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.