Plea to Sarepta and Arrowhead: Just Focus on Aberrant Exon 1 Transcript in Huntington’s Disease

Last week, uniQure achieved a breakthrough in developing disease-modifying therapies for Huntington’s Disease by showing that AMT-130 slowed disease progression by 75%.  AMT-130 is a DNA-directed RNAi construct targeting a sequence in the triplett-expanded exon 1 of the 67 exons-containing huntingtin gene.  Since other approaches aimed at lowering full-length huntingtin, but leaving exon 1 mRNA untouched, have fallen short (e.g. tominersen by Ionis and Roche) this is very strong support that targeting the aberrant exon 1 transcript is essential for success.

The aberrant exon 1 transcript, and likely the protein produced from that, is loaded with polyglutamine-encoding CAG tripletts, has been shown in animal studies to be exceptionally toxic and is also produced in patients (see previous blog entry 'Huntington's Disease Therapeutics Finally Breaking Through!'), the amount of which is correlated with triplett expansion size based on comments by uniQure CMO Walid Abi-Saab at a conference earlier this year.  Add to this the observation that a phase 3 trial with tominersen had to be stopped due to the oligo performing worse than control, maybe it is time for a heretical new idea:

accept that the old notion of Huntington’s Disease being an autosomal dominant disease caused by toxic full-length triplett-expanded huntingtin has been wrong, and full-length huntingtin indeed is an important protein to have. Instead, leave full-length huntingtin alone and exclusively target the aberrant exon 1 ‘fragment’.   Hey, have you ever considered the possibility that the polyQ htt protein aggregates are not cleavage products from full-length Htt?  Why always so complicated?  Why consider both exon 1 and full-length Htt contributing meaningfully to disease?  Shouldn't the properties of a short protein largely made up of polyglutamine be radically different from that of a much larger one containing the same polyglutamine stretch?

Sarepta and Arrowhead have shared plans to enter the clinic with a systemically administered RNAi conjugate for Huntington’s Disease.  A systemically administered option would be a very valuable one since, especially in light of the one-time administered AMT-130, repeat intrathecal administration is less attractive.   The Tfr1-targeted ARO-HTT being prepared for clinical trial application this year apparently does not affect exon 1 transcript.  The excuse given is that it is difficult to find an effective RNAi trigger in the merely 102 nucleotide exon 1 target space.

The good news: by focusing exlusively on aberrant exon 1 transcript, 7000+ nucleotides of additional target space (from 5’ end of intron 1) opens up.  

What good is it to have a trigger that is great at knocking down a transcript when it is aimed at the wrong target?  It is not too late and can be explained to the market.

Vinay Prasad's Days as Head of CBER May Be Numbered

Many industry insiders expected Sarepta CEO Doug Ingram to be out by now.  Instead, a new dynamic is gathering steam: Vinay Prasad, the recently hired head of CBER at the FDA, is coming under increased pressure as he is being identified as sabotaging Republican pro-choice philosophy. 

Last night’s latest panicky press release by ‘the FDA’ (= Prasad) that it is investigating the death of a Brazilian boy with Duchenne’s muscular dystrophy (DMD) who had received the AAV Elevidys gene therapy from Sarepta, speaks volumes.  According to a recount by FirstWordPharma, the death of this boy occurred on June 7 and was logged onto the agency’s FAERS adverse event reporting system on June 18.  The death was deemed unrelated to Elevidys by the reporting physician and caused by flu, possibly exacerbated by immunosuppression.  It remains to be seen whether the immunosuppression was part of the boy’s normal treatment for the disease (corticosteroids are standard of care) or whether aggressive immunosuppression was practiced to control acute liver injury related to Elevidys.

In any case, given that shipments of Elevidys had been halted in the US, press releasing such an 'investigation' into a death deemed unrelated to the gene therapy smacks of desperation…the desperation of Vinay Prasad clinging onto his post.

 

Trump loyalty enforcers zeroing in on Vinay Prasad

When Vinay Prasad was appointed to head CBER 3 months ago, it caused a sell-off in biotech stocks relying on accelerated approval pathways aimed to ultimately provide patient choice in a timely manner.  Gene therapy companies in particular rely on these pathways (e.g. biomarker-based, natural history comparisons) with their ongoing programs and Prasad has been public with his disdain for the Elevidys approval under Peter Marks.

In subsequent weeks and months, the investor and sector panic subsided as Vinay tagged along FDA Chief Makary advocating regulatory flexibility (approval at the slightest sign of efficacy or plausible biological mechanism).  After recent rejections of Capricor’s DMD cell and Replimmune’s oncolytic viral therapies and now the controversy around Elevidys and Sarepta, this view is changing quickly again.

Unsurprisingly, Trump’s loyalty enforcer Laura Loomer has picked up the scent and identified Prasad as a Bernie Sanders-style progressive, a wolf in sheep’s clothing undermining Republican healthcare philosophy.  

To wit, like so many of Trump’s current and past allies, Prasad has made fun of the President in the past.  More importantly, he has been a strict advocatefor healthcare rationing, limiting tests and drugs to circumstances of the highest medical certainty with Father State, not the patient or physician, being the one to decide.  His tone deafness for patient views, also on risk taking, is further illustrated by his statement that the instance you are dead, nobody will ever think of you again.

Yesterday, former Republican Senator Rick and Newsmax contributor Rick Santorum tweeted the following, citing an article that specifically attacked Prasad for his handling of Elevidys (the intro to that piece is quite the read).

 

How we got here

When Sarepta announced its company reorganization last Wednesday, shifting its focus away from gene to synthetic RNAi therapies, it did not mention the death of a non-ambulatory man treated in one of its AAVrh74 gene therapy trials for limb girdle muscular dystrophy (LGMD).  AAVrh74 is also the serotype of Elevidys. 

This death occurred and was reported to the FDA in early July.  To be clear, it was an unforced error by Sarepta to not publicly disclose that death for transparency reasons as it claimed that it retiring most LGMD gene therapy programs had nothing to do with safety.  It is, however, becoming increasingly evident that pre-existing poor liver health such as cholestatic disease (à XLMTM-related AAV gene therapy deaths) is the major risk factor for dying from AAV-related liver failure.  The fact that none of the ~800 ambulatory and therefore younger DMD subjects have died due to high-dose AAVrh74 gene therapy, but 3 out of ~140 non-ambulatory (à hepatic steatosis risk) patients treated with AAVrh74 fits neatly, making it the hypothesis that needs to be disproven first.

Sarepta has made itself many enemies and having just laid off 500 employees added to this pool.  According to BioCentury's Steve Usdin, the reporter who broke this story, there were numerous messages sent to journalists on Wednesday night and Thursday following the re-organization call enquiring why the LGMD patient death went unmentioned.  This resulted in numerous sensationalist media reports and put the publicity-minded FDA in a tough spot.  So after this AI-powered agency had sat on the Brazil and LGMD deaths for almost a month without (understandably) seeing a reason to pull Elevidys for ambulatory DMD patients from the market, it felt it had to act and be seen as a tough regulator.

Two press releases (here and here) and constant Signal chat leaks from an ‘unnamed senior FDA official’ attacking Sarepta followed, one less coherent than the other reaching yesterday’s low point.    

 

What is next

To me it is clear that this is coming down to a showdown between Sarepta, and in particular its CEO Doug Ingram and Board of Directors, and Vinay Prasad.  The company is taking the high road saying that it is working to resolve the shipment halt ‘within the ordinary and well-established FDA channels and procedures’.  You bet, however, that Sarepta is also working through unofficial political channels and Vinay Prasad has not taken into account how Sarepta got to where it is today in the first place and, being tone deaf to patient concern, certainly not the strength of DMD patient advocacy which clearly wants Elevidys to remain a choice for them.  With Replimmune and Capricor showing that Elevidys is part of a broader pattern, his fate has been sealed in my opinion.  If not, expect a Jesse Gelsinger-type scenario for gene therapy.

 

Scientific Post-Scriptum

There are less widely discussed strategies that the systemic AAV gene therapy field in general may want to explore more to increase safety besides refining immunosuppression regimens which, as we all know, carry their own risks.

One is to use RNAi, highly potent for directing gene knockdown in hepatocytes, to downregulate the processing and presentation of AAV antigens attracting the sometimes fatal T-cell response.  Sarepta with their partnership with Arrowhead Pharmaceuticals, is ideally placed to do so.  Another option could be to inhibit hepatic uptake of AAV by co-administering heparin, which should inhibit hepatic AAV uptake via HSPG and may shift the balance towards non-hepatic uptake.   

Related to that and highlighting the failure of Sarepta to have run a proper dose escalation study, with Elevidys doses being sky-high at 1.33 × 10¹⁴ vector genomes per kilogram and muscle delivery being subject to a step function (similar to LNP delivery to hepatocytes), using significantly lower doses will likely not impact microdystrophin expression much, but further decrease liver failure risk.  This and excluding patients with pre-existing liver issues, especially hepatic steatosis in DMD, may be the most rapid way to get Elevidys back onto the US market in a scenario where everybody can save face.  DMD patients and their families do not deserve to be held hostage as Prasad is clinging onto his job.  

RNAi Companies Ought to Look Beyond Their Platform

This week, Alnylam announced in their roundabout way that it has discontinued the development of ALN-HBV for the treatment of chronic HBV infection.  Instead, it has come to an agreement with well-funded start-up Vir Biotechnology to select a new GalNAc-based RNAi compound, ALN-HBV02, which Vir will largely take control over clinical development.  Alnylam meanwhile retains considerable back-end loaded opt-in rights, milestones and royalties.  

As alluded to in a post last week, ALN-HBV seemed always doomed to fail due to target site selection issues. Also, Alnylam’s heart never really was into ALN-HBV with project leader Sepp-Lorenzino functioning as a one-woman show while competitor Arrowhead mobilized considerable internal and external resources.  She’s, of course, left the company not long ago.

We may never find out the real reasons for their decision although Alnylam would like us to believe that it was a specificity issue since ALN-HBV02 will now be ESCplus-based. Please don't fall into that trap.

My expectation is that ALN-GO1 for primary hyperoxaluria (PH) will suffer the same fate as the initial biomarker data from clinical studies have indicated that likely prohibitively high doses would be necessary to achieve robust oxalate lowering (a modest ~2x increase in glycolate biomarker at a single dose of 6mg/kg).

Once again, a much smaller competitor, Dicerna, has been running circles around RNAi juggernaut Alnylam.  Importantly, it has conducted detailed work on the biochemical pathway of oxalate metabolism and closely studied the natural history of the disease.  The reward was the discovery that the lactate dehydrogenase A (LDHA) is in all likelihood a much better target.

In hindsight it is quite clear that both HBV and PH were only chosen by Alnylam to scare investors away from their competition to cement RNAi dominance.  Not only that, it then filed a frivolous lawsuit against Dicerna alleging trade secret violations.

Will RNAi platform companies have to broaden their tech base?

Given the need to immerse oneself into individual diseases, and also as RNAi is about to open the commercialization chapter, frequently developing drugs for entirely new disease categories served by medicines, a new question arises for the industry: do I remain a platform company or do I have to open my technology base to best serve patient communities and shareholders?

While I do like to criticize Alnylam for shamelessly behaving like the industry's big bully, TTR amyloidosis is one of the diseases they deeply care about.  With 10-20k TTR amyloidosis patients in developed markets, a number that should grow due to increased disease awareness, better diagnosis tools and access, and hopefully prolonged lives, this could well develop into a $5B+ annual market over the next decade.

As the company behind what promises to be the first dominant drug for this disease and a next generation product candidate (ALN-TTRsc02) that should remove any doubt who owns TTR gene knockdown following on its heels, Alnylam would be irresponsible not to try and control the whole patient experience, including complementary non-RNAi treatment options.  In other words, Alnylam would cease to be a pure-play RNAi Therapeutics company while continuing to rely on RNAi for conquering new markets.

This evolution of corporate strategy, of course, is not new to biotechnology and has played out at companies like Vertex Pharmaceuticals (small molecules à cystic fibrosis focus), Biogen (recombinant proteins à multiple sclerosis/CNS focus), and indeed in the Oligonucleotide Therapeutics space before.

Sarepta, once singularly based on morpholino antisense technology, for example has been on a Duchenne muscular dystrophy (DMD) business development spree as their first antisense drug (EXONDYS51) was getting approved.  Sarepta sports a solid >$3B market capitalization illustrating that the financial markets reward such commitment to commercialization and category dominance.

On the other end of the spectrum is Ionis Pharmaceuticals.  

This company is happy to cease control over its groundbreaking compounds and disease insight quite early in their drugs' development paths. Consequently, it finds itself in situations where it either only gets a pittance from breathtaking medical and commercial successes such as SPINRAZA (for spinal muscular atrophy) or it gets caught with their pants down when a partner returns a compound late in the game as has just happened with IONS-TTR/Inotersen and GSK.

The market cap of Ionis, a company that shames $100B+ Big Pharma in terms of its pipeline and which will have close to a dozen compounds on the market and/or in pivotal clinical trials in a year or two, is comparatively paltry: $8B.

And the reason for this?  They are considering themselves a scientific company with a mindset of ‘we are better than organizations with corrupt sales departments’ that is difficult to find even in academia nowadays.  OK, management keeps drawing nice salaries and humongous stock/options rewards, so its really only shareholders that suffer.  

So as much as it hurts me as an RNAi scientists, when it comes to maximizing shareholder returns, the platform serves the purpose of capturing dominant footholds in new disease categories of high unmet needs, but this position has to be fortified by deep relationships with the patient community and an equally deep understanding of disease pathology.  And if necessary, develop and/or license complementary non-RNAi compounds.  

A word on the Arcturus-Janssen HBV deal

In addition to the Alnylam-Vir deal, another HBV RNAi-related deal was announced this week, namely between newly public Arcturus and Big Pharma player Janssen, a unit of Johnson & Johnson.

According to the agreement, Arcturus will work together with Janssen to develop an RNAi drug for HBV utilizing Arcturus’ intravenously administered RNAi triggers formulated in LNPs.   Arcturus uses fancy names for these components, but this is what's behind 'LUNAR' and 'UNA Oligomer Chemistry'. 

Given the availability of potent, but subcutaneous GalNAc-conjugation options in the industry, this interest by Janssen may come as a surprise to some and will serve Arbutus bulls as ammunition in claiming that Arbutus’ HBV RNAi LNP formulation has a future.  A Big Pharma deal after all is a knighthood in the industry.  

Still, I highly doubt it.

It is more likely that similar to Merck which had used LNPs internally as a disease interrogation and target validation tool, but not for commercial development before- even before GalNAc had come to prominence- J&J may view LUNAR-RNAi as a relatively speedy, but inexpensive way to test RNAi for its potential as a backbone therapy in HBV.  If it likes what it sees, watch out for the real deal with Dicerna or Arrowhead.  

FDA Hard-Pressed to Approve Biomarin, But Not Sarepta Drug

When Biomarin late last year bought Prosensa for its experimental exon skipper drisapersen for the treatment of Duchenne Muscular Dystrophy (for $680M plus potential milestones), it exuded confidence about the likelihood of getting approval for the 2’-O-methyl phosphorothioate antisense molecule.  This, despite of the fact that drisapersen failed in a pivotal phase III trial of 186 patients which prompted the old partner GSK to dump the drug and walk away.

Tenuous early evidence for drisapersen in earlier trials

The confidence is largely based on some supposedly successful earlier trials, especially a multi-center, randomized, blinded 53-patient phase II study which had seen improvements in the 6 minute walk distance (6MWD) at week 25, the primary endpoint of the study (Voit et al. 2014).  

This, however was statistically significant only the case in the subgroup of patients that received drisapersen continuously (à treatment in 10 out of 10 weeks with 6mg/kg), but not in patients which were treated identically, except for the small difference in skipping the last week in a 10-week treatment cycle.

At week 49, the difference with placebo failed to reach statistical significance and Prosensa had to resort to pooling both subgroups to claim victory for that time point. Similarly, drisapersen failed in obtaining statistically significant outcomes for other muscle function endpoints.

Since the mechanism of action for the DMD exon skipping candidates is to change splicing of the mutated dystrophin transcript to a form in which the reading frame is restored with recovery of partial activity, it is important to understand the relationship between drug treatment and dystrophin production.

Here, too, the evidence was less than robust.  For example, even when applying the sensitive immunofluorescence technique, no increase or even a decrease in dystrophin was seen in almost half of treated subjects.  With the less sensitive Western blot, an increase in dystrophin was seen in only a third of treated subjects (0 for placebo).

Therefore, given the failed phase III trial and the less than robust earlier evidence in favor of the drug, I struggle to understand Biomarin’s confidence in obtaining approval in 2016.   

The importance of dystrophin as a surrogate endpoint

Part of the difficulty of obtaining statistically significant results for muscle function endpoints is most likely due to the small patient size (orphan disease affecting ~1 in 3500 male births) and the consequent need to pool boys at various stages of the disease together in a given trial.  It would thus not be surprising if say obtaining 10% levels of normal or Becker-type dystrophin will translate into very meaningful clinical benefit in some, but not other boys.

This will be an even more challenging problem for the DMD subgroups that are not amenable to exon 51-based exon skipping which is targeted by drisapersen.  Probably insurmountable for first-generation chemistries like drisapersen.

Accordingly, in both the drisapersen and the competitive PMO-based eteplirsen trials, it has not been possible to correlate dystrophin production with functional outcomes.

For that reason, I strongly support the importance of establishing reliable, quantitative methods to measure dystrophin in clinical trials (there was an FDA workshop related to this last week).  Dystrophin-dependent markers may also be acceptable if they can be measured by means that do not involve taking painful muscle biopsies.  For example, serum-based microRNAs as developed by Rosetta Genomics and Marina Biotech would be of interest here.

Eteplirsen before drisapersen

I thus find it difficult to grasp the notion of rejecting the current crop of exon skippers like drisapersen or eteplirsen should they be found to produce functional dystrophin with few side effects.  After all, it is the loss of dystrophin function that causes Duchenne Muscular Dystrophy and one has to wonder how generating additional dystrophin cannot be beneficial to patients, especially since the principle behind drisapersen and eteplirsen is strongly supported by human genetic evidence (à Becker’s Muscular Dystrophy).

In this world, it has got to be eteplirsen that should be first in line for regulatory approval.  This is because there is overwhelming evidence (e.g. Heemskerk et al., 2009; Sarepta's Barclays presentation March 12, 2015) that the PMO-based drug is much more potent than drisapersen which, let’s face it, is based on stone-age antisense chemistry (2’-O-methyl phosphorothioate).  Such chemistry is characterized by minimal efficacy and dose-limiting toxicities, especially renal in the case of drisapersen.

In a paper comparing 2’-O-methyl to PMO chemistry for DMD exon skipping conducted by researchers close to eteplirsen, it was found that at same doses in mice, PMO chemistry is moderately to vastly more potent than 2’-O-methyl phosphorothioate antisense compounds of a size comparable to drisapersen.  The extent of the difference depended on whether the human or mouse dystrophin were targeted and the target sequence.   Unsurprisingly given the acrimonious competition between the two parties, Sarepta has also picked up on this and continued along these lines by showing that in addition to chemistry, eteplirsen has the edge over drisapersen in terms of the targeted sequence:

Sure, there is the theoretical caveat that PMO and 2’-O-methyl scale differently from mice to humans and that what is the most potent target sequence for one chemistry does not necessarily have to be the most potent one for the other.  Intuitively, however, the differences are too big for these factors to compensate the preclinical evidence.  Also, keep in mind that in the clinic, eteplirsen is being given at 5 to almost 10-fold increased doses than drisapersen and, on top of that, is much safer and better tolerated than drisapersen.

Because of this and the competition, it is not surprising and disingenuous when Biomarin would now suddenly like to de-emphasize the importance of dystrophin as a surrogate biomarker (see last week's workshop).

Dear regulatory agency, if you approve drisapersen, you cannot deny eteplirsen.  Sure, drisapersen has been tested in more patients than eteplirsen and Sarepta has conducted a clinical trial in the worst possible manner and probably ‘embellished’/overstated some of their results, including the dystrophin evidence.  However, given that eteplirsen almost certainly generates more dystrophin than drisapersen, the highly favorable side effect profile of eteplirsen (also in comparison to drisapersen), and in light of the 6MWD issue that applies to both drug candidates, the question is whether the bureaucratic application of rules should trump scientific evidence and patient interests.

Disclosure: I am long SRPT based on the notion that Biomarin, with its orphan disease savvy, will turn out to be the biggest supporter of eteplirsen getting approval this time around.  Additionally, the agency is partly responsible for the long duration of the ongoing eteplirsen trial (close to 4 years soon) and the repeated taking of muscle biopsies, and after all this taking away hope from patients and their close ones is difficult to fathom.

Sarepta, Biomarin Move Over- Here Come tcDNAs

In a head-to-head comparative study in NatureMedicine, Goyenvalle and colleagues claim that tricycloDNAs (tcDNAs), a relatively unexplored constrained nucleic acid analogue commercialized by SYNTHENA have superior efficacy over 2’-O-methyl oligos and PMOs for therapeutic splice modulation in mouse models of Duchenne Muscular Dystrophy (DMD). 

In addition to ~3 to 5-fold enhancements in dystrophin exon skipping in various muscles compared to the competing chemistries, the authors report that only with tcDNAs there was splice modulation also for the dystrophin isoform expressed in the brain.  This was accompanied by improvements in the behavioral and cognitive abnormalities in this model.   Apparently, these neurological defects are another important treatment goal in DMD.

Splice modulation was accompanied by ~2 and 20ug/g oligo concentrations in brain and muscle, respectively, following the administration of relatively large weekly doses of 200mg/kg that were necessary particularly for the correction of the neurological symptoms.

Interestingly, such delivery and target modulation was achieved without encapsulation or further modification technologies.  Whereas a 2012 publication by some of the same authors in collaboration with ISIS Pharmaceuticals explored the use of tcDNAs for RNaseH-mediated gene knockdown in conjunction with the phosphorothioate backbone known to greatly improve biodistribution and cell uptake, tcDNAs here were applied in their naked form.

The authors attribute this to an apparently spontaneous tendency of tcDNAs to self-assemble into ~100nm nanoparticles.   

The results seem to warrant the development of tcDNA for DMD exon skipping.  Critical to their success in the clinic will be the safety and tolerability of tcDNAs in Man with the kidney predicted to be the dose-limiting organ.  

The study is also a reminder that there are now a number of other chemistries, including the high-affinity CRN chemistries by ISIS Pharmaceuticals and Marina Biotech, the latter with a stated focus on DMD, which similarly promise improvements over the trail-blazing 2’-O-methyl and PMO chemistries.  

Ebola Emergency Tests Oligo Manufacturing Readiness

With officials scrambling to put in place measures to contain the further spread of Ebola, it is highly likely that RNAi Therapeutic TKM-EBOLA will be on their shopping list.  This, however, requires the ability to manufacture the oligonucleotide-based medicine in quantities sufficient to treat at least 10 thousand or so either infected or potentially infected persons.  In the absence of commercial Oligonucleotide Therapeutics success stories this could prove to be a challenge, although in this case, I believe it's doable.

Manufacturing, an advantage of TKM-EBOLA over PMOs and antibodies

When the US Department of Defense selected TKM-EBOLA as its preferred Ebola development project a few years ago, manufacturing, and not just efficacy and safety should have been part of the equation.  The reason is that e.g. currently about 0.3mg/kg*70kg/day*7 days= i.e. approx. 140mg of TKM-EBOLA RNA oligonucleotide is required per treatment course.  Since its simple chemistry (a couple of spiked-in standardd 2’-O-methyls in an otherwise unmodified RNA) makes it one of the cheapest RNA oligonucleotides conceivable, let’s ballpark its manufacturing cost at $300 per treatment course at some of the largest possible manufacturing scales possible today (kilograms).  Note that the cost of the lipids in TKM-EBOLA is negligible compared to the oligo component.

1.4kg oligo and $3 million for 10,000 treatment courses.

Taking into account that these are the pure manufacturing costs when protocols have been established, and other monies will have to be spent when starting from sequence design---let’s say conservatively $5 million. I believe given the gravity of the current situation, this is a number we can live with (1/200 of investment in the response).

If you do the same Gedankenspiel for Sarepta’s Ebola therapeutic morpholino antisense candidate, you have to multiply the $3M number first by a factor of 30 for the much larger amount of oligonucleotide required and then by another factor of 6 or so for the greatly increased costs of making morpholinos over standard RNAà $500M+.  A non-starter not just for the increased costs, but also because the manufacturing capacities for that amount of oligonucleotides are not readily available today, let alone for the morpholino chemistry where Sarepta has experienced significant delays in obtaining sufficient oligo supplies for a relatively small (~100 patients on drug at 30mg/kg/week for 48 weeks) phase III program in an orphan diseases indication (DMD).

When we move to Tekmira’s antibody competition, in particular ZMapp, the situation is not all that different from Sarepta’s with the slight advantage that ZMapp could somewhat tap into the established know-how of monoclonal antibody production.  But in the end, we’d be talking about years of process development and scale-up compared to a few months TKM-EBOLA.

Wake-up call for oligonucleotide manufacturing

Although TKM-EBOLA should be within current oligonucleotide manufacturing capacities, the Ebola situation should get oligonucleotide manufacturers wondering whether they are ready for the upcoming surge in Oligonucleotide Therapeutics approvals and sales.  Following years of disappointment about the lack of big manufacturing requests, CMOs have been loath to build the plants that can churn out hundreds of kilos or even tons of oligonucleotides.

Consequently, I attribute the decisions of first ISIS and Sanofi/Genzyme, then Alnylam (for GalNAc conjugates), and more recently also Sarepta, to shift oligonucleotide manufacturing in-house, to this lack of outside manufacturing capacity.  While having manufacturing in-house may sound attractive for a number of reasons, spreading manufacturing risk across multiple vendors is an accepted risk reduction strategy in this and other industries. 

I am therefore hopeful that the ongoing Ebola outbreak will end up increasing oligonucleotide therapeutics manufacturing capacities to rule out a situation that manufacturing constraints could limit commercialization of agents like ISIS-ApoCIIIRx or the HBV agents with potentially very large patient populations. 

Dear Chris Garabedian,

I am writing to you today to introduce myself as a potential candidate for the CSO position at Sarepta.  Based on media reports last week (see here for the Wall Street Journal version), it seems that this position has become vacant after you had a little spat with Art Krieg.  If the anonymous sources close to Sarepta cited so abundantly in those reports were to be believed, at least some of the disagreement related to Art’s failure to push and expand the morpholino-based product pipeline the way you would have liked.  Art maybe be an icon in the oligo industry, but I believe that I am the right man for you as I know where the gold mine for morpholino oligonucleotides lies.

First, however, a few words about myself.  You may have read or at least heard about my critical writings of your company’s pursuit of getting eteplirsen, your exon-skipping drug candidate for Duchenne Muscular Dystrophy, on the market.  In case that there was some truth to speculations that some of the disagreement related to the accelerated approval strategy for eteplirsen, rest assured that I will ignore all my concerns about the conduct of the rather small trial and biochemical assays used, and tow the party line anyway.  Even better, I would be prepared to cede any involvement in the experimental conduct and interpretation of data generated in preparation of filing for accelerated approval although these activities would naturally call for my involvement as the CSO of the company.

Coming back to my prior criticisms of the company….to be frank it was Janet Yellen and the adverse impact her comments have had on biotech stocks that now forces me to apply for employment.  Nothing personal, no hard feelings, we will all be able to get along and laugh about it later.  I am running out of options as I have burned down bridges with virtually all other RNA Therapeutics companies in the field as I have freely criticized them before when I still had money.  As you know from reading the message boards carefully, I have been desperately applying for jobs there only to be rejected and attack them even more venomously.  Given my track record and financial situation, regardless of the fates of both Krieg and Linsley before that, please forgive my youthful follies and I fully expect you to be a difficult and demanding boss and I will put up with it.

I know that you have long made up your mind to not hire me and the only reason for humoring me and reading on is the thing about the gold mine.  I genuinely believe there is one and- you are right- it’s got to do with splice modulation and by extension the modulation of RNA processing by masking sites for RNA binding proteins.  In particular, I find morpholinos highly attractive for splice modulation in pharmacologically contained compartments such as the CNS where the risk- and please excuse my language here- that the expensive oligo is pissed out immediately is limited.  There is a reason why morpholino has become such a popular tool to study developmental biology in zebrafish embryos and the like: they cannot pee!!!  So with the ability of maintaining high tissue concentrations and the amazingly safe morpholino chemistry, you may be able to generate invaluable medicines for awful diseases such as spinal muscular atrophy (maybe even better/safer than what ISIS is generating currently) and Huntington’s disease.

Kind regards,

Dirk. 

Tekmira Expands Biodefense Efforts to Cover Both Ebola and Marburg Virus

Following the successes with its Ebola biodefense program, Tekmira has started to present increasingly promising data for treating a related filovirus, the Marburg virus.  With 100% survival rates in non-human primate models and new RNAi triggers that should cover a broad spectrum of strains and possibly related viruses, the company is well positioned to also take away the Marburg indication from competitor Sarepta which in turn has become preoccupied with its exon-skipping drug candidate for DMD.  

For the Ebola program alone, the $100M market cap company estimates that the successful development of a drug under the so called Animal Rule could result in stockpiling orders from the US government worth about $100M annually.

From guinea pigs...

In a publication last month, Tekmira and their collaborators from the UTMB in Galveston, Texas, reported the successful treatment of guinea pigs infected with a number of different strains of the Marburg virus.  Because the development of treatments for rapidly mutating viruses and viruses with a multitude of divergent strains is hampered by sequence diversity, a broader strain coverage was achieved, like in Tekmira’s Ebola approach or in Arrowhead’s chronic HepB strategy (ARC520), through the concurrent use of two siRNAs in a single formulation.

Given that rodents have limited predictiveness for anti-filoviral efficacy in Man and given that the formulation was apparently a 1^st generation D-LinDMA-based SNALP, I was not all that excited about the publication due to the apparent early stage of development.

...to non-human primates

In a positive surprise therefore, the Chief Scientific Officer of the company, Ian MacLachlan, presented gold standard non-human primate data of SNALP RNAi Therapeutics for Marburg virus at the ongoing OligoDIA regulator-industry conference.  Accordingly, Tekmira’s newer LNP formulations (‘SNALP-G’) were shown to fully protect monkeys from death due to Marburg infection when given at 0.5mg/kg (=the magic safety threshold for SNALP).

As an important comparison, Sarepta last year reported ‘83% to 100%’ protection rates in comparable models when treatment was initiated up to 96 hours after infections with its newer PMOplus morpholino chemistry.   It is therefore of interest to test the impact of further delaying treatment with SNALPs.

As I have been following Tekmira’s biodefense program over the years, one development I noticed, especially in the era of budget cuts, was the apparent push by the Department of Defense for biomergency treatments that can address not just multiple strains of a virus, but multiple viruses all-in-one.  Intriguingly, one of the bullet points in Tekmira’s slide presentation on goals for the Marburg program (slide 36) talks about the ‘Design broad spectrum siRNA to target multiple MARV and Ebola’.  Does this mean that the ultimate prize here are stockpiling contracts for a single drug addressing both Marburg and Ebola?

Having a better ear for and adjusting to the needs of the DoD is also one reason why I believe Tekmira will continue to have the upper hand over competitor Sarepta with its DMD distractions and an investor base that could not care less about the Marburg program.  Although the targets were explicitly not disclosed, I wonder whether there are host factors one could target and which would be beneficial for various viral applications.  This would be an alternative to trying to come up with target sequences that are conserved across the viruses.

Finally, serving as yet another example of illustrating the value of biodefense contracts for platform technology companies, Tekmira disclosed plans that a first clinical trial with a lyophilized SNALP formulation, TKM-EBOLA, is planned for the first quarter of 2014. 

Sarepta Ditches Dystrophin Assay Getting Closest to Being Quantitative

I am aware that my interpretations of Sarepta’s exon-skipping data for Duchenne Muscular Dystrophy are not universally embraced.  But one point that I believe all of us can agree on is that it would be important to determine whether the antisense oligo eteplirsen can restore sufficient Becker-type dystrophin to have a therapeutic effect.  Although falling short of a controlled clinical outcome study, one way to gain accelerated approval for this devastating disease would be to first establish what this ‘magic’ level is through historical outcomes studies and then compare this value to the levels actually restored following drug treatment. 

Western blot not worth the film

But when you listened to last week’s presentation by Sarepta CEO Chris Garabedian (link to transcript), the company appears to have dropped the idea of demonstrating such altogether by declaring dystrophin Western blots as unreliable.  I agree that Western blots do not lend themselves to quantitation.  Moreover, I have sharply criticized a number of technical aspects with the eteplirsen Western blot: spliced gels, uneven loading controls, cropped band of unknown identity shown.  

The latter point is particularly bothersome as any molecular biologist would know that even two supposedly identically processed protein samples will show numerous bands that are present in just one of the samples if only you expose the Westerns for long enough.  Ergo, the particular Western blot does not live up to being even merely ‘supportive’ as the company had claimed until recently because it now seems that even the company is only guessing that the cropped band shown is the one based on predicted size (note: proteins do not even migrate on Western gels according to known size...charge etc).

All this begs the question of why Sarepta never developed a reliable quantitative assay (e.g. an ELISA) before entering clinical development and making the case for biomarker-based accelerated approval.

Counting dystropin-positive fibers by immunofluorescence does not address magic number

This alas leaves us with the immunofluorescence (IF) assay data.  Sarepta and their collaborator have counted and presented numbers, so this surely must mean that the IF data are ‘quantitative’?

Wrong.  

As I keep pressing the point, the IF assay (at best) can only determine if there was a increase in dystrophin.  Manipulate the exposure times and picture contrasts enough and you can magnify any difference in dystrophin levels out of proportion. Again, this is no revelation to the molecular biologists and hobby photographers here, and I won’t even start to discuss the issues arising out of comparing IF numbers from experiments conducted at different dates and from different muscles.

By way of example, say you obtain an average signal level of 1.5 due to naturally occurring alternative splicing noise when measuring background, with the vast majority of fibers being below 2.0 and with some rare revertant fibers exceeding that value manifold.  Then you treat with eteplirsen and you find that the signal level increased to between 2.2 and 2.8 in 70% of fibers.  You then set the background arbitrarily to be 2.0, subtract the few revertant fibers, et voila, you determine that 65-70% of fibers have started to express dystrophin following drug treatment although the absolute increase in dystrophin produced is well below what we would predict to be therapeutic: from 1.5 to about 2.5. 

    

Leaving it open what that ‘magic number’ actually refers to

Consequently, IF is ill suited to address the issue of the ‘magic number’ as Mr Garabedian called it.  It would have been incredibly helpful for him to have elaborated what he actually meant with the ‘magic number’.  Does he really believe that counting fibers allows you to get at this issue, as he implied in his answer*, or would he agree that it should be with respect to the absolute amount of dystrophin?  I hope the company will clarify that point almost as quickly as they declared the image duplication event an honest mistake.

What do you think?  Should the ‘magic number’ refer to counting dystrophin-positive fibers or quantitating the absolute amount of dystrophin?  Participate on the poll on the right hand top.

* Making a circular argument, the CEO said that ‘yes’ they know they have surpassed the magic number because of the claimed 6MWT improvements.  Note that in making the case for a biomarker like dystrophin, it should be the amount of biomarker that supports functional outcomes like 6MWT e.g. through some quantitative correlation- not the other way around. 

FDA Questions around Dystrophin Quantitation by Sarepta Delay DMD Drug

As most of you know, yesterday was the day that morpholino antisense company Sarepta would provide guidance on whether they would be seeking accelerated approval for the Duchenne Muscular Dystrophy (DMD) exon skipping drug eteplirsen based on their interactions with the FDA.  This was a widely watched event not just for eteplirsen and boys with DMD, but also because regulators highly supportive of Sarepta’s meager data package would indicate a new lowered standard for the approval of rare, severe diseases.

Dystrophin as surrogate biomarker

Accelerated approval may be granted by the FDA in the absence of full clinical evidence of drug efficacy if the results from surrogate biomarkers that are thought to reasonably predict clinical outcome pointed in the right direction.  For dystrophin exon skipping this is an important potential avenue given that for most genetic lesions causing the disease it would be difficult to conduct full-fledged placebo-controlled clinical trials.

Although this is not really the case for exon 51 candidates, the most frequently affected exon in DMD and that eteplirsen is addressing, Sarepta ran one of the worst designed and conducted clinical trials for this indication involving just 12 boys (in case you are asking again why I hold this view: minute size of study when larger patient numbers were clearly feasible as the Prosensa/GSK competition shows; single-center; key supportive data miraculously collected with unblinding of the trial; key secondary endpoint- 6MWT- added with unblinding of trial; exclusion of patients in results analysis as company saw fit etc).  As a result, obtaining approval by simply showing dystrophin production has been Sarepta’s best hope with the data at hand.

Hardly anyone would argue that dystrophin was an inadequate surrogate marker.  The loss/absence of dystrophin is what causes the muscle wasting, and there is strong evidence that restoring dystrophin or even a somewhat less functional Becker’s type truncated version would be beneficial.  It would also be no surprise to see some increase in dystrophin with a gain-of-function approach such as this exon skipping one. 

No, the key questions are how much of Becker’s type dystrophin production was needed to be clinically meaningful to patients and how you actually quantitatively measure the increase in such dystrophin.

Questionable science

I don’t want to argue here about the amount of Becker’s type dystrophin needed to be clinically meaningful.  The reason is that if you do not have reliable numbers, such a question is moot.  As I had explained in a post a few months ago, the data presentation by Sarepta on the amount dystrophin produced reminded me of some of the worse cases of ‘data management’ and misinterpretation that you sometimes come across in the literature: there was apparently unequal loadings between drug-treated and control samples which suggested a high amount of skipped dystrophin by Western blot, a molecular technique looking at protein content; the RT-PCR that was supposed to support meaningful skipped dystrophin production by looking at the RNA (not protein) level actually suggested the opposite as the PCR cycle number seemed inappropriately high. 

Moreover, choosing percent dystrophin positive fibers instead of the absolute amount of dystrophin produced as the primary endpoint in the clinical trial always seemed wrong to me as for this gain-of-function approach you could imagine a very low amount of dystrophin that can be detected by the particular assay could yield very high percent positive fiber numbers, but of which the clinical relevance would still be highly uncertain (e.g. if sensitivity of assay could pick up 1% of normal, and the restored dystrophin was homogeneously distributed between the fibers).

And these are only the obvious questions.  What I would also like to know (and the FDA may or may not know) following the piecemeal data release by the company and the surprising finding that after 12 weeks no dystrophin restoration could be observed, yet after 24 weeks the company apparently could, would be the comparability of the data (e.g. the immunohistochemistry/IHC) across the different time-points, who performed the analysis, the appropriateness of the antibody used in the IHC and the Western blots, the collection method of the biopsies etc etc

FDA also doubtful

It is becoming clearer with each report on the company’s interaction with the agency that this methods issue is a real sticking point before a proper evaluation can be conducted.  This can also be seen from today’s press release: ‘The Agency, however, requested additional information related to the methodology and verification of dystrophin quantification.’

Alas, this should come as no surprise.  It is still good to see confirmation that the FDA is taking a scientifically sound approach, but as the discussion around eteplirsen had become quite politicized, one could have been excused for falling for the belief (as I’m sure countless retail shareholders did) that politicians and not scientists have taken over the eteplirsen approval process.  I had even considered whether we were about to witness a landmark event lowering the evidence bar for the approval of orphan drugs to unprecedentedly low levels.  Apparently not.

PS: The fact that Sarepta would decide to file for accelerated approval sometimes in the first half of 2014 should not come as a surprising.  Even if they themselves had considerable doubts about the success of accelerated approval or even about acceptance for review following their FDA interactions, there was nothing to be gained from not stating such intent: the stock price would have plummeted much more than the roughly 20% it did yesterday.  The interim now can be used to exercise their ATM (=issuing new shares on the actively trading markets) at still much elevated levels.

Disclaimer: The above was written based to the best of my knowledge and belief. Nevertheless, no guarantees can be given as to the factual accuracy of the content. I should also add that I am not 'against' eteplirsen.  I just don't think that there is sufficient evidence yet to decide either way. And for parents eager to get their kids on eteplirsen now, I believe, given the apparent safety of the drug, that a way could be found in which Sarepta were to reciprocate the support by the patient community and provide the drug at cost until a possible full approval.

FDA Proactively Questions Eteplirsen Biomarker Methodology

Orphan drugs tailored to address defined genetic lesions account for a significant portion of the current value of RNA Therapeutics.  One of the reasons is that they promise much more economical approval pathways, including shorter timelines, smaller patient populations, and biomarker-based approval.  Following RNAse H antisense Kynamro by ISIS Pharmaceuticals/Genzyme Eteplirsen for homozygous FH, eteplirsen by Sarepta for Duchenne Muscular Dystrophy (DMD) offers us another front-row view of this process.

Currently, the question on most observers' minds is whether the biomarker data gathered so far, most notably from a phase IIb trial in just 12 patients is sufficient for accelerated approval (AA).  Towards this end, Sarepta and the FDA had a meeting a few weeks ago to discuss whether the FDA would accept for review such an AA submission.  According to the Minutes of the meeting which were discussed in a press release by Sarepta on Monday, the FDA does not believe the data it has seen warrants an AA submission and asked the company to come back with more data.

I am one of the very few open critics of eteplirsen and have explained my rationale why I have little confidence in the phase IIb data that have catalyzed much popular support: a manipulated trial conduct where essentially all the supportive evidence was collected after unblinding of the trial and a cherry-picking way of presenting them.

What emerged from the recent FDA interaction appears to be largely consistent with my concerns.  Today, I would like to highlight a critical issue when it comes to pursuing biomarker-based approval strategies and that was the focus of the recent interaction.

How you measure biomarker is as important as the biomarker per se

It does not reflect well on a company seeking accelerated approval when, after two decades or more of morpholino-based drug development, it has yet to establish sufficient manufacturing capacity to satisfy even the small patient population that would be eligible for eteplirsen.  The same poor planning becomes evident when the FDA appears to be questioning the method by which the biomarker (dystrophin) data was collected: what is the value of a biologically strong biomarker if you can’t reliably measure it to draw comparisons?

The dystrophin expression by immunofluorescence and Western blot were from biopsy samples.  Obviously, muscle tissue is everywhere in the body and any drug efficacy will vary depending on the muscle.  Even within a given muscle, (revertant) dystrophin expression is known to be variable in DMD patients, so despite of consistently taking biopsies from the same muscle before and after treatment, you may not be able to tell 2 or 3-fold differences.  This problem was noted by the investigators in the first phase II trial of eteplirsen in discussing the discrepancies of Western blot and immunofluorescence data.

Of course, this sampling issue could be addressed with large patient numbers, but not with the 12 as in this trial.  Given the importance of methodology, it is surprising that Sarepta missed the opportunity to provide the FDA with sufficient related information to be in a position to more conclusively tell whether it’s worth submitting for AA or not.

Issues the FDA might want to know more about could be the selection strategy of biopsy location and whether the antibody was appropriately chosen also for taking into account revertant fibers.  Revertant fibers are a phenomenon due to either secondary mutations or alternative splicing which leads to dystrophin expression in many DMD patients.  This might also shed light on for example why after 12 weeks on drugs, no dystrophin expression was seen, but at 24 weeks it was. 

Another important question that the detailed methods might answer is the quantitation of the absolute amount of dystrophin expression (not percent fibers expressing).  Based on the phase II study publication, it appears to be quite difficult to do even just semi-quantitative calculations on fuzzy Western blots.  Of course, once the real expression is established, the question is how functional such alternatively spliced (Becker-type) dystrophin is.  Maybe what you need is at least 30% of such dystrophin to have a functional impact on DMD.  

Sustained 6 MWT stabilizations common

Supporters of eteplirsen like to point out that the proof that it works is in the apparent disease stabilization over 52 weeks or more when measured in terms of the 6-minute-walk test.  However, it appears from natural history studies of DMD that such walking stabilizations are common.  In fact, depending on the age, you would even expect an increase in walking ability over 52 weeks.  Thus, arguing that the 6 MWT data are proof for drug efficacy is like arguing that Stable Disease in an uncontrolled cancer trial is evidence that a drug works.

Confounding the 6 MWT data is the fact that it is an effort-based test and that the separation between drug and placebo was only observed after unblinding of the data and dropping out the worst performers. In the very words of Sarepta itself and clinical collaborators when discussing clinical data from the competing exon-skipping drug by Prosensa and GSK:

'In terms of the clinical efficacy, the PRO051 study claims that eight of 11 boys who were ambulant at entry to the extension study showed improvement in the 6-min walking test of 35·2 m (SD 28·7) after 12 weeks' treatment; however, this change was not significant. Moreover, several of these children were younger than 7 years and, according to longitudinal observation, boys younger than 7 years with Duchenne muscular dystrophy gain motor function. Additional confounding factors are the variability in the walking test (SD 36 m) and the powerful placebo effect of open-label studies. Despite these limitations, this observation is encouraging.'

I don’t hold out myself to be an expert in DMD, but as a skeptical scientist I believe there remain too many open questions around the eteplirsen data and trial conduct to make me feel comfortable.  Add to this the almost religious support by a strange alliance of investors, patient groups, politicians, and journalists.  Spending half a million per patient per year on a drug that may turn out to be ineffective and have side effects would neither protect patients nor help the cause of RNA Therapeutics.  If patients are so keen on the drug, and I'm all in favor of patient choice, then why not provide them with eteplirsen based on compassionate use?

Last but not least, why newly Cambridge, Mass-based Sarepta had to issue the press release on the Minutes of their FDA meetings just minutes after the Boston marathon bomb blasts, is a mystery to me.

Disclosure: no position in Sarepta.

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