Wave Life Sciences to Focus RNA Editing on Gene Upregulation

Yesterday, oligonucleotide therapeutics developer Wave Life Sciences provided a high-level preview on how it will deploy its RNA Editing technology.  Accordingly, modulating protein-protein interactions and, even more so, increasing gene expression will be the declared mechanisms of action of development candidates following its lead candidate WVE-006 for alpha-1-antitrypsin disease (AATD).

WVE-006 was recently licensed to GSK and should be the first RNA Editing candidate to enter clinical development later this year.  A big milestone for the field.   WVE-006 corrects a common single nucleotide mutation in the alpha-1-antitrypsin gene, Z-AAT, that causes both liver and lung manifestations of AATD. Z-AAT is retained in liver hepatocytes to cause cellular stress instead of being secreted to do its job and protect the lung.  As such, WVE-006 can be considered both a mutation corrector and gene function booster.

 

Mutations often scattered across genes

More often than not, however, mutations causing rare genetic diseases are scattered across a gene and precision genetic medicines targeting small segments of a gene at a time may thus only address a subset of patients.  A prime example is Duchenne Muscular Dystrophy where even exon 51 skipping which is the approach with the largest addressable patients still only serves 11-13% of the overall DMD population.

                                DMD patient segmentation according to skipped exon (from Wave Life Sciences presentation)

A very interesting indication for ADAR RNA Editing is Rett Syndrome (affects 1 in 10000 girls by age 12 in the US).  Here as well are the mutations scattered across the MeCP2 gene.  Almost half of those would be addressable by RNA Editing (including eliminating stop codons), but each individual target would be quite small.

So instead of targeting the specific mutations, ADAR Editing may also be used to screen all adenines in the MeCP2 transcript to identify those that lead to an increase in protein abundance and thus function either by stabilizing the resulting mRNA or by increasing MeCP2 stability.  While this approach would not apply to Rett Syndrome caused by 2 null mutations on the X chromosomes, a say 3x increase in activity of the chromatin CpG-binding protein may be enough to alleviate disease in a large fraction of Rett Syndrome patients with MeCP2 versions having reduced activity.  Or consider mutant CFTR proteins in cystic fibrosis with reduced channel activity. Increase the abundance of those CFTR mutant proteins and it should increase the overall desired activity.

The screening approach would also facilitate finding potent RNA editing oligos due to the flexibility and increase in targeting space as opposed to having to optimize the editing oligo around a small defined target site.

 

mRNA technology

Wave Life Sciences likened the gene upregulation approach as a simpler version of mRNA therapeutic technology.  Simpler, because it does not involve the delivery of long mRNAs which necessitates the use of LNPs and similar larger nanoparticle formulations due to mRNA stability requirements.  By contrast, RNA editing can be mediated by oligos ~30 nucleotides in length, short enough to be amenable to conjugation and oligo chemistry strategies already applied in RNaseH and splice modulation ASO and RNAi.

Smaller also means better tissue penetration and delivery to more target tissues.

Moreover, meaningful expression from an mRNA only occurs in short bursts so that the frequency of repeat administration is dictated by protein half-life.  Meanwhile, the administration frequency for oligo-mediated editing, due to the longer persistence of highly stabilized oligos, can be expected to be in the weeks and months.

It should be noted though that RNA editing would essentially upregulate what is already present in the cell (with the exception of the one editing change), whereas mRNA therapeutics in sensu strictu can generate entirely new proteins.

RNA editing would also not be the first oligonucleotide approach to mRNA upregulation.  RNA activation, the targeting of promoter-proximal regions using RNAi-type double-strand RNAs, and the targeting of upstream 5’ UTR mRNA elements with steric blocking antisense molecules as developed by Ionis Pharmaceuticals are competing approaches.  These, however, have so far either lacked the robustness or the flexibility in terms of sequence choice that AàI editing should afford.  

 

Now more than ever in biotechnology, companies need to carefully tease out the unique, differentiating advantages of a platform technology when selecting an indication.  RNA Editing leaders ProQR and Wave Life Sciences are in the fortunate position that they can apply the new biotech paradigm starting with their first RNA Editing candidates.  Biotech is ripe for a reboot and RNA Editing should have every ambition to be part of it.

 

Disclosure: I own both ProQR and Wave Life Sciences shares, though ProQR considerably more. 

Small Molecules and Gene Therapy Muscle Out Oligonucleotide Therapeutics

Over the last two days, breathtaking data were reported for the treatment of two severe, inherited muscle-wasting diseases affecting children.  The investigative agents were a small molecule splicing modulator and a gene therapy both of which appear to achieve superior results compared to approved Oligonucleotide Therapeutics agents. 

The developments highlight the risk that while gain-of-function changes (here by splice modulation) may have proved to be low-hanging fruits for Oligonucleotide Therapeutics, they, unlike gene knockdown approaches, face increased challenges from other technology platforms.

Small molecule splicing modulation for Spinal Muscular Atrophy (SMA)

In 2011, Roche started collaborating with PTC Therapeutics on small molecule splice modulators for the treatment of SMA.  The idea is to screen small molecules for their ability to bring about changes in RNA processing that would hopefully be gene-specific enough so as not to cause widespread off-targeting.

I had always considered this to be a monumental, if not insurmountable task.  This is because a given splicing event brings together a set of proteins that each in turn also function at other genes.  So surely a small molecule that may bias splicing from SMN1 to SMN2, as does antisense oligonucleotide SPINRAZA from Ionis and Biogen through highly specific base pairing, would also affect a range of other genes.

If that were not enough of a challenge, a small molecule carries the extra baggage of being more widely available across tissue types such that off-targeting is a risk to not just the CNS as with SPINRAZA, but many other cell types where there may be no benefit from SMN upregulation.

Accordingly, the first compound in the PTC-Roche collaboration to enter clinical development, RG7800, had to be discarded last year due to retinal tox concerns.  Another small molecule competitor, branaplam from Novartis, had similarly been put on hold due to tox concerns although this compound has resumed development late last year.

It was therefore amazing to see updated results from the FIREFISH study of the follow-on compound RG7916 in type I SMA infants.  They show that 90% of children had an improvement in the CHOP-INTEND measure of physical functioning after 6 months on the drug.  The results are particularly impressive considering that treatment had been initiated relatively late compared to the new standard of care with SPINRAZA and the soon-to-be-approved gene therapy by Novartis (àAvexis).   

Not only that, there had been no treatment discontinuations due to safety issues with RG7916.

Given that SPINRAZA has to be given intrathecally while RG7916 can be given orally, and given that both the gene therapy and the orally available RG7916 appear to be somewhat more efficacious than the oligonucleotide, the focus of Biogen and Ionis should now be on testing combinations of SPINRAZA with both modalities.  Ideally, there is added efficacy from using the agents together either because due to higher achievable SMN protein levels and/or due to complementary biodistribution (note: the value of SMN increases outside motor neurons is debated).  If not, the SPINRAZA franchise may have a limited shelf-life.

Fake-it-‘til-you-make-it Sarepta with gene therapy breakthrough

The other piece of great news for families dealing with neuromuscular disease came yesterday at the Sarepta Therapeutics R&D Day. 

To wit, Sarepta had used dubious data and a lot of political lobbying to get the controversial exon skipper eteplirsen approved under accelerated approval.  While delaying the confirmatory study that is supposed to be part-and-parcel of an accelerated approval, Sarepta has been raking in billions in sales and added market capitalization.  This has allowed the company to build a veritable DMD powerhouse with a number of candidates that look much more promising than ordinary PMO-based eteplirsen.  They include peptide-conjugated PMOs and especially gene therapies.

If you are involved in drug development, better get used to the dubious morals of the industry.  If things go well, you behave like the paragon of virtue, if things don’t go so well you fake it until you get another chance at succeeding.  I digress…

Before the initial gene therapy data were to be presented by Jerry Mendell from Children’s Nationwide of SMA fame, I had dreaded the thought of having a hyped-up R&D Day being about divining the meaning of a biopsy slide or two on the barely-above-background expression of the microdystrophin transgene.

However, what was presented was anything but borderline.  Unlike with eteplirsen where we were dealing with debatable 1%-type absolute expression levels, there was robust microdystrophin expression: ~75% of cells expressed the transgene (by IF) with roughly 30% absolute expression of microdystrophin relative dystrophin from a normal person (by Western blot).

Not only this, the microdystrophin was functional at the molecular level as judged by restoring dystrophin-related protein complexes serving to protect the muscle from damage by acting as shock absorbers.  Accordingly, CK levels in the blood, a marker of muscle damage and elevated in children with DMD, were robustly (9x) and uniformly lowered in all 4 boys between the ages of 4 and 7.  Add to this the obligatory before-and-after videos and there is little doubt already at this relatively early stage already (~1-3 months after gene transfer) that AAVrh74.MHCK7.microdystrophin is a powerful agent applicable to essentially all types of DMD.

On the safety side, there were considerable, but transient and manageable increases in liver enzymes.  This was to be expected, however, considering the very high doses of AAVRh74 needed to achieve widespread transgene expression in muscles throughout the body and treating physicians know to look for it.

If the safety holds up and expression continues to be long-lived, AAVrh74.MHCK7.microdystrophin could render many exon-specific oligonucleotide splice modulators obsolete. The duration of action is the most concerning issue to me at this point given the attendant cell turnover and attendant risk of losing episomal gene therapies in patients with muscle damage.    

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.  

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 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.

Register for the GTC RNAi Research and Therapeutics meeting in San Francisco today (June 20-21).  Get a free RNAi Therapeutics blog T-shirt and 20% discount on registration by entering discount code 'RNABLG13'.

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. 

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.