipomersen population.

To be clear, at least the liver fat accumulations (not sure about  the ELF score) is very likely a target-related side effect and cannot be attributed to the phosphorothioate antisense platform per se, but it is an important issue for mipomersen and raises suspicions about the way ISIS Pharmaceuticals has dealt publicly with platform-related safety issues.

2) Partial Clinical Hold: Vasculitis

Were you surprised by the fact that, according to the Briefing Doc, the FDA issued a Partial Clinical Hold for the non-severe LDL patient population in January 2008?  I certainly was taken by surprise and would also think that this should have been disclosed in an SEC filing as a Partial Clinical Hold for your lead program should be considered a material event.  Not only can shareholders be pitied, but also Genzyme which less than a month before invested more than $300M in mipomersen! I cannot imagine that they would have closed such a deal had it known that there was data that would trigger such a Hold (I do not exclude that the data was there under their nose, but in that case it was probably either buried under a pile of paper, or Genzyme just did not do their job well).

Apparently (à Briefing Doc), the ‘final interim report’ from the 52-week monkey tox study that was submitted in June 2007 showed vasculitis (inflammation of the vasculature which can impair blood supply and therefore organ function, possibly leading to organ failure) at clinically relevant doses of 3mg/kg and up, mainly, but not exclusively, in the gastrointestinal tract. Although the Hold was lifted after the actual clinical safety data did not indicate vasculitis to be an issue, subsequent skin biopsies around the injection sites showed an accumulation of inflammatory cells around the vasculature.

Although similar to the liver fat issue, the clinical significance and degree of systemic vasculitis due to phosphorothioate oligonucleotides remains unknown in Man, the potential for sustained inflammatory processes in the entire vasculature is a serious safety concern that needs to be studied in more detail before the technology can be applied to less than the most severe, 'orphan' patient populations- if at all.

3) Liver Enzyme Increases

That mipomersen increased liver enzymes in the serum, a measure of liver tox, has been widely known.  But ISIS used to claim that in a given individual these were one-off findings that could just as well have come after a night of heavy drinking.  By contrast, it seemed to me new news that there were cases where elevated (>3x ULN) liver enzymes were found on at least two subsequent investigations.  In fact, the FDA feels that the liver enzyme increases warrant them be tightly monitored as part of a REMS program should mipomersen be approved in the homoFH population.

4) Injection-Site Reactions and Discontinuations

One of the major marketing tools, and in fact actual draws, of ‘gymnotic delivery’ has been that it can be subcutaneously administered, while the leading RNAi Therapeutics delivery technologies, foremost Tekmira’s SNALP, still rely on intravenous infusion (for most applications at least).  Turns out that injection site reactions ranging from pain, redness, swelling, to skin discolorations and haematomas were a major factor for study drug discontinuations, especially in the open-label phases of the studies which should be indicative of what will happen in a real-world setting.  Overall, 61% of HoFH patients (23 of 38) discontinued study drug during the open-label phase, and 77 of 141 discontinued in the pooled Phase 3 population (including non-hoFH subjects).  Other adverse events attributable to the inflammatory potential of the technology, such as flu-like symptoms, contributed to the high discontinuation rate.

A curious, hitherto undisclosed finding, were injection site recall reactions with mipomersen (but not control).  This refers to an inflammatory reaction at an old injection site when the drug was administered at a new site.  The molecular basis and importance for such recall reactions seem unknown.  In fact, my literature searches suggest that they have almost exclusively been described for the TNF-alpha blocking antibody Enbrel.  However, to me, they once again indicate the systemic inflammatory potential of phosphorothioate antisense chemistry, likely involving adaptive immunity.  Accordingly, 30/50 patients (60%) in the CS5 and 6 studies exhibited mipomersen antibodies compared to none in the control population- another ‘surprise’ that should be applicable to the phosphorothioate platform in general.

5) Carcinogenicity

While there were ~2-3x more cases of tumors in the mipomersen-treated population compared to control, I agree with the reviewers that they are not the basis for concluding that mipomersen increases the risk of developing cancer.  This is because of the nature of the reported cases and the relatively short treatment duration after which many of them occurred.

However, what would worry me more is that in the preclinical carcinogenicity studies in rodents (mice and rats), mipomersen clearly increased cancer risk- despite ISIS’ claim that they have been ‘clean’.  Among the findings were hepatocellular adenoma and subcutaneous tumors.  Having a report by your CRO stating the findings are likely species-specific issues and not applicable to humans, does not mean that the findings are not material in a regulatory sense, especially as they were made during a period at the FDA when the agency was much more risk-averse.  I expect the clinical and preclinical findings to cause carcinogenicity to be included in a REMS program.    

6) Kidney Damage

The liver, spleen, and kidneys are the major sites of phosphorothioate accumulations.  Consequently, it is not surprising that kidney damage has been an important safety concern of such antisense technology.  Although the clinical safety findings did not demonstrate that mipomersen impaired kidney function, the study drug did increase the amount of protein found in the urine (proteinuria).  This finding supports that kidney function remains a safety concern and such monitoring should be included in a REMS.

In summary, the safety findings were not all that surprising given what we know about phosphorothioate safety in animals and study discontinuations by competitor Santaris, very likely due to safety.  However, if you indeed have taken the reassurances by ISIS CEO Dr. Crooke literally, you may be justified in feeling misled.  Even on that count, however, I am not all that surprised, and his salesmanship may indeed be one of ISIS' great assets.  

Disclosure: The author is 'long' the stock ($ISIS).

Clarification: The term 'gymnotic delivery' in the linked paper refers to a specific protocol for optimizing gene knockdown in tissue culture in the absence of assisted delivery.  However, the term has been more broadly used by Santaris to refer to unassisted delivery in general (including clinical applications), obviously in an effort to express that in contrast to phosphorothioate oligonucleotides, RNAi needed assisted delivery.  I found it suspect, however, that they used a new term to refer to an over decade-old industry practice.  In a further clarification, I have not seen ISIS Pharmaceuticals adopt the term either. But clearly, not using (in most cases) assisted delivery has been a greatly exploited marketing tool of the antisense industry built on the back of RNAi Therapeutics.

Genzyme Taps RNAi Therapeutics for TTR Amyloidosis

Orphan disease gold miner Genzyme has just partnered Alnylam’s promising TTR amyloidosis drug candidate, giving it the right to commercialize resulting medicines in Japan and ‘other Asia-Pacific countries’.  In addition to a healthy $22.5M upfront, Alnylam stands to receive the customary development milestones and actually high royalties for a post-phase I program, plus Genzyme bears responsibility for the regulatory process in the licensed territories.

The basis for this deal, as also expressly stated in the press release, were the potent, ~90% knockdown efficacies obtained in the (single) dose-ranging ALN-TTR02 phase I study (for review, see 'Simply SNALP').  Critically, it was Tekmira’s SNALP delivery technology that made these results possible and catapulted Alnylam’s market cap by ~300M (50% increase in share price) at the time the phase I results were announced this summer.  I should add, however, that in addition to ALN-TTR02, the preclinical subcutaneously administered candidate ALN-TTRsc has been included in the deal. 

Clearly, if they are really serious about the competitiveness of the subcutaneously administered GalNAc-siRNA conjugate, Genzyme has got to be hoping that there won’t be a repeat of the mipomersen (antisense) injection site reaction fiasco which, in addition to the liver fat and immune stimulation issues, is endangering the approval for mipomersen even in the very high unmet medical need population of homozygous familial hypercholesterolemia.  In this case, the adverse event that could drastically lower the deal value soon after its close comes in the form of the Tekmira litigation risk (note: Genzyme is ISIS' partner on mipomersen and should know, also about the strength of the competing subQ ISIS-TTRRx program which is partnered with GSK).  

In case you are wondering what I am alluding to: less than a month after Genzyme invested more than $300M in ISIS and their hypercholesterolemia candidate mipomersen, the FDA issued a Partial Clinical Hold due to significant safety signals with the systemic phosphorothioate oligonucleotide, according to last week’s Briefing Documents for the mipomersen Advisory Committee meeting.   The risk in the TTR case, of course, is the pending litigation with Tekmira, involving the critical RNAi delivery technology enabling the ALN-TTR02 results, its manufacturing and other (e.g. 2’-O-methyl) IP associated with the TTR amyloidosis program.  You would expect that Genzyme would have at least waited for another month or so before closing the deal (trial date in November).  

I doubt that the deal value would have been much higher without the overhang given the size of the Asian TTR market.  But then, of course, nothing can surprise me here any more and a resolution to the Alnylam-Tekmira troubles may already have been hammered out.  A clue may be how Tekmira responds to the news.  If there is tonight a we-are-pleased-that-Genzyme-has-partnered-ALN-TTR02-for-which-we-provide-the-delivery-technology-PR, then it's probably nothing to get excited about.  But if there is silence, then maybe the real news may come with a slight delay.  

Talking of Tekmira, this company, as it is customary in biotech sub-licensing, is likely to receive a partial payment out of the $22.5M upfront (plus a cut of the future income stream related to ALN-TTR02).  Another partnership implication is that Alnylam has talked to Takeda, and Takeda declined on ALN-TTR.  As you remember, in 2008 Takeda forked over $150M to Alnylam, also for being the exclusive Asian partner for 5 years with a right of first refusal for Alnylam drug candidates in the Asian territories.   You’ve got to wonder how comfortable that conversation was, especially after handing over some of that for the ‘technology transfer’.  There might be even less room to hide from Genzyme, as I believe Genzyme and Alnylam may be sharing one building now.

Arrowhead Research Breaks Up Dynamic PolyConjugates into Two

Arrowhead Research announced yesterday that it had received Notice of Allowance from the USPTO for a Dynamic PolyConjugate-related patent application (for systemic RNAi delivery).  Instead of it being your run-of-the-mill patent PR involving known technology that finally received patent protection, it was really about revealing Arrowhead’s fundamentally new approach towards DPC delivery (for my take on the original form of DPC, see here).

The patent, part of a series of patent filings that have come out recently, shows that under Roche’s ownership, the technology has morphed through a number of iterations from the original complex polyconjugate chemistry combining endosomolytic polymer, masking groups, RNAi trigger, PEG, and cell targeting ligand all in one molecule (schematic shows such molecule and presumed mechanism of delivery), into one where a RNAi trigger and the masked polymer, both targeted individually, are administered as separate agents. 

One problem with the original design has been that combining all the functional groups, including negatively charged RNAi triggers and positively charged polymers, into one molecule was not particularly easy.  The tendency to aggregate and poor yields made it a quite expensive and difficult-to-scale proposition.

Turns out that such complicated chemistry wasn’t needed after all.  As long as the RNAi trigger and the masked endosomolytic agent end up in the same place, it does not make much of a difference whether they are getting there as one molecule or separately.  In the example provided, namely for gene knockdown in hepatocytes, the RNAi trigger could be conjugated to either cholesterol or a cluster of galactose sugars, whereas the masked endosomolytic polymer was targeted to the hepatocytes by galactose.  Viewed differently, the polymer allowed the cholesterol-siRNA that apparently gets trapped in the endosomes when alone to be released into the cytoplasm.  Hence, the multi-fold increase in potency (Arrowhead Research says it’s 500-fold) over Alnylam’s original 50mg/kg cholesterol-siRNA report (Soutschek et al., 2004).

Another potential advantage of this separated approach is that it makes each component smaller, perhaps 'one day' enabling subQ dosing.  However, as mipomersen's FDA AdCom meeting briefing docs show, when it comes to subQ dosing, be careful what you wish for.

Manufacturing appears to have been one of the issues delaying the clinical translation of DPCs for quite some time (Arrowhead Research says they are ready to file an IND in Q2 2012 for Arc520 in HepB).  Toxicity, mainly due to premature unmasking in the blood instead of in the target cell endosome, seems to have been the other main reason.  Such premature unmasking also adversely affected circulation times, thereby rendering attempts to get beyond the liver, one of the original promises of DPCs, futile.  We will probably get word from the company soon what solutions it found for this challenge, but it seems that, based on the emerging patent literature (including non-human primate data; e.g. WO 2012/083185), masking the membrane penetrating peptide mellitin with endosomal protease-sensitive groups, is a promising approach.

Sarepta Discloses Dystrophin Expression Data at World Muscle Society Congress

Last week, I criticized Sarepta Therapeutics for failing to disclose critical dystrophin expression data regarding their exon-skipping drug candidate eteplirsen for the treatment of DMD.  Instead, they merely disclosed the percentage of muscle fibers staining positive for dystrophin, i.e. fibers that expressed some dystrophin, which, of course, would be a much more impressively sounding number.  Adding to what I perceived as dubious data presentation was 6-minute-walk test (6MWT) data of which the integrity seemed compromised based on cherry-picking the patients and the fact that the apparent improvement occurred in the open-label extension phase of the study.

Admittedly, the percentage positive fiber number was a previously defined primary endpoint whereas the amount of overall dystrophin relative to normal/healthy was not.  Therefore, like other biotechs frequently do, it might always have been Sarepta’s intention that only the primary endpoint data be presented initially ('top-line data'), with the balance to be disclosed at a future scientific conference or peer-reviewed presentation.

Accordingly, Sarepta today presented additional data at the World Muscle Society Confress in Perth, Australia.  I was pleased to see that at least some dystrophin expression Western blot data were revealed (slide 12)- with a direct comparison to ‘normal’ at that.

First of all, you can see on the left the ‘normal control’ (healthy) comparison.  The labeling seems to be somewhat off as I believe the outermost left lane represents the sample.  You can also see that the normalization control ‘actin’ was less abundant in the control sample compared to the patient samples (by naked eye about 3-5 fold).  This means that the 'normal' dystrophin signal is an under-estimate relative to the patient samples. What is more, because the dystrophin signal in the ‘normal control’ is over-saturated (big black blotch), the ‘real’ relative dystrophin amount should be even more compared to the patients'.  Because there is no standard curve and because of the apparent loading differences (à actin), it becomes an art to estimate the relative dystrophin amount restored by eteplirsen treatment, but with 7 years of doing the same type of experiments at the bench under my belt, I would guesstimate that the true value is about 2-5% of normal.  Certainly not the 20% believed to be necessary to start impacting the disease phenotype. [Correction 10/13/2012: This was an inadvertent mis-characterization of the quoted source, please see the discussion in the Comments section].

Supporting the notion that the Western Blot overestimates restored dystrophin levels, is the RT-PCR experiment on the right.  Again, the larger band which represents the mutated transcript is much more abundant than the re-spliced signal below, indicating that only a minor fraction of the mutant transcripts were re-spliced.  Moreover, smaller PCR products amplify more efficiently compared to larger ones, meaning that the apparent splice correction is an over-estimate.  Similarly, because the mutated and ‘normal’ PCR signals are almost the same (you would expect an mRNA with premature stop codons to be destabilized relative to wild-type), it is likely that the normally spliced bands have reached saturation, further over-estimating the splice correction in the RT-PCR experiment.

Overall, whatever Sarepta’s motives were behind failing to disclose the absolute dystrophin data last week, they clearly provided some of the necessary transparency at the WMSC presentation.  Having said that, I believe the data support the underlying concern that the splice correction may not be enough to be therapeutic.  I would not exclude it, however, as the 20% value, of course, is also an estimate.  Blame a less than rigorous trial design for not knowing for sure for quite some time to come. 

The Regulus IPO: A Reason to Invest in MicroRNA Therapeutics

Many of you will already have seen it: With little fanfare, Regulus Therapeutics (ticker: RGLS) is now a publicly traded company.  Rather than re-hashing the financials of the IPO and whether the first (depending on whether you count Rosetta Genomics in) public exit was good for the microRNA Therapeutics sector or not, I want to remind readers of one key selling point of the technology, one that I believe explains why Regulus has been so successful in  generating partnership revenues from the likes of Sanofi-Aventis, GSK, and AstraZeneca: microRNAs are a major class of genes and their products can only be targeted by oligonucleotide approaches.

Big Pharma companies are a conservative bunch and small molecules habits die hard.  While the industry has embraced monoclonal antibodies with all their benefits, but also warts, oligonucleotide therapeutics are still considered outcasts in the industry.  Maybe a therapeutic area group in Big Pharma will take a look at it when nothing else seems to work (hello respiratory disease groups).   While this could change given therapeutic (and vaccine) oligonucleotide candidates in TTR amyloidosis (Alnylam and ISIS/GSK), HBV (Dynavax), and hypercholesterolemia (mipomersen/ISIS) and the excitement around the exon-skipping drug candidates from Prosensa and Sarepta, the precise timing of a lasting turnaround in sentiment would probably depend on whether and when sales of any of these potential products really take off.  Money does not smell to Big Pharma either.  But until then, it's likely to continue being a roller-coaster ride.

Despite falling in the oligonucleotide therapeutics category and despite being younger and technically less validated, microRNA Therapeutics have an important marketing advantage over RNAi and RNaseH antisense.  Unlike RNAi and RNaseH which often have to hide in the ‘undruggable’ space, i.e. in addition to the desperate research groups they may be considered by Big Pharma if small molecules and monoclonal antibodies cannot address a biologically attractive target, microRNA Therapeutics do not stand in such direct competition.  Biologically too important to ignore and implicated in many diseases, oligonucleotides, at least for the foreseeable future, are what it takes to tap this critical group of genes.  

Because of that, I expect industry interest (= partnership $$$) in microRNA Therapeutics, and as a leading company in that area, in Regulus Therapeutics, to be less volatile than what has been the case for its two older siblings.  Having a strong balance sheet, should also help in that regard.  So welcome, Little Emperor, to the public markets.

Sarepta Fails to Report Meaningful Dystrophin Expression Data, Falls Behind Prosensa

Restoring truncated, yet largely functional dystrophin in Duchenne Muscular Dystrophy (DMD) by exon skipping is a promising approach to this devastating disease.  For this, however, to have any meaningful therapeutic impact, it has been estimated that one would have to achieve about 20% of wild-type (probably somewhat higher for truncated versions) dystrophin levels in all muscle fibers.

Releasing 48-week data from one of the most manipulated clinical trials that I have seen, Sarepta Therapeutics just reported that treatment with their exon-skipping antisense morpholino eteplirsen resulted in 34-52% dystrophin-positive muscle fibers.  Impressive? I can’t tell, but what is more worrisome is that I don’t know why the company does not tell either.

Percent muscle fibers expressing dystrophin does not mean equal amount of dystrophin restored.

In a New England Journal of Medicine paper last year, Sarepta’s competitor Prosensa (with partner GSK) reported in a similar-sized study that in 10 out of 12 patients, the percent of fibers expressing dystrophin following treatment with their exon skipper PRO051 was between 60 and 100%.  Yes, 60-100% and nost just 34-52% as in Sarepta's case.

What is more, the immunofluorescence intensity and Western blot data in the Prosensa study showed that those fibers expressing dystrophin did so at much reduced levels compared to healthy muscle, on the order of 5-20% (overall bulk levels).  In other words, it is quite likely that the ~50% of fibers that express dystrophin in the Sarepta study together express at most 3-10% of normal dystrophin, well short of what is expected to be therapeutic.  It is also possible, however, that Sarepta’s assay is very sensitive such that even fibers with just 1% or so of wild-type signals were counted in. 

Given that stating the percent fibers expressing any dystrophin is almost meaningless, you have to wonder why Sarepta has not presented the data.

But then you ask, then why did the lame start to walk in the study (à 6 minute-walk-test data)?  Actually, I don’t believe that this was shown in the study either.  Just because this is a devastating disease in children does not mean that rigorous trial design and execution can be entirely done away with.  Yet, in this already tiny 12-patient, single-center (!), and open-label (!) study, the company wisely discarded the worst responders early on allowing the 6 MWT numbers to be 'statistically significant'.   There seemed to be more subgroup analyses presented than patients in the study.  If I were a regulatory agency, I would strongly consider refusing to even just look at the 6 MWT data.

Why am I writing this?  First of all, I am obviously trying to find any shares to short.  And second of all,  what really irks me is that as orphan drugs become more and more popular in drug development, a number of companies are doing away with rigorous science, drive aggressive patient recruiting and disease awareness campaigns (best if a pediatric indication is involved), and expect the rest of us to subsidize the enormous costs of these drugs.  Fine, if the drugs worked, but not for placebos.  Wait another 5-10 years and there will be an enormous backlash and those that need access to these drugs will suffer the most.

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."

Roche Reveals Antibody-Targeted DPC and SNALP Data

Arrowhead Research has stated that it is about to lift the veil on the RNAi Therapeutics research at Roche through a number of publications.  As you remember, Arrowhead Research made a daring splash a year ago when it acquired much of the Roche RNAi assets for about a cent on the dollar invested in RNAi Therapeutics by Roche.  I will be covering the revelations on this blog as I had once considered Dynamic PolyConjugates one of the more promising systemic RNAi delivery technologies- albeit at least 3-4 years behind Tekmira's SNALP technology in terms of clinical translation and validation.

It was actually Roche (and not Arrowhead/Madison) that fired the opening shot with a study on the use of bispecific antibodies for targeted siRNA delivery (Schneider et al. Molecular Therapy- Nucleic Acids: Targeted siRNA delivery and mRNA knockdown mediated by bispecific digoxigenin-binding antibodies). 

Antibody-targeted siRNA delivery has been a concept that has been around for a while.  The overall industry sentiment on this topic is that while it is still worthwhile pursuit, there have been problems with replicating some of the early data concerning simple antibody-siRNA conjugates and electrostatic complexes.  Stability and cytoplasmic penetration have been the two main issues.

The latter obstacle was also encountered when Roche simply added siRNAs conjugated to digoxigenin (DIG) to a bispecific antibody recognizing both the small molecule DIG (thereby binding the siRNA) and a cell surface protein: the siRNA was specifically delivered to the cell expressing the cell surface protein, but there was no gene silencing, presumably due to lack of endosomal escape into the cytoplasm.

This, of course, is not all that surprising, although, in all fairness, it is not just the old antibody-siRNA literature, but also work from the aptamer-siRNA field and the GalNAc data by Alnylam that suggest that certain cell surface receptor uptake pathways allow for such simple delivery.

To further facilitate gene silencing, the bispecific antibody technology was then applied to DPCs and SNALPs by  linking DIG to the polymer backbone (DPC) and PEG-lipid (SNALP), respectively, with the siRNA either covalently bound to the backbone or enclosed in the aqueous SNALP interior.  The masked cationic polymer in the DPC and the cationic liposomes were thus tasked with overcoming the endosomal release challenge.

Targeted SNALPs for Gene Knockdown in Vascular Endothelia

Indeed, gene silencing could now be observed in tissue culture with both approaches.  Next, the authors tested out the concept in the much more challenging in vivo animal setting.  Data for the targeted SNALPs were reported.   

Mouse seeded with human tumor cells were administered SNALPs (employing Tekmira’s ’57.1’ formulation ratio, KC2 ionizable lipid, and in-line mixing) targeted towards the VEGFR2 receptor that is abundantly expressed on the endothelial cells of the vasculature.  Impressively, such constructs not only maintained the expected endothelial silencing efficiency of SNALPs, but greatly enhanced it: a 40% versus almost 80% gene silencing of the CD31 endothelial marker gene.

Endothelial cells are a good initial target cell population for this approach as these complex, and rather large (>100nm) nanoparticles have easy access.  The approach also does not rely on positive charge for cellular uptake which could be a safety advantage over competing approaches.

The reason why the performance of DPCs in this model was not described is unclear.  It is possible that DPCs simply did not work.  It is also possible that Roche did not want to steal Arrowhead’s thunder and held off on publishing the data.  Pointing in that direction was the fact that the siRNA sequence and, more importantly, modification was not described in the paper.  Not only would this have been crucial for interpreting the innate immune stimulation data (it seemed to be absent), but also an important factor when it comes to uptake that involves the direct exposure of the siRNA to endosomal endonucleases.

Overall, it was a fun paper to read and quite a bit more innovative than many of the RNAi research that has been conducted by Big Pharma bar Merck. Antibody-targeted RNAi delivery is certainly not dead. The cationic lipoplexes by Silence Therapeutics could get competition, although Atuplex enjoys a considerable, multi-year head-start.

Comment on Last Week’s ‘Patent Victory’ by Alnylam

Last week, a US judge ruled that, based on the contracts between Alnylam and Tekmira, Alnylam has standing in enforcing certain exclusively licensed patents.  Tekmira tried to duck this infringement lawsuit by claiming that as the licensor of these patents, it was immune from such enforcement.  The judge disagreed saying that such immunity should have been explicitly stated in their agreement.  In my opinion, this technical decision is not entirely surprising.  All it means is that the case will now be tested in more detail, representing another cash drain on Tekmira.  That financial pressure is Alnylam's main aim in filing the lawsuit is also shown by the company expanding the case into Canada, where Tekmira actually conducts its business.

The ruling does not, however, predict whether Alnylam will prevail on the merits.  This should boil down to the question of whether Alnylam’s right to the patents extend to target validation and not just RNAi therapeutics.  As the Alnylam field is defined as “the treatment, prophylaxis and diagnosis of diseases in

humans using an RNAi Product or miRNA Product", it obviously does not.  

The decision also does not reduce the damage that Tekmira could claim as part of the much more important Trade Secret litigation where Tekmira has charged financial damage arising from Alnylam trying to cut out Tekmira from the financial benefits of SNALP, e.g. when dealing with Big Pharma companies such as Takeda and Novartis.  I do not recall that Tekmira accused Alnylam of making it impossible for Tekmira to close RNAi Therapeutics deals directly with Big Pharma, without Alnylam.  An important distinction.