Oligonucleotide Strategies beyond the Liver

As the first major wave of pivotal trial successes involving gene knockdown in the liver has reached the shores, oligonucleotide therapeutics are quickly establishing themselves as the dominant modality in this important target organ for new drug development.  While the industry is milking this organ for therapeutic applications, my thoughts are directed towards the next tissue opportunities that should further feed this revolution in drug development we are witnessing now.

After not having been part of the conference circus for 2 years, this week’s Oligonucleotide Therapeutics Society annual meeting in Bordeaux, France, was just what the doctor ordered for me to get a clear perspective on this issue.  Importantly, lessons learned from GalNAc-targeted oligonucleotide delivery to hepatocytes, but also LNP delivery to the liver prior to this, now allow the field to take the next step in extending delivery beyond the liver.

Chemical stability

All these efforts essentially share their use of highly modified oligonucleotides which have particularly changed the philosophy around the RNAi modality.  This allows the oligonucleotide to not only reach the target tissue intact, but also to remain trapped in endosomal compartments which serve as slow-release depots for long duration of action.

Chemical modification also has greatly reduced the immunogenicity of RNAi triggers and obviated the need for protective nanoparticle formulations.  These often came with the added liability of amplifying the immunostimulatory potential of these molecules.  Consequently, decade-old approaches are now being revisited with more fully modified RNAi trigger versions (e.g. self-delivering RNAi trigger structures as pursued by RXi Pharmaceuticals and the Khvorova group at UMass).

Ironically, after all the song and dance by RNAi bellwether Alnylam about the utility of exotic modifications in their conference presentations (one can also call it willful misleading of the field- not really the purpose of scientific conferences), the strong trend is towards maximizing 2’-O-methyl content, in addition to some 2’-F and phosphorothioation at the RNAi trigger termini.

PK enhancers

One of the reasons why the liver became the first major oligonucleotide target organ is that it is readily accessible from the blood.  This allows it to soak up oligonucleotides before they get removed by renal filtration.  In an effort to fight this tendency, the use of PK enhancers, in particular lipophilic groups is frequently seen.  Ionis and Alnylam are testing these for example for getting better distribution to the muscle and potentially also better functional uptake.

Receptors

PK enhancers, however, are largely about shifting around biodistribution, but it is hepatocyte ASGPR-type receptors that are the most valuable assets the industry is striving to identify.  My highlight of the conference therefore was a talk on a collaboration by AstraZeneca and Ionis demonstrating strikingly selective and effective targeting of beta cells in pancreatic islets. Think diabetes! 

It is the GLP1-receptor that does the magic here and which can be targeted by GLP1-peptides for effective oligonucleotide uptake.  While the in vivo validation was limited to rodent models, including an elegant GLP1-receptor knockout mouse model, I am convinced that the findings will translate to larger animals and humans.  It is one of those things you just know when seeing such data.

This example illustrates the value of knowing your target cell type really well, as this may allow you to identify additional ASGPR-type receptors which had been thought of elusive.  But even if they are lacking in some tissues, a nice, yet simple strategy to overcome this was illustrated by MPEG LA and Axolabs: by linking more than one RNAi trigger to a small scaffold, they were able to show that cellular oligonucleotide uptake capacity can be increased beyond the limits of receptor amount on the cell surface.

 

While the liver certainly does not need this strategy, it should definitely be applied to new targets like the beta cells.  Let free market competition do its magic and have oligonucleotide therapeutics solve diabetes now that you can effectively reach hepatocytes, adipocytes, and now also beta cells. Yes, I love the free markets, but I digress… 

After beta cells, it was a collaboration between Alnylam and Johnson & Johnson on overcoming the long-held dream of oligonucleotide therapeutics addressing gene regulation in cancer cells.  Here, small and stable ~2nm peptide scaffolds referred to as centyrins were coupled to the RNAi trigger and directed towards different receptors like PSMA and EGFR.  Perhaps the most striking aspect of centyrin-siRNA conjugates was their effective tumor penetration where prior RNAi delivery attempts like LNPs had fallen short.

Endosomal release

Sometimes getting to the endosomes alone is not enough when the rate of cytosolic release therefrom is insufficient.  So despite of the DPC fiasco last year and despite of aborted arginin-based endosomal release attempts prior to this, active endosomal release is still embraced in some delivery efforts.  Most notably, Sarepta has shown dramatic increases in dystrophin exon skipping in non-human primates with new peptide-PMOs (PPMOs) compared to their unconjugated parent molecules.

Of course, everybody now wants to know what the therapeutic window really is.  While the Sarepta representative at OTS was a bit cagey when asked about it, Sarepta’s CEO noted in a recent investor presentation that the filing of an IND by the end of the year would be a major positive signal in that regard.

Finally, all of the above developments are aided by more general progress in technologies interrogating biology such as single cell technologies (cell type isolation from complex tissues like the kidney), reduced chemistry costs allowing for much larger numbers of oligonucleotides to be screened, and ubiquitous low-cost and high-throughput sequencing.

 

Sometimes I pinch myself asking whether all this is real and not just a figment of my imagination, but at least in my mind the stars just keep aligning allowing for RNAi and oligonucleotide therapeutics to take the next step up the value ladder.

RNAi Therapeutics Become Real

It’s been a long ride.

The lives of quite a few of us has been consumed now for close to two decades dreaming about a world in which RNAi Therapeutics have real-world clinical impact.  Yesterday, that dream has officially materialized.

TTR knockdown improves disease state

With the conclusion of the most comprehensive clinical study conducted to date in the severe orphan disease ATTR amyloidosis, Alnylam and investigators have found that prolonged knockdown of the causative transthyretin (TTR) gene improved both objective (mNIS+7) and subjective (quality of life) measures of the main manifestation of the disease, peripheral neuropathy.

There is no doubt that this was the result of on-target TTR gene knockdown since this was a strictly controlled study in which the only difference to the placebo group was the administration of the RNAi formulation.  Furthermore, results from a similar study for this indication (NEURO-TTR) by Ionis earlier this year in which related RNaseH antisense oligonucleotide technology was utilized for TTR knockdown also demonstrated disease benefit, albeit a ‘mere’ halt of neuropathy progression in QoL as opposed to the improvement seen here (note: mNIS+7 was reported only with regard to placebo, not versus baseline).

Importantly, this extra benefit is likely explained by the fact  that the Patisiran RNAi formulation had been shown to be slightly more potent than antisense drug candidate Inotersen in earlier-stage studies (here and here).  While considering a ~80-85% vs ~70-75% knockdown may not seem much at first glance, protein deposition, clearance and in particular misfolding that is at the root of the disease are higher-order concentration-dependent processes (think about crystallization) so a difference of ~17.5% vs ~27.5% (>50% more remaining than with Patisiran) remaining insulting protein may well be highly meaningful. 

Of note, phase II data from Patisiran have demonstrated that the ~80-85% knockdown was able to somewhat tilt the tables in favor of TTR tissue clearance, but also that it was not able to fully do so.

It is therefore of utmost importance to push forward with the development of the even more potent GalNAc-enabled RNAi candidate ALN-TTRsc02.  Preliminary phase I data from that candidate suggest that it should be able to reduce TTR levels to 5% or less with subcutaneous dosing as infrequently as every 3-6 months (Patisiran: intravenous every 3 weeks; Inotersen: weekly).

Why this is such a big deal for RNAi Therapeutics

As I had indicated in last week’s post, the phase III APOLLO results represented a make-or-break moment for RNAi Therapeutics.  If it turned out that RNAi- and delivery-related side effects were to outweigh the benefits of gene knockdown at the conclusion of this decade-long, high-visibility program, the financial markets would have reacted violently and starved the industry of the cash necessary to more fully develop the technology (Alnylam with its cash reserves at least would have mounted a comeback eventually).

If it’s one thing I’ve learned about biotech money matters over the last 15 years of riding the RNAi rollercoaster, it is that the perception of a technology is as important for its continued development as its intrinsic technological validity.

Personally, I am most relieved in that the small, but often pervasive transcriptomic off-target changes introduced by an RNAi trigger (à microRNA-type off-targeting) did not have an apparent detrimental effect on the target organ (here: liver) after prolonged, 18-months treatment.  This should not be entirely surprising based on what we’ve learned about microRNA biology- not all targets of a microRNA are biologically relevant- but is still a great relief to see play out in practice.

To wit, the RNAi industry has been overly focused on potency when selecting RNAi trigger sequences for clinical development and mostly relied on bioinformatics for specificity. Indeed, Alnylam has paid the price for this negligence in that certain RNAi triggers, such as the one for its original alpha-1-antitrypsin program, have caused liver tox most likely due to microRNA-type off-targeting.  It is therefore finally employing chemical strategies such as the incorporation of modified nucleotides in the seed region of the trigger to bias the RNAi apparatus towards RNAi cleavage instead of microRNA-type message destabilization. 

This strategy was first reported by Rosetta Inpharmatics (now Merck) and had then been developed further in the commercial realm by Marina Biotech which then licensed the IP to Roche (then acquired by Arrowhead) and possibly others. 

It will be some time until we have complete certainty that microRNA-type off-targeting won’t rear its ugly head again, but the odds should be getting better and better with the employment of best practices. More generally, Patisiran is only the beginning of a long string of real-world impactful RNAi Therapeutics.

The Wait is Finally Over

For some time now, I’ve been fascinated by how the RNAi Therapeutics sentiment cycle has been progressing in 3-year intervals.  Before I left off for my baby-break, I referred to 2014-17 as The Wait. The Wait was a reference to the ‘market’ having been convinced by early clinical data that RNAi can bring about deep and sustained gene knockdowns in WoMan, but that the ultimate clinical utility and subsequent commercial value remained to be proven.

We are now on the cusp of finding out with results from the registrational phase III study of the TTR-lowering Patisiran in familial amyloidotic polyneuropathy (FAP) imminent.

The wind is clearly in RNAi’s back with supporting developments in the related oligonucleotide therapeutics and, indeed, drug regulatory spaces.  In particular, the successful commercial launches of splice modulators EXONDYS51 (for Duchenne muscular dystrophy) and SPINRAZA (for spinal muscular atrophy) and promising phase III data from the triglyceride-lowering Volanesorsen (gene target: ApoCIII) should have removed the long-held doubt that oligonucleotides can be clinical practice-changing and profitable drugs.  And boy, are EXONDYS51 and SPINRAZA changing the clinical practice of two devastating, previously orphan diseases!

Equally important, drug regulation is strongly moving infavor of patient choice and access.  As a result, we are going to see many more drug candidates benefiting from accelerated approval pathways similar to EXONDYS51.  A number of RNAi Therapeutics programs such as Givosiran by Alnylam (àmetabolite ALA lowering as reasonably predictive endpoint) and DCR-PHXC by Dicerna (àoxalate lowering) are poised to benefit from this development.

But let’s not get ahead of ourselves and await the Patisiran APOLLO study outcome for which sponsor Alnylam has guided a ‘late September/summer’ read-out.  Drug development is full of surprises, so despite all the data* pointing towards a positive outcome, a number of variables (e.g. steroid use, lipid nanoparticle delivery) remain unknown in how they impact overall outcomes.  I for one will breathe a big sigh of relief should Patisiran be able to overcome this Make-or-Break event for RNAi Therapeutics.

 *

à positive open-label extension data from phase II Patisiran;

à positive phase III data from IONS-TTRRx targeting same gene and indication;

à the high roll-over rate into the open-label extension) indicating a positive outcome