Thursday, August 22, 2013

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’?


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. 


Anonymous said...

Garabedian said that he was told to go ahead and file an NDA for regular approval....and that once it's filed the FDA can chose to grant it accelerated approval or anything else it wants. So the issue of a surrogate marker and all the well worn complaints you now discuss are moot. If a surrogate marker and te means for measurement are ever settled on by the FDA I'm sure they will let you know.

Dirk Haussecker said...

Garabedian essentially discarding the Western blot (which nevertheless had been presented in scientific settings before) as useless, is news to me. Worth discussing IMO given the importance of eteplirsen. Oligo society journal to even devote special issue on the 'challenges' of developing DMD drug.

Anonymous said...

So what if it's news to you that Garabedian repeated your own scepticism about Western Blot? And devoting an issue to the challenges of DMD drug development also has nothing to do with any general discussion of Western Blot's effectiveness. If they mentuion it at all it would be in passing. The challenges in 'developing a DMD drug lie elsewhere.

Maybe had you chosen to add Sarepta or AVII to your portfolio before the news of the drug's effectiveness came out last October, you'd no where the interesting problems in DMD drug development really lie, and you'd find more interesting ways to attack the company than quibbling over unreliable measuring techniques.

Anonymous said...

The drug works. Have you seen videos of Max and Billy doing things that normally wouldn't have been possible for kids with DMD, almost two years since they enrolled in the trial? Or are you blind to that?

Anonymous said...

Everyone--the scientists at GSK and Prosensa, I mean everyone, even blogger Dirk who got his feelings hurt when SRPT succeeded and TKMR failed--knows eteplirsen works. That's not the issue. The issue is that Dirk is insinuating that Sarepta has manipulated signal levels and background levels. He has no evidence to back up these allegations. Real scientists base their arguments on evidence. Dirk has no evidence, so he makes false accusations based on hurt feelings and bitterness.

The Romans ordered the death penalty for those who made false accusations. I wonder what the penalty will be in civil court today?

Anonymous said...

An Oxford (UK) based company are close to completing a biomarker programe to measure Utrophin. Hope this helps.

Anonymous said...

I read the interesting details about product information from this Canaccord Genuity's 33rd Annual Growth Conference Transcript. You can read it too @

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