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.