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. 

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 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.