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.

Alnylam’s Imminent PCSK9 Partnership Deal

A lot of the business development and corporate communications strategy that the 17 VPs at Alnylam come up with these days is, you guessed correctly, directed at building investor confidence so that it may survive the Trade Secret case with Tekmira.  So also the planted interview by John Maraganore a few weeks ago in which he suggested that a PCSK9 deal (ALN-PCS) may be imminent.

Needless to say, without having resolved the important questions about ownership and control over SNALP delivery, chances for a real deal are pretty slim.  Instead, expect a VSP-type deal with minimal upfront, and where the press release will state that development efforts will be focused on the subcutaneous GalNAc conjugates at the expense of (Tekmira’s) SNALP delivery.  

It would, of course, be extremely convenient to announce such a deal just in time for trial, as Alnylam will be able to look the jurors in the eye and state that TTR, PCS, and hemophilia are all now employing a non-Tekmira technology.   A minimal upfront would have the further advantage of depriving cash-strapped Tekmira financial ammunition for the litigation.

Of course, no IND has been filed for any of these yet and, most importantly, clinical validation has not been established leaving a big question mark behind the value (and safety) of GalNAc-conjugates.  I’m wondering what type of messaging the 4-person PR team will come up with to explain, after things have calmed down, that they are still favoring the SNALP-delivered versions.

Note: To put (the estimated) 17 VPs and 4 PR employees in context, Alnylam had 115 employees after the latest lay-offs earlier this year.

Novartis Sells $30M Worth of Alnylam Stock

Last time I’ve checked, Novartis was not desperate for cash.  The cash-flow of Big Pharma is still one of the most impressive features of their businesses, maybe more so than their innovative juices. 

It therefore strikes me as very odd that Alnylam has just provided notice that Novartis last week sold approximately $30M worth of their stock, and has a remaining holding of $80M.  If Novartis wanted to get out of Alnylam, and worse still, RNAi Therapeutics altogether, why not sell the entire stake at once and get it over with?

For Novartis, Delivery is Now or Never

Novartis has RNAi trigger IP rights from Alnylam for 31 targets.  However, the IP, especially the Tuschl II 3’ overhang one which I consider the most valuable, albeit not gate-keeping part of that decimated estate, is ageing rapidly.  Novartis therefore needs access to some clinically viable delivery fast.  Tekmira’s systemic SNALP delivery, of course, is currently industry-leading and, also considering the IP clock (~2020-2021), is an obvious technology for Novartis to utilize.   

Indeed, Tekmira’s Complaint against Alnylam includes a charge that Alnylam allegedly manufactured SNALP LNPs for Novartis, despite the Tekmira/Protiva-Alnylam Agreement that clearly state that Tekmira is the exclusive liposome manufacturer until late-stage clinical development commences.  This would seem an easy charge to verify (note: this is a Count that is independent from the MC3 story). If so, Alnylam’s very survival would also depend on the silence of Novartis (and its other current and former Big Pharma partners).  It is also known that the Head of RNAi Therapeutics at Novartis, David Morrissey, is intimately familiar with Tekmira’s technology from a hepatitis-related collaboration when still at Sirna Therapeutics (~2004-5).

Since neither Alnylam nor Tekmira have a clinically-focused hepatitis program, I suspect that at least HepC has been picked by Novartis exclusively; HepB may be co-exclusive with Arrowhead/Mirus..hence 31 and not 30 targets.

All this leads me to the following speculation (and I emphasize that this is just my speculation, nothing more): Novartis is shifting their RNAi Therapeutics budget to invest $30M or more in delivery technology.  If that involved Tekmira's technology it could also be part of an effort to resolve the hard-fought litigation.  

Novartis’ delivery strategy is one of the industry’s best-kept secrets, but with the August 2012 Marina Biotech oligonucleotide modification deal and an estimated 60-100 employees working on RNAi Therapeutics at that company and having played the game relatively smartly compared to its Big Pharma peers Merck and Roche, which leads me to believe they are not getting out of RNAi Therapeutics (also in light of the recent clinical results), some of that secret may soon be lifted.