ProQR and Partner Eli Lilly Demonstrate Oligonucleotide-induced RNA Editing in the CNS: A Major De-risking Event for the Industry

With every new oligonucleotide therapeutics modality that feeds into an endogenous cellular mechanism comes uncertainty as to whether the mechanism is sufficiently robust to be of therapeutic utility.  

This is especially true for RNA Editing as in its early days targeted AàI editing was only shown with the concomitant DNA-directed overexpression of ADAR along with an targeting RNA or the introduction of recombinant ADAR-antisense conjugates of little direct therapeutic use.  Similarly, simply introducing into a cell a chemically synthesized antisense oligonucleotide hybridizing to the area surrounding the target adenosine in an mRNA will only give you minute editing efficiencies in cell culture without further structural and chemical optimization.

The liver and CNS, due to their gene target richness and the demonstrated clinical feasibility of delivering oligonucleotides to these organs, are of particular importance to the RNA Editing industry.  The demonstration byscientists from Wave Life Sciences of oligonucleotide-directed RNA Editing in non-human primates was therefore an enormous de-risking event in that it showed that RNA Editing is sufficiently robust in living primate livers.

Of similar importance was the revelation by ProQR and their partners from Eli Lilly last week that this also holds true for the primate nervous system following the intrathecal administration of an editing oligonucleotide.  10-30% editing were seen in the brain depending on the anatomical location investigated.  In both the mice (intracerebroventricular delivery) and cynomolgous monkeys, editing was highest in the cortex.  Even higher editing levels, up to 50%, were observed in the spinal cord of non-human primates.

The spinal cord (motor neurons) also happens to be the location of the most successful oligonucleotide therapeutic currently on the market: SPINRAZA (nusinersen) for spinal muscular atrophy.  Since RNA Editing is quite new and many do not fully appreciate what 10-50% editing efficiencies mean, SPINRAZA can serve as a good example for how impactful such target engagements can be particular for gain-of-function approaches.

SPINRAZA is a splice modulator and works through gain-of-function by obscuring an intronic splice silencer element in the SMN2 pre-mRNA.  Typically, only 10-20% of SMN2 mRNA is ‘correctly’ spliced to yield a functional full-length protein.  With 12mg of SPINRAZA in infants (same dose used for the RNA editing studies in cynomolgous monkeys), this increases 2-3x.  This means that an approximately 10-40% successful target engagement can save babies from certain death and, if given early enough, may allow children with the type I SMA mutations to grow up almost normally.

In the case of the (undisclosed) target gene that Eli Lilly is looking at, these types of target engagements with RNA editing resulted in 5-25x increases in protein function.  Because of the above and because gain-of-function is a particular competitive strength of RNA editing, this application should be prioritized in target selection of industry pipelines.

Landmark Chemical Modification Study Shows RNA Editing Ready for the Clinic

At this stage, providing investors and the pharmaceutical industry with a clear line of sight that RNA Editing can be readily translated from concept into therapeutic reality is key to unlocking the next step-up in valuation.

A landmark study in March earlier this year by scientists from Wave Life Sciences (Monian et al, Nature Biotech) on chemically modifying ADAR guide RNA oligos (I will abbreviate them from now on AgRNAs due to missing consensus nomenclature) should go a long way in this regard.  It shows that applying a plethora of standard oligonucleotide stabilization chemistries (e.g. PS, PN backbones, 2’-O-methyl-, 2’-F-ribose) which are critical to enabling delivery and desirable durability do not compromise endogenous ADAR enzyme activity.

In fact, backbone stabilization for example via phosphorothioates, especially when in the SP stereopure conformation can actually greatly increase activity.  In a luciferase model system, editing activity of a fully (stereorandom) PS-modified AgRNA was 10x that of a corresponding AgRNA with an unmodified PO backbone.

Accordingly, when GalNAc-conjugated AgRNAs were tested in non-human primates, ~40% editing rates were observed for at least 2 months.  For this, a loading dose of 5mg/kg per day for 5 days was used.  This is on the higher end of what should be clinically acceptable, but as we know from experience with RNAi, what GalNAc works in non-human primates works even better in humans.

Illustrating the value of further refined chemical optimization of high-value candidates, impressive ~70% mRNA editing efficiencies were seen for a AgRNA against mutant SERPINA1 in primary mouse hepatocytes resulting in a concomitant increase in corrected protein.  SERPINA1 is also the target for Wave’s and possibly the industry’s first clinical RNAEditing program and addresses alpha-1-antitrypsin liver and lung disease.

What piqued my interest was that this AgRNA involved a 8-oxo-deoxyadenosine mismatch base opposite the adenine to be modified and a nearby inosine.  What this means will be addressed in my next blog entry...

So congratulations Wave Life Sciences on this study, but also they will admit that the study still only scratches the surface of what gains in potency will be possible with more detailed structure-activity studies.

Tekmira Expands Biodefense Efforts to Cover Both Ebola and Marburg Virus

Following the successes with its Ebola biodefense program, Tekmira has started to present increasingly promising data for treating a related filovirus, the Marburg virus.  With 100% survival rates in non-human primate models and new RNAi triggers that should cover a broad spectrum of strains and possibly related viruses, the company is well positioned to also take away the Marburg indication from competitor Sarepta which in turn has become preoccupied with its exon-skipping drug candidate for DMD.  

For the Ebola program alone, the $100M market cap company estimates that the successful development of a drug under the so called Animal Rule could result in stockpiling orders from the US government worth about $100M annually.

From guinea pigs...

In a publication last month, Tekmira and their collaborators from the UTMB in Galveston, Texas, reported the successful treatment of guinea pigs infected with a number of different strains of the Marburg virus.  Because the development of treatments for rapidly mutating viruses and viruses with a multitude of divergent strains is hampered by sequence diversity, a broader strain coverage was achieved, like in Tekmira’s Ebola approach or in Arrowhead’s chronic HepB strategy (ARC520), through the concurrent use of two siRNAs in a single formulation.

Given that rodents have limited predictiveness for anti-filoviral efficacy in Man and given that the formulation was apparently a 1^st generation D-LinDMA-based SNALP, I was not all that excited about the publication due to the apparent early stage of development.

...to non-human primates

In a positive surprise therefore, the Chief Scientific Officer of the company, Ian MacLachlan, presented gold standard non-human primate data of SNALP RNAi Therapeutics for Marburg virus at the ongoing OligoDIA regulator-industry conference.  Accordingly, Tekmira’s newer LNP formulations (‘SNALP-G’) were shown to fully protect monkeys from death due to Marburg infection when given at 0.5mg/kg (=the magic safety threshold for SNALP).

As an important comparison, Sarepta last year reported ‘83% to 100%’ protection rates in comparable models when treatment was initiated up to 96 hours after infections with its newer PMOplus morpholino chemistry.   It is therefore of interest to test the impact of further delaying treatment with SNALPs.

As I have been following Tekmira’s biodefense program over the years, one development I noticed, especially in the era of budget cuts, was the apparent push by the Department of Defense for biomergency treatments that can address not just multiple strains of a virus, but multiple viruses all-in-one.  Intriguingly, one of the bullet points in Tekmira’s slide presentation on goals for the Marburg program (slide 36) talks about the ‘Design broad spectrum siRNA to target multiple MARV and Ebola’.  Does this mean that the ultimate prize here are stockpiling contracts for a single drug addressing both Marburg and Ebola?

Having a better ear for and adjusting to the needs of the DoD is also one reason why I believe Tekmira will continue to have the upper hand over competitor Sarepta with its DMD distractions and an investor base that could not care less about the Marburg program.  Although the targets were explicitly not disclosed, I wonder whether there are host factors one could target and which would be beneficial for various viral applications.  This would be an alternative to trying to come up with target sequences that are conserved across the viruses.

Finally, serving as yet another example of illustrating the value of biodefense contracts for platform technology companies, Tekmira disclosed plans that a first clinical trial with a lyophilized SNALP formulation, TKM-EBOLA, is planned for the first quarter of 2014. 

Arrowhead Research Lifts Veil on Recent Progress in DPC Delivery

Four years after Mirus scientists published their seminal paper in polymer-mediated siRNA delivery, and 3 years after the acquisition of the Dynamic Polyconjugate (DPC) technology by Roche, Arrowhead Research, the new owner of the technology, finally lifted the veil on recent progress in bringing DPC technology to the clinic.

The White Paper reveals, for the first time, that DPCs have been successfully delivered to the livers of non-human primates, and moreover that they may also have use for applications outside the liver, one of the theorized attractions of DPCs based on their small size. Reading between the lines, however, it is also possible to identify some of the factors that have delayed clinical translation, and despite the progress and substantial investments by Roche, it remains an open question when the first DPC-based RNAi Therapeutic will enter clinical development.

Short circulation times were one of the deficiencies of 1^st generation DPCs. In order to reach tissues besides the liver, it is critical to achieve circulation times that are long enough so that the drug gets a chance to find and accumulate in its target tissues. Although DPCs on paper seemed to incorporate the features necessary for achieving such long circulation times, it came somewhat as a surprise when a DPC imaging paper last year showed DPC circulation times to be quite poor (Mudd et al., 2010). Although the particles were still able to accumulate in the liver, the data also raised questions whether the causes of the unexpected pharmacokinetics could have other consequences besides impacting biodistribution, for example in terms of safety.

It turns out that the short circulation times were the result of the premature exposure of the chemical groups that were supposed to shield the membranolytic functionalities of the DPCs outside their target cells. This would also explain why there seem to have been toxicity issues not just due to the poor biodegradability of the polymers, but also because such premature exposure renders DPCs as troublesome as many of the positively charged first-generation polymer approaches.

The White Paper indicates that the instability (and biodegradability) issue has been remedied to some degree such that the longer-circulating DPCs now show first promise for delivery outside the liver. But DPCs may also be competitive for delivery to the liver considering the following performance in non-human primates (excerpt from the White Paper):

‘Latest generation DPCs are remarkably efficacious in rats and non-human primates with ED80 values of ~0.1 mg/kg siRNA after a single dose. Increasing the dose two-fold in non-human primates results in >99% knockdown with a duration of effect of nearly 7 weeks. This is a 10-fold increase in efficacy compared to first generation DPCs containing PBAVE polymer. Latest generation DPCs are also better tolerated and have therapeutic indices of >10 in non-human primates as calculated from ED80 and NOAEL values.’

A 99% knockdown with 0.2mg/kg for 7 weeks- I’m impressed! It will be important to publish these data so that it is possible to see which model system was used, whether the 99% knockdown was seen for pretty much the 7 weeks, what the tox/tolerability profile was, the route of administration, and finally an explanation why a simple 2-fold increase in dosage had such a dramatic effect on knockdown efficacy.

The White Paper is also a reminder that for RNAi delivery technologies to be viable, there needs to be efficient scale-up. Apparently, manufacturing was, and possibly still is, a major issue with DPCs. At least the initial formulations had to be purified so extensively such that the yield became unacceptable for clinical translation and commercialization. Besides general liposomal expertise, quality manufacturing, of course, is what has always differentiated RNAi delivery company Tekmira from its competition.

My sense is that DPCs still have the potential to become an important delivery alternative. However, it is also clear that the path of DPCs was a tough one and, in the absence of guidance from Arrowhead Research, I expect additional delays (2-3 years?) before we will see the first DPC-based RNAi Therapeutic candidate in the clinic. This view is possibly shared by Alnylam as $10M in upfront and an increase of about $10M in annual operating costs would have been a small price to pay for Alnylam if DPCs were as advanced as Alnylam’s current systemic delivery workhorse, Tekmira’s SNALPs.

Until then, Arrowhead Resesarch needs to hit the ground running on the business development front given the increase in expenses that come with the 40+ research team in Wisconsin. The $15M facility with Lincoln Park Capital at least provides Arrowhead with increased financial flexibility. Ironically, it is positive clinical data from the RNAi Therapeutics candidates that are based on Tekmira’s SNALPs, DPCs most direct competitor that would greatly aid in that goal by re-igniting interest in the RNAi Therapeutics platform.