Stabilizing RNAi Triggers against Cytoplasmic Degradation Pays Dividends

In describing the preliminary phase IIa results of ARC520 for HBV, Arrowhead Research noted that the duration of gene silencing (2 months and more) was surprisingly extended in Man compared to the preclinical experiences in rodents and non-human primates.  Alnylam hasnoticed the same with its GalNAc-siRNA conjugates, especially the highly modified ESC version. 

The extended gene silencing activities, of course, bode very well for RNAi Therapeutics in general when in the early days (~2002-2003) I was a bit apprehensive when gene silencing in my transfections of cancer cell lines persisted for only 2-3 days (as we now know largely due to their rapid cell division).  To maximize the duration of gene silencing, thereby opening up RNAi Therapeutics to new applications and increasing its competitive profile, it is important to understand the factors underlying it.

Alnylam explained the differences to the preclinical experience because rodent and monkey hepatocytes seem to have a more hostile, degradative cytosol compared to human hepatocytes (hypothesis 1).  In one experiment, only 6% full-length ESC-GalNAc-siRNA remained after a given time in rodent and monkey cytosolic extracts while in human liver cytosol extracts more than 60% persisted.

This, however, was only a correlation and I have considered it equally likely that the difference in gene silencing duration might be a function of more stable RISC complexes in humans (hypothesis 2) or increased stability in the endo-lysosomal compartment (hypothesis 3).  Especially for GalNAc-siRNAs, I would think that the reason that it works in the first place is due to them being able to accumulate in endo-lysosomes from which they only get released in the wake of natural vesicle membrane turnover.  So chemical stability here would be a critical factor since the endo-lysosomal compartment is known to be highly degradative.

DPC and SNALP: two endosomolytic technologies with different durations of gene silencing

While I still consider that endo-lysosomal stability of the naked RNAi trigger is critical for approaches like GalNAc-siRNA conjugates, the new DPC-enabled ARC520 results strongly indicate that another critical factor lies downstream of endo-lysosomes.  This is because in the DPC approach which involves strong endosomolytic activities that should activate soon after endocytic uptake, the risk of the RNAi trigger being degraded in the endo-lysosomes should be low.  Similarly, there should be little contribution to gene silencing from RNAi triggers that get released into the cytoplasm in a delayed fashion.

SNALP is another delivery technology where the RNAi triggers that become active in gene silencing get released into the cytoplasm soon after endocytic uptake.  However, while clinical data supporting 3-4 week dosing frequencies have been obtained with SNALPs (e.g. ALN-TTR02), the silencing does not appear to be as extended as with DPCs.  So given that one marked difference of the payloads used with SNALPs and DPCs is the modest degree of chemical modification historically used with SNALPs, this, too, points towards cytosolic stability of the RNAi trigger being important for the duration of gene silencing.  Parenthetically, it also suggests that Tekmira may want to similarly explore heavily modified RNAi triggers while being mindful not to step on the McSwiggen patent toes of Alnylam.

RISC-optimized ultra-stable single-strand RNAi triggers

In the case of traditional double-stranded RNAi triggers as e.g. used with DPCs, the stabilized RNAi triggers get used up over time as they are recruited into RNAi effector complex RISC.  Part of this process involves their unwinding into single-strand RNAs with the guide strand being retained.   It is known that once used, a 'normal' guide strand (or microRNA) is not recycled into another RISC complex and will likely suffer metabolic destruction once the protein components of RISC have become degraded as part of natural protein turnover.  And even if the guide strand had been stabilized, because a standard single-strand molecule that had relied on being part of a double-strand structure for RISC recognition, old age will eventually catch up here, too. 

What a waste after all this effort of getting the RNAi trigger into the cytoplasm.  So why not take a cue from the single-strand RNAi practitioners who optimize single-strand RNAi triggers also based on being able to be recognized by RISC?  If a corresponding dsRNA contained corresponding recognition elements, then the guide strand could contribute to another round of gene silencing, thus extending and enhancing knockdown.  On the other hand, the lessons learned from stabilized dsRNAi triggers should also benefit the single-strand RNAi approach as increased cytosolic stability should also increase their duration of activity: RISC-optimized ultra-stable single-strand RNAi triggers.

Big De-Risking of ALN-TTR02 and SNALP Delivery Platform

Today, Alnylam reported (PR here, presentation here) first data from its phase II open-label extension study of ALN-TTR02.  ALN-TTR02 is developed for the treatment of TTR amyloidosis (FAP form) and with a phase III study underway is the industry’s lead blockbuster candidate.  There is no doubt that its results will have a big impact on the perception of the technology.   Importantly, the new data revealed at the International Symposium on Amyloidosis show that ALN-TT02 not only maintains consistent knockdown potency following multiple (>2) administrations, but even more importantly, indicate that the biggest risk, long-term safety, will not be a problem.    

Phase II promising, but with efficacy and safety questions

The new data follow on from a 2-dose phase II study (somewhat euphemistically labeled 'repeat administration) showing that ALN-TTR02 can achieve ~80% TTR knockdown levels not only following a first dose, but also following a second dose.  This was good to see because there was theoretical concern that SNALP LNPs could provoke generation of neutralizing antibodies to the PEG component which might compromise efficacy following repeat dosing.  Indeed, TTR levels seemed to return back to normal more quickly following the second compared to the first dose and was responsible for Alnylam switching from a 3-week to a 4-week dosing interval.

On the safety side, infusion-related hypersensitivity reactions are the major concern with SNALP LNP as with most other infused therapeutics, nucleic acids, protein, or small molecules alike.  While the steroid pre-treatment may have ensured that no major immune reaction was seen in the phase II study, infusion reactions had been recorded in 3 of 20 infusions at the key 0.3mg/kg dose.  Intriguingly, when a micro-dosing pre-treatment strategy was employed (increasing the length of infusion from 60 to 70 minutes) no such reactions were seen in the 18 infusions thus administered.  This is probably related to the fact that infusion reactions in general are essentially restricted to the first dose and are dose-related.

Phase II open-label extension data provide highly positive answers

In thinking about a headline for today’s blog entry ‘spectacular’ was on my mind a lot.  However, I refrained from using this term as the scientific results show very comforting consistency. 'Spectacular consistency' may be too oxymoronic.   In stock market terms, however, this consistency may very well be called ‘spectacular’ both for the much increased likelihood that ALN-TTR02 will make it across the finish line and for the safety demonstration of Tekmira’s SNALP delivery platform in general for which the major concern has always been safety, not efficacy.

Importantly, the results revealed today show that the ~80% knockdown potency is maintained following an aggregate of 119 doses of 0.3mg/kg ALN-TTR02 compared to an aggregate 38 doses before.  In the patient who has been on therapy the longest, this was the case following 9 doses over half a year.  The integrated knockdown level over time may have indeed been slightly higher than 80% since TTR levels were measured at their nadir, just before the next dose.  One can therefore conclude that the enhanced recovery from gene knockdown following the second dose did not intensify over time and that sustained and consistent knockdowns can be achieved over the long-term.

Regarding safety, just 3 instances of mild-moderate adverse events were reported in the extension phase that were deemed possibly treatment-related.  Probably only the 2 instances infusion reactions in a single subject call for attention.  Even so, none of them were deemed severe enough that the infusion rate had to be slowed as it is normally done.  The other mild-to-moderate AEs were an ‘increase of diarrhea’ and an 'impairment of taste'.  Maybe not all that bad in a disease like FAP.

Further indicative of the tolerability of ALN-TTR02, Alnylam projects 27 out of the initial 30 patients to participate in the open-label extension study.  First functional results from this OLE are expected to be reported by the end of 2014.

Disclosure: I could not resist but buy back some TKMR today based on the recognition that today’s data represent a great de-risking of the SNALP LNP delivery platform the company depends on.

Further Possibilities for Arrowhead Phase I Dose Extension

Last night’s speculations on the reason for the phase I dose extension of the HBV RNAi phase I trial by Arrowhead Research was actually positive: they are tackling the potency issue presented by the 2mg/kg dose. 

In addition to further increase dose, it could also involve prolonging drug infusion times which we know, based on Alnylam’s GalNAc data, could optimize hepatocyte uptake by the GalNAc-targeted DPCs.

Regardless, you have got to question why extending beyond 2mg/kg had not been part of the original plan and why the study extension has not been publicly discussed, e.g. in the latest conference call.   

Extending the study to further potency is the most rose-colored scenario that one can draw.  The other scenario would have to do with safety concerns, possibly raised by regulators.  As I had discussed before, for DPC, especially the 2 molecule version, the tox-limiting element is the melittin-like peptide.  Melittin is derived from bee venom and although the MLP is not the identical sequence as melittin, there are theoretical concerns around allergic reactions.  In general, having peptides involved raises a new set of immune issues, especially when you require 2mg/kg of them.

Of note, one of the exclusion criteria for the phase IIa HongKong trial is excluding those with a history of allergy to bee venom, indicating that this has been an issue with regulators:

·         Has a history of allergy to bee venom or history of hypersensitivity reaction requiring an emergency visit to a physician or hospital and/or requirement for treatment with steroids and/or epinephrine.

So how about adding transient immune suppressant to the mix- e.g. an anti-histamine?  Nothing spectacular, a safety precaution, but once again highlighting the benefit of Arrowhead Research making advances with the single-molecule version, also for applications outside the liver (see today’s positive news around Endocyte and folate targeting for cancer as just one example of where such research could be directed at).

Potency matters, and wouldn’t it be ironic that as Tekmira is weaning itself off immune suppression (à dose-intensive TKM-EBOLA trial), on the back of developing more potent formulations, Arrowhead Research, not known as a public supporter of liposomal RNAi delivery, is adopting such?  

The Ebola Clinical Trial a Real ‘Haertetest’ for SNALP LNP Delivery

In German, a Haertetest is a test that challenges the performance of something under the most exacting conditions.  

It is therefore an apt description for the Ebola clinical trial now underway which is testing the safety and tolerability of SNALP LNP delivery following multiple consecutive daily administrations.  This compares to once-weekly and once-a-monthly regimens tested so far.  If SNALP LNP comes out positive from this, it would provide great comfort around the safety and tolerability of the industry’s most potent delivery technology for gene knockdown in the liver, in addition to paving the way towards more biodefense applications.

Teething problems with 1^st gen SNALP LNP

In January 2010, Tekmira reported clinical results from the phase I study of TKM-ApoB, the first clinical candidate employing SNALP LNP technology.  This trial was terminated prematurely due to immune stimulations observed at 0.6mg/kg, a dose at which only minimal target knockdown was observed at that.

This event triggered a healthy amount of soul searching and meant that subsequent SNALP LNP trials had to employ transient immune suppression.  A number of important practical lessons were learned from this.  

Firstly, it showed that the preclinical immune stimulation tests had been inadequate and this prompted the development and adoption of a more predictive test to better screen out candidates that are immunostimulatory before they enter humans.  Secondly, the phase I results were consistent with the notion that the DLinDMA lipid itself (and not just the RNAi trigger), the basis for the 1^st gen SNALP formulations, was immunostimulatory and that new lipids were needed.  And lastly, it highlighted the need to develop more potent SNALP formulations, including new lipids.

Research rises to the challenge

Three years following these events, all three challenges have been successfully addressed.  Today, the potency of SNALP LNP has increased ~30 fold from 1^st gen to 3^rd gen formulations (based on measuring gene knockdown in the liver), with the 2^nd gen MC3-based SNALP generation already with a confirmed 10-fold improvement in clinical potency based on phase I and II results of ALN-TTR02.

Tekmira not shy to ask the tough questions

Despite the progress, detractors of the technology like to point out that (transient) immune suppressions has still been employed in subsequent clinical trials.  With the recent initiation of the phase I study of TKM-Ebola by Tekmira, this has changed however.  Not only that, transient immune suppression has been dropped even as administration intensity is ratcheted up to 5-7 daily administrations. This compares to once-a-month and once-a-week dosing schedules anticipated for SNALP LNP applications involving gene knockdown in the liver and solid cancers, respectively.

The reason why TKM-Ebola necessitates daily administrations is due to the rapid progression of the otherwise fatal Ebola virus.  Therefore, in order to get it approved under the Animal Rule, Tekmira needs to demonstrate the safety and tolerability of SNALP LNP in human volunteers under conditions where they are able to rescue monkeys from succumbing to otherwise fatal infections.  

Moreover, while 0.2mg/kg already provided good protection in the monkey studies (2/3 survival), it was the 0.5mg/kg dose that complete protections were achieved.  0.5mg/kg also happens to be the ‘magic’ dose for SNALP LNPs following which idiosyncratic immune stimulations and other side effects may be expected.

The reason why I am pretty sure that TKM-EBOLA, despite the daily administrations and up to 0.5mg/kg dose, should not involve immune suppression is because it would be incompatible in a viral infection.  There is also no evidence that such pre-treatments are planned in the clinicaltrials.gov entry nor when Tekmira reported the monkey results.  The same considerations apply to the HBV candidate by Tekmira for which an IND is expected by year-end.

Should Tekmira come out of this trial unscathed, it would further de-risk the entire SNALP LNP platform and solidify its position as the most potent oligonucleotide therapeutics technology for gene knockdown in the liver, for hemorrhagic fever applications, and possibly oncology.

Where some see risk, others see opportunity.    

Alnylam Solidifies Lead in TTR Amyloidosis Race

Over the weekend, Alnylam announced expanded data from the phase II study of ALN-TTR02 (aka PATISIRAN) in the FAP form of TTR amyloidosis at the ISFAP meeting being held in Brazil.  The data confirm the consistent knockdown potency of ALN-TTR02 and, most interestingly, suggest that a new dosing regimen can address the most rate-limiting safety issue encountered so far. 

Compared to the 19-patient dataset revealed at the Peripheral Nerve Society meeting at the end of June, the latest update includes data from 9 more FAP patients that received the highest dose (0.3mg/kg) in the once-every-3-week treatment regimen.  Previously only 3 patients received such dosing whereas the data from the 6 patients with the once-every-4-week dosing indicated an undesirable TTR rebound effect towards the end of the 28-day treatment cycle.

Data from the 12-patient once-every-3-week cohort now show that a ~80% persistent knockdown of TTR can be achieved although there still seemed to be a minor rebound effect at the end of the 21-day treatment cycle (down to ~75% knockdown).  It therefore needs to be watched whether the apparent rebound effect worsens or maybe gets better with dosing for more than the two infusions as performed in this study (open-label extension study ongoing).   

The most exciting revelation, however, from the new data was that there were no apparent infusion reactions in the 9 additional subjects.   Previously, 3 out of 7 in the once-every-4-week cohort of 0.3mg/kg experienced such reactions.  Infusion reactions thus far tends to rank as the rate-limiting safety issue in the development of SNALP-related RNAi Therapeutics and although they appear to be readily managed by slowing the rate of infusion, are dose-related, and generally occur only during the first infusion, it would put everybody at ease if there were no such risk.

Rather than being the result of a streak of luck, the absence of infusion reactions coincided with the institution of a 70-minute microdosing strategy which Alnylam labeled as ‘proprietary’.  If it holds up, it would definitely be good news for Alnylam and FAP patients.  Beyond that, Tekmira may actually be the major beneficiary of such progress as its pipeline rests on i.v.-administered SNALP RNAi Therapeutics such as ALN-TTR02.


By initiating a phase III study of Patirisan and further announcing fast-track designation for this product candidate, the company remains on track for an approval in 2016/7.  Data from the competing RNaseH antisense compound by ISIS and GSK at the same meeting remains to be publicized.

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. 

A First Clinical Step Towards Subcutaneously Administered RNAi Therapeutics

Today, Alnylam reported more detailed results from their phase I study of ALN-TTRsc for the treatment of the cardiomyopathy form of TTR amyloidosis (press release here, data presentation here).  As telegraphed by COO Barry Greene at the recent Morgan Stanley Healthcare Conference, 5mg/kg and more of the subcutaneously administered ALN-TTRsc were required to obtain a persistent knockdown of 80% (5mg/kg) and more (10mg/kg dose).  

As these doses fail the 2.5mg/kg challenge the company had set itself earlier, increased injection volumes and injection numbers are being evaluated.  A phase II trial with ALN-TTRsc is planned to start at the end of the year with a pivotal phase III study slated to start in 2014.

Small potency differences to monkey studies matter for ALN-TTRsc

As I had indicated earlier, the preclinical monkey studies with ALN-TTRsc (see slide 7) left open the possibility that with an 80% target knockdown, ALN-TTRsc can be sufficiently effective at 2.5mg/kg which corresponds to the magical injection volume of 1ml for safe and convenient subcutaneous administration.  However, the phase I human data are shifted by 1 dose cohort meaning that 5.0mg/kg were required for an approx. 80% sustained knockdown (see slide 11 where 2.5mg/kg clearly missed).

If you are confused about the press release prefers which talks about an 87.5% 'mean' (n=3) knockdown ‘at nadir’ for the 5.0mg/kg dose, it is more appropriate for this indication to refer to the average knockdown level over time which was more like 80-82% (slide 11).  

Nevertheless, oozing with optimism, Alnylam set itself an even higher bar for ALN-TTRsc for future studies with a sustained target knockdown of 90%.  Such a level of knockdown was only achieved in this study at the very high dose of 10mg/kg.  Indicative that 10mg/kg is not a desirable dose, the company amended its study to evaluate 7.5mg/kg given as two 1.5ml injections in addition to 5.0mg/kg and 7.5mg/kg every 2 weeks to determine whether less frequent dosing is feasible.

Personally, I believe 5.0mg/kg twice a week would be a more promising dosing schedule as with higher doses there should be diminishing returns due to receptor saturation.

Safety OK, but questions raised

The reported safety profile did not yield apparent nasty surprises.  This is obviously very good in a phase I study of a new chemistry.  However, it has also become obvious that injection site reactions could become an issue.  Unlike ISIS Pharmaceuticals which has declared such reactions to be a cosmetic problem in the past, injection site reactions have to be closely watched as they could indicate systemic immune issues.

Despite the small numbers, the injection site reactions appear to be dose-related with 3 out of 3 subjects at the 10mg/kg exhibiting them during the 5 weeks of the study.

 

These observations are also relevant to the competitive DPC delivery technology by Arrowhead Research because both involve galactose conjugations.  In the case of the intravenous version of DPC, the galactose has been added to the endosomolytic peptide, while a cholesterol is added to the RNAi trigger administered separately. 

Initially, I had thought IP issues could have prompted Arrowhead to put the galactose on the ‘less precious’ peptides.  However, an alternative explanation could be that galactose conjugated to nucleic acid might be more prone to immune stimulations.  Due to the apparent dose-relatedness of the reactions, it will be important that future subQ candidates (both Alnylam's GalNAc and Arrowhead's subQ DPC which have galactose and the RNAi trigger on the same molecule) have therapeutic knockdown potencies at 2.5mg/kg and below.  

A very perplexing observation, albeit not discussed at all, is the fact that elevated immune cytokine levels were reported in the placebo cohort, but none in the ALN-TTRsc cohorts (see slide 15 of the presentation).  I really don’t know what to make of this. 

Summary

Despite some of the blemishes above, the results are a decent start for subcutaneously administered RNAi Therapeutics for liver-expressed gene expression.  In addition to the safety profile, this thus renders also the last argument for phosphorothioate-based RNaseH antisense technology in such indications obsolete, namely the ‘patient-friendly’ route of administration.  Patients with TTR amyloidosis and their relatives can now look forward to two attractive RNAi options, and I expect there to be continued RNAi improvements for this indication.

For those interested in Tekmira, the results are positive in the sense that they increase the value of ALN-TTR02 given the modest potency of ALN-TTRsc, and support SNALP to be the clinically validated gold standard in RNAi delivery.  For those interested in Arrowhead Research, the results are positive as they underline the value of more potent subQ RNAi delivery technologies and because they lower the safety risk around the galactose chemistry.

Alnylam Comments Suggest SubQ Tech Failed to Achieve Efficacy Goal

At today’s Morgan Stanley Healthcare Conference, the President and COO of Alnylam, Barry Greene, told the audience to assume that it took a dose of 5 to 10mg/kg of ALN-TTRsc to achieve the 80% knockdown that had been reported in July.  This suggests that ALN-TTRsc has failed to meet the stated goal of limiting the subcutaneous injection volume to 1ml or less as this is predicted to correspond to a dose of 2.5mg/kg.

If it holds up, this would have a number of important implications.  Firstly, it would affect the profile of ALN-TTRsc itself.  I don’t believe it to be necessarily terminal in a disease like the FAC form of TTR amyloidosis for which ALN-TTRsc is being developed.  Nevertheless, squeezing 2 or even 4ml into the subcutaneous space, or alternatively administering multiple 1ml shots in one sitting, could put some needle-phobic patients off, especially as TTR amyloidosis is set to become a competitive marketplace.

Secondly, such doses would not bode well for Alnylam’s GalNac delivery platform on which the company has pinned its future.  For example, one of the big draws of the hemophilia drug candidate ALN-AT3 which is about to enter clinical development was supposed to be its subcutaneous, instead of intravenous route of administration. However, as I speculated last week (Alnylam's GalNAcs as Cholesterol-siRNA 2.0), I believe ‘GalNac-siRNAs’ have advanced somewhat since ALN-TTRsc with the addition of lipophilic moieties such as cholesterol and the jury could therefore still be out regarding the capabilities of the GalNac delivery platform.

And finally, a weak potency of ALN-TTRsc would further increase the value of competing delivery technologies, especially SNALP (Tekmira) and the subcutaneous version of DPCs (Arrowhead Research). In fact, if the full results that are to be presented in about a week at the American Heart Failure Meeting confirm the 5-10mg/kg doses, it would explain why Alnylam chose not to have ALN-TTRsc compete with and potentially replace SNALP-based ALN-TTR02. Instead, the intravenous ALN-TTR02 will likely be applied to the earlier onset, more rapidly progressing form of TTR amyloidosis (FAP), whereas the subcutaneous ALN-TTRsc will be used for the more slowly progressing, later onset form of the disease (FAC).

Alnylam’s GalNAcs As Cholesterol-siRNA 2.0

That Alnylam bets its future on GalNAc-siRNAs is remarkable and indicates that the company feels the technology is clinically viable.  Accordingly, the preclinical animal data leaves open the possibility that the required volume of an effective dose of ALN-TTRsc, the first GalNAc-based clinical candidate, can be administered in a volume of 1ml or less, the stated goal of the company (results to be presented at HFSA, Sep22-25).  

While I view ALN-TTRsc as a cliffhanger in terms of commercial viability, not just because of the volume issue but also in light of the likely more effective SNALP option in the form of ALN-TTR02, the potency of GalNAc-based ALN-AT3 for hemophilia looks quite adequate.

Alnylam’s claim that a simple ligand-siRNA conjugate was able to mediate gene silencing in vivo required a rethinking of RNAi delivery by many since similar efforts involving especially aptamer-siRNA conjugates have been ‘viewed with skepticism’ to put it nicely.

Did Alnylam mislead the competition?

If you are a student and your funding body just spent $3000 to send you to a conference to learn about your subject, you might be forgiven if you came away with the notion that simple siRNA-conjuates such as Alnylam’s GalNAc-siRNAs can be effective (see e.g. slide 16 of the presentation by John Maraganore at the OTS last year).  The trick apparently is that with a magic ligand-receptor pair such as GalNAc-ASGPR all you need is to conjugate an RNAi trigger chemically modified for stability.  

Of course, the imaginary student here is only collateral damage as the real target audience of a presentation by Alnylam is its investors, potential partners, and competition.

As such, it may not surprise that according to patent application US2012/0136042A1, a simple GalNAc-siRNA conjugate has no activity on its own.  

Instead, silencing activity critically depends on the molecule also having a cholesterol (=lipophilic group) attached to it.  Since Alnylam had worked on such cholesterol-siRNA conjugates before (Soutschek et al. 2004), what now appears to be the actual GalNAc-siRNA structure can be viewed as an evolution of the cholesterol-siRNA which showed knockdown activity only at doses of 50mg/kg and higher.

DPCs by Arrowhead Research Take another Step

The 10-20 fold gains in potency with the addition of the GalNAc ligand shows how comparatively inefficient ApoE/LDL-based uptake systems are for liver-directed RNAi delivery.  The reason why SNALP is still more effective than GalNAc-siRNA is because cholesterol-siRNAs do an even poorer job when it comes to the next rate-limiting step in delivery: endosomal release.

With Arrowhead’s Dynamic Polyconjugates (DPCs) for liver-directed knockdown, you combine the best of both worlds: effective attachment to hepatocytes via multivalent GalNAc-ASGPR interactions and effective endosomal release through endosomolytic polymers/peptides.  

In fact, because of the potent chemistries used for the latter step, the endosomolytic agent has to be masked which initially made me a bit wary about the safety of DPCs (note: clinical safety data for ARC520 are about to be released, probably in October, and the preclinical safety performance looks good so far). 

According to Arrowhead’s research, this combination allows for more than 500-fold potency gains compared to cholesterol-siRNAs (Wong et al. 2012).  

Because the ratio of siRNA to endosomal-release peptide has to be held constant in the clinical studies of ARC520 which is based on the 2-molecule DPC version, you will not see these improvements in potency translated into clinical practice.  This is not all that critical anyway since ARC520 is administered intravenously and cholesterol-siRNAs should not be toxic even at elevated dosages.  Still, while I’m excited about ARC520 for all the stated reasons (see the HBV Knockdown Blog), for the sake of Arrowhead’s platform, I really would like to see the elegant single-molecule DPCs enter development.  This should not take much longer given the advanced data presented at OTS 2012.

But hey, maybe they have already advanced into development, but they are just being referred to as 'GalNAc-siRNA'.

Alnylam Moves to Increased Dosing Frequency as Knockdown Duration Shortens

The ALN-TTR02 phase II results have finally been announced.  The results are consistent with the view that ALN-TTR02 is a strong candidate for becoming RNAi’s first marketed drug.  Having said that, the knockdown efficacy results came short of what one could have expected based on the phase I and non-human primate results and explain why the company has amended the protocol to test increased dosing frequencies.

Non-human primate studies show cumulative dosing

Alnylam had long telegraphed the results for the phase II studies.  They stated that we should expect the clinical results to be essentially the same as what they are seeing with the drug in non-human primates.  I understand that non-human primates are an immensely insightful model system in RNAi Therapeutics development.  Still, making such strong suggestions just weeks before clinical data release seemed a bit odd in that it risked stealing its own thunder.

Anyhow, the non-human primate studies showed the peak and the terminal knockdown (terminal knockdown defined as the knockdown just before repeat dosing) increased with each additional dose administration.  Whereas the peak/terminal knockdown in cycle 1 was ~85%/47% at 0.3mg/kg, they were ~90%/58% in cycle 2 and so on.  This is what Alnylam refers to as with ‘cumulative’ efficacy.

Unfortunately, this was only partially repeated in humans.

Knockdown not as sustained with repeat dosing

The ALN-TTR02 phase II study represented the first opportunity to rigorously test gene knockdown of a SNALP-delivered RNAi Therapeutic following repeat dosing.  Whereas the phase I study in healthy volunteers was a one-dose study, this phase II study was a 2-dose repeat-administration trial in European patients with TTR amyloidosis (FAP form).  In both studies, target gene knockdown could easily be determined by measuring TTR levels in the blood.

In my analysis of the data, I will stick with slide 10 of Alnylam’s slide set which seems to provide the clearest overview of the data.  At the critical 0.3mg/kg dose level, the peak knockdown either remained +/- the same (~80à80% for once-every-4-weeks) or showed evidence of cumulative activity (~80à88% for once-every-3-weeks) when comparing the first with the second dose administration.  While this looks quite good, albeit somewhat less than what was observed in the phase I study, slide 5), the downside surprise comes with the terminal knockdown values.  Here, the knockdown for cycle one (day 28; n=6) was around 75% with tight error bars, but dropped to 60% for cycle two (day 56; n=5) with much more considerable inter-patient variability.  The same was observed for the 0.15mg/kg dose level with ~53% and ~45% reductions after cycle one and two, respectively.

I have no idea where Alnylam is getting the 93% knockdown from that is so prominently mentioned in the press release.  Are they referring to a single patient that achieved such knockdown?    

Once-every-3-weeks to stay on top of competition

You can safely assume that reduced knockdown duration and the fact that the critical phosphorothioate antisense competitor by ISIS/GSK has a ~70% knockdown have driven Alnylam’s decision to amend the ongoing phase II study by including once-every-3-week regimens.

Data for the first such cohort was provided.  As noted above, once-every-3-weeks showed evidence of cumulative peak knockdown activity thus staying on top of the antisense competition.  It is a shame though that the data provided coincidentally ended on day 35, only a week before the important repeat terminal knockdown point.  It is thus not possible to tell whether diminished terminal knockdown is also an issue for the once-every-3-week regimen.

Safety findings largely related to route of administration  

Safety findings were only reported at the 0.3mg/kg level (4 events in 9 patients).  The infusion-related reaction and the 'nuisance' side effect of fever/chills are known for SNALP delivery, but do not appear to be prohibitive.   Unfortunately, there was a serious adverse event due to a misplacing of the infusion needle.  New to me is the finding of polyuria (peeing a lot). 

Based on this profile, Alnylam will try to lower the amount of immune suppression given with the drug.  Immune suppression is given as a precaution to avoid hypersensitivity reactions seen with SNALP-enabled TKM-ApoB.  It seems to me that in introducing the once-every-3-week regimen, Alnylam wants to dial up the product profile by lessening the burden of the immune suppression.

The future of ALN-TTR02

Given the high unmet medical need for FAP patients, ALN-TTR02 should remain on track of making it to the market provided reduced TTR levels indeed translate into a clinical benefit (a low risk at 80% knockdowns).  Some additional warts, however, have been added to the product profile and my main interest as to the future data will be how efficacy and safety is maintained not after just two, but after 3, 4, 5 doses and so on.  

It would also be good to find an explanation for the diminished knockdown duration (e.g. some neutralizing antibodies or a peculiarity of TTR gene regulation?).  Tekmira, the delivery partner which should receive a $5M milestone on the initiation of ALN-TTR02 phase III studies at the end of this year, would be an important ally in answering this question.  This could therefore be an important test of whether the two companies can put aside past differences.

  

The ALN-TTR02 Phase II Results...Abstract Today, Presentation Tomorrow

The abstracts for the Peripheral Nerve Society Meeting in France have been published.  The abstract on ALN-TTR02 does not reveal the results, but I post them anyway as a warm-up for tomorrow.  You can notice though that there is a relative shift towards the once-every-3-week regimen and the dose escalation started at the same point as the phase I study did (0.01mg/kg).  Check back for more tomorrow.

INTERIM RESULTS FROM PHASE II TRIAL OF ALN-TTR02, A NOVEL RNAi THERAPEUTIC FOR THE TREATMENT OF FAMILIAL AMYLOIDOTIC POLYNEUROPATHY

Adams D1, Coelho T 2, Suhr O3, Conceicao I4, Waddington-Cruz M5, Schmidt H 6, Campistol J7, Pouget J8, Buades J9, Falzone R10, Harrop J10, De Frutos R10, But- ler J10, Cehelsky J10, Nochur S10, Vaishnaw A10, Gollob J10. 1Centre Paris-Sud, APHP, Hopital de Bicetre, INSERM U788, Service de Neurologie, and Centre de Reference des Neuropathies Amyloides Familiales Le Kremlin-Bicetre, France; 2Unidade Clinica de Paramiloidose, Hospital de Santo Antonio, Porto, Portugal; 3Department of Public Health and Clinical Medicine, Umea University, Umea, Sweden; 4Centro Hospitalar Lisboa Norte-Hospital de Santa Maria, Lisbon, Por- tugal; 5Hospital Universitario Clementino Fraga Filho, Rio de Janeiro,Brazil; 6TheUniversityHospitalofM¨unster,M¨unster, Germany; 7Hospital Clinic, Barcelona, Spain; 8Centre de Ref- erencedesMaladiesNaueromusculairesetdelaSLA,Hopital de la Timone, Marseille, France; 9Hospital Son Llatzer, Palma de Mallorca, Spain; 10) Alnylam Pharmaceuticals, Cambridge, MA, USA.

Familial amyloidotic polyneuropathy (FAP) is a fatal, autosomal dominant, multi-system disease caused by abnormal tissue deposition of mutant and wild-type transthyretin (TTR). Almost all circulating TTR is synthesized by hepatocytes, and in FAP this liver-derived TTR is responsible for amyloid accumulation in the main target organs, including peripheral nerves, gastrointestinal tract and heart. Treatment approaches have focused on reduction of amyloidogenic TTR monomer either through elimination of hepatic production of mutant TTR (liver transplantation) or stabilization of the TTR tetramer (tafamidis). While both of these approaches have been shown to slow neuropathy progression in a limited subset of FAP patients with the V30M mutation and early disease, there remains a significant unmet need for new therapies that can impact FAP patients with different TTR mutations across a broader range of disease severity. ALN-TTR02 is a systemically administered lipid nanoparticle (LNP) formulation of a small interfering RNA (siRNA) targeting wild-type and all mutant forms of TTR. This formulation predominantly delivers the siRNA to the liver, thereby inhibiting synthesis of TTR at the primary site of production. In non-human primates, repetitive dosing with ALN-TTR02 every 3–4 weeks at doses up to 0.3 mg/kg resulted in an average relative TTR suppression of up to 90% with no tachyphylaxis of the pharmacodynamic effect. In a randomized, placebo-controlled, Phase I dose-escalation trial in healthy volunteers, a single intravenous infusion of ALN-TTR02 administered over 60 minutes was shown to be safe and well-tolerated, and potently suppressed serum TTR levels by 82-94% at doses of 0.15–0.5 mg/kg. TTR reduction of up to 67% was observed out to 28 days post- dose, suggesting that sustained TTR knockdown could be achieved with a monthly dosing schedule. Based on these promising results, a multi-national Phase II trial of ALN-TTR02 in FAP patients was initiated in mid-2012 to evaluate the safety/tolerability and pharmacodynamic effect of multiple doses of ALN-TTR02. Patients receive two doses of ALN- TTR02 administered every 3–4 weeks at dose levels ranging from 0.01 to 0.3 mg/kg, with a total anticipated enrollment of approximately 27 patients. In this presentation, we will provide an update on the results of this ongoing clinical trial.

Alnylam Reports Potential Advance in SNALP Safety

One concern with the use of any drug is that it ought to be eventually eliminated from the body.  This is a particular issue with nanoparticulate drugs for which efficacy have been the main design criterion.  To address biodegradability for Tekmira's SNALP delivery, a technology that routinely involves un-natural lipids which may not be easily biodegradable, Alnylam has just published on SNALP lipid-derivatives containing readily degradable ester bonds.   The results show that such SNALPs are rapidly eliminated from the body and indicate that their use can increase the therapeutic index.

In the paper, Maier and colleagues took the (once fiercely contested) MC3 lipid, which is part of the ALN-TTR02 formulation for which phase II results are imminent, and tested whether the incorporation of ester bonds at various positions in the lipid tail would retain knockdown efficacy.  The ester bond was chosen because it was predicted to maintain the MC3 lipid geometry and can be degraded by the ubiquitous esterases in the body.  Consistent with the idea that the apparent pKa of the head group is important for efficacy, positions were identified that left the pKa, and consequently knockdown activity in mice largely unchanged (ED50 of <0 .01mg="" kg="" o:p="">

To confirm the prediction that the resultant SNALP particles, economically formulated with microfluidics, were less stable than their parent particles, plasma and tissue elimination rates as well as the generation of turnover intermediates were tested.  Indeed, the new SNALP particles were much less stable.

What is more, at doses of more than 3mg/kg SNALP-siRNA there was a trend towards slight elevations with the MC3 SNALP, but not with a derivative.  The authors stopped short, however, to test concentrations at which much more toxicity can be seen.

The major downside of the study, however, was that in non-human primates, the new SNALPs had less activity than expected for MC3 SNALPs.  At 0.3mg/kg, the dose at which 80-90% knockdown was observed in the phase I study of ALN-TTR02, only a 70% TTR knockdown was achieved.  Nevertheless, it is likely that other biodegradable SNALPs optimized for primates will achieve MC3-type efficacy.    

I do not expect SNALP biodegradability to become an issue for the upcoming ALN-TTR02 phase II results.  The longer-term dosing experience with cancer RNAi Therapeutic ALN-VSP02, however, suggested that there could be spleen toxicity with extended dosing and at higher dose levels.  The last word from Alnylam on that issue had been that the spleen toxicity was due to on-target activity.   The paper by Maier et al., however, leads me to believe that both explanations are still on Alnylam’s table.   

ApoC III Confirmed as High Potential Gene Knockdown Target

Small molecules, monoclonal antibodies, and fish oils move out!  Combining the power of genetics and therapeutic gene knockdown, ISIS Pharmaceuticals presented last night by far the most profound reduction in serum triglycerides which are thought to be an important risk factor in cardiovascular disease and other less common conditions such as pancreatitis: a 72% reduction of serum triglycerides following an 88% gene knockdown of ApoC III with a bonus 40% elevation of the good HDL cholesterol in a phase II study of ISIS-ApoCIIIRx presented at the Amercian Diabetes Association. This confirms in Man that ApoC III antagonizes the metabolism of triglycerides.

The ApoC III knockdown results were not unexpected.  In the preceding phase I study, 71% and 78% ApoC knockdowns were seen at the 200mg/week and 400mg/week dose levels, respectively.  The enhanced, 88% knockdown seen in this phase II study at the 300mg/week level can be explained by the fact that the study drug in the first study was only given for 4 weeks, at which point the phosphorothioate oligonucleotide may not have reached saturation in the liver, whereas in this study it was given for 13 weeks.  

More surprising was the deep 72% reduction in serum triglycerides.  In the phase I studies, 'merely' 43-44% reductions were observed, although this to my knowledge is still superior to e.g. Amarin’s glorified and controversial fish oil.  It is possible that this result is due to a non-linear relationship between ApoC III and serum triglyceride lowering.

Obviously, questions remain unanswered following this small and still early-stage study.  I was surprised to learn that data were reported for only 11 patients with 200 and 500 mg/dL serum triglycerides and type 2 diabetes (the enrolment criteria) although the clinicaltrials.gov entry indicates that 24 was the originally planned number for the blinded, placebo-controlled study.

Secondly, it will be important to learn about the safety and tolerability profile of ISIS-ApoC IIIRx, also in light of the clinical trial experience with the ApoB-targeting, LDL cholesterol-lowering mipomersen (aka KYNAMRO).  To wit, ‘nuisance’ side effects such as injection site reactions and flu-like symptoms contributed to frequent discontinuations in the trials and likely explain what appears to be a very slow uptake after marketing approval.  Of course, Dr. Stan Crooke, the ever-so optimistic CEO of ISIS Pharmaceuticals is convinced that ISIS-ApoCIIIRx has no such issues due to improved screening  methods.  As a reminder, in the phase I study with ISIS-ApoCIIIRx, one out of six injections were associated with injection site reactions.

Thirdly, the link between ApoC III and cardiovascular risk is still debated.  Moreover, similar to ApoB, ApoCIII is thought to contribute to VLDL efflux and inhibiting it may lead to an elevation of liver triglycerides.  This is particularly problematic given that the target patient population is already at an increased risk of hepatosteatosis.

While phosphorothioate antisense company ISIS Pharmaceuticals clearly has a head-start on ApoC III, given the ability of various RNAi technologies to potently knock down genes in the liver, ApoC III is an attractive target for the RNAi Therapeutics industry.  Such a candidate could either be positioned as a best-in-class alternative (à safety; I particularly like here the prospect of a subQ DPC version) or possibly as part of a multi-targeting cocktail against cardiovascular disease (attractive for SNALPs).  It is also one with an attractive partnering potential for some of the smaller companies in the space (early clinical POC, maybe partnering even before clinical development). 

ALN-TTR02 Phase II Results Preview

Although the phase I results for ALN-TTR01 in late 2011 and ALN-PCS02 in first half of 2012 should have caught the attention of the diligent biotechnology investor, it was the highly potent knockdown (>90%) reported for ALN-TTR02 in July of last year that put the spotlight back on RNAi Therapeutics. The results for the TTR amyloidosis drug candidate not only benefited Alnylam, they were a blessing to the entire field of RNAi Therapeutics allowing it to raise ~$300M over the ensuing 12 months.  It is therefore an understatement that the phase II results for ALN-TTR02 to be disclosed in about two weeks will be closely watched across the industry and investors.

Possibly hoping to relieve some of the tension around the event, Alnylam has long telegraphed what we should expect the results from this open-label study to be: just look at the multi-dose non-human primate data and this is what you will see repeated in humans.  Nevertheless, I doubt that the generalists are buying the argument that non-human primates studies are highly predictive of clinical results, at least in terms of gene knockdown.   Just witness the valuations of other RNAi Therapeutics companies that have reported impressive non-human primate data (one could call that a buying opportunity).

Study primarily designed for safety…

The phase II study for ALN-TTR02 is primarily one of safety for repeat administration of ALN-TTR02.  The initial two-dose study design seems an overly cautious approach given that products enabled by Tekmira’s SNALP delivery technology have already been dosed for several months in patients with cancer.  This could be due to differences in how the different divisions within the FDA view the safety of SNALP and/or RNAi product candidates. 

With regard to hypersensitivity reactions, which are probably the most critical hurdle ALN-TTR02 needs to overcome, multi-dosing should actually the lower the incidence of infusion reactions (desensitization) which were the major safety issue in the phase I single-dose study.  With regard to spleen toxicity, multi-dosing is predicted to increase risk (cumulative).

Overall, given the dose (study geared towards the 0.3mg/kg dose) and history of ALN-TTR02, I am quite optimistic that no nasty surprises will emerge on the safety front.  This would also be consistent with plans by Alnylam to reduce the use of immune suppressants in the extension phase of the study.

…but efficacy could drive volatility

Although we have already been told what the efficacy results should look like and that Alnylam is aiming for an 80% knockdown, I expect analysts to pay close attention to efficacy given that Alnylam is in an arms race with ISIS/GSK.  As a reminder, in the 300mg/week regimen that has been chosen for the ongoing phase II/III study, a ~70% knockdown was reported with the ISIS/GSK antisense compound.  However, while a phosphorothioate antisense compound for liver-targeted gene knockdown given weekly has a sustained knockdown effect over time, there are wider fluctuations between peak and trough knockdowns for a SNALP-delivered RNAi Therapeutics administered only once a month.  In other words, watch the time-course of the knockdown, especially after Alnylam announced that it is considering a once-every-3-weeks regimen for the extension phase of the study.

Wall Street ascribes most of Alnylam’s value to TTR amyloidosis program

The reason, of course, why the TTR amyloidosis data is expected to cause major volatility is because two thirds if not more of Alnylam’s market value is based on a discounted cashflow analysis for ALN-TTR02 and ALN-TTRsc in the polyneuropathy and cardiomyopathy markets, respectively.  It is estimated that with premium orphan pricing (you will be surprised how precisely Alnylam’s pharmacoeconomic models will arrive at a fair price of $300,000 per patient year), peak sales revenues for Alnylam’s TTR products will be somewhere between $1.3-$2.0 billion.

I am optimistic that the TTR product candidates will make it to market and dominate over any phosphorothioate-based antisense competitor.  However, there remain important uncertainties as it relates to pricing and reimbursement, especially in some of the European markets where many of the FAP patients reside.  Furthermore, there is always an element of chance when it comes to avoiding prohibitive toxicities from sequence-specific off-targeting in a chronic treatment setting.

Over time, however, it does look like the rest of Alnylam’s pipeline is finally gathering steam.  With the porphyria and AAT deficiency candidates ALN-AS1 and ALN-AAT, respectively, and the intriguing complement inhibitor program, additional differentiated, high-value orphan opportunities are emerging.  It remains to be seen, however, whether the GalNAc-siRNAs are potent enough to exploit the potential of RNAi Therapeutics for these diseases or whether other delivery technologies enabling deeper knockdowns would have been appropriate.  This concern e.g. applies to the hemophilia program where my feeling is that only deep knockdowns will allow you to see clinically meaningful improvements in clotting times.  Unfortunately, I am still trying to find results from clotting assays (which are standard in the field) and not just the thrombin measurements that Alnylam is providing.

Trading the event

As Alnylam's share price has tripled since last year's announcement of phase I results and given that the two-dose design won't do that much for additional de-risking, I am speculating that only in a very bullish, confident biotech market that seizes on every news opportunity there is much upside from the phase II top-line results.  Because of the nervousness that has been creeping into the markets the last two weeks, I am taking a small bearish position ahead of the event, in a way hedged by much larger long positions in other RNAi Therapeutics companies (Arrowhead Research, Tekmira, RXi Pharmaceuticals) which are more attractively valued on a relative basis and should benefit from renewed RNAi Therapeutics investor interest as a result of the data release.

Last reminder that the GTC RNAi Therapeutics conference will start this week.  Don't forget to mention 'RNABLG13' for 20% discount on registration.

RNAi Therapeutics Ebola Candidate Achieves Critical Milestone

Yesterday at the TIDES meeting, Tekmira reported new efficacy data for its Ebola RNAi Therapeutics development program.  The non-human primate data suggest that the main remaining hurdle before regulatory approval for this biodefense agent (on which any government stockpiling contract would hinge), safety in healthy human volunteer studies, is now more realistic than ever.     

Is second time's the charm?

In 2010, Tekmira and collaborators from the Geisbert lab and the US Army reported in The Lancet of a breakthrough in the post-exposure treatment of Ebola infection.  In the gold-standard rhesus monkey model for Ebola infection, their were able to demonstrate that SNALP-RNAi could effectively rescue Ebola-infected animals from near-certain death. 

The efficacy, however, depended on doses of 2.0mg/kg is likely too high a SNALP dose to be considered safe.  Based on the SNALP human clinical trial experience to date, first dose-limiting toxicities are observed at 1.0mg/kg (note: SNALP potencies are very comparable between humans and monkeys).  Moreover, at doses higher than 0.5mg/kg, sporadic signs of immune stimulations (flu-like symptoms) can be observed.  These caused Alnylam and Tekmira to adopt precautionary transient immune suppressions with corticosteroids in some of their other SNALP-based clinical studies.   

It therefore became important to take advantage of the improvements in SNALP potencies and develop more potent Ebola SNALP-RNAi candidates.  Fortunately, Tekmira was able to get the blessing from the US Department of Defense for this, and announced in April that it would receive up to ~$7M in additional funding on top of the up to $34M initially agreed upon in order to bring such a candidate to the end of phase I clinical studies. 

Importantly, since Ebola efficacy trials cannot be conducted in humans, the regulatory approval of TKM-Ebola will be according to the Animal Rule.  The Animal Rule states that for indications where human efficacy trials are not possible due to ethical considerations, it is sufficient to show efficacy in accepted animal models and then establish drug safety at corresponding doses in healthy human volunteer studies.  

With the new formulation, 12 out of 12 monkeys treated with the magic dose of 0.5mg/kg RNAi stayed alive in a post-exposure treatment setting, compared to only 1 out of 6 treated with placebo.  This means that should the human volunteer trials show good safety at the same 0.5mg/kg dose level, Tekmira will stand a good chance at gaining regulatory approval under the Animal Rule.  Of note, the first Ebola trial were likely conducted in the absence of corticosteroid pre-treatment and it is very likely, also in light of Alnylam's recent announcement of tapering off corticosteroids in the ALN-TTR02 trial, that the future TKM-EBOLA studies will also be conducted without them.

Since 0.5mg/kg is right on the border, however, there still remains an element of risk before victory can be declared on the safety front.  It is therefore good to hear that 4 out of 6 monkeys stayed alive at the 0.2mg/kg dose. This should provide a good safety cushion despite the aggressive daily treatments that will be required in this demanding setting.

Critical human safety data, ideally from repeat-dosing studies, should be forthcoming in 2014 as Tekmira has guided that it will commence clinical studies with the new candidate by the end of 2013.

Update 16/5/2013: One issue on the efficacy side of the equation that I forgot to mention in the original post is that the DoD might ask the company to test TKM-EBOLA with longer delays after the infection in monkeys.

SNALP Shows Good Potency with Subcutaneous Dosing

Also at the TIDES meeting, Tekmira presented efficacy data for SNALP when given via the subcutaneous route of administration.  To wit, SNALP has largely been administered intravenously which may not be suitable for a number of indications, especially the lower risk, chronic diseases.  Pfizer once famously criticized the state of RNAi delivery as being limited to intravenous options.

Comments like these (which are very relevant for partnering purposes) and trends in the industry to adopt subQ dosing, most notably Arrowhead’s DPCs and Alnylam’s GalNAc-conjugates, probably motivated Tekmira to explore this route of administration with SNALP further.

 

Based on an early ApoB patent application, Tekmira had clearly had some familiarity with subQ, and I had always wondered why the company did not pursue this further.  It could be that the more potent SNALP chemistries now allow for much more potent and robust subQ RNAi gene knockdown.

The new data show a very impressive 96% knockdown of transthyretin (TTR) with a single subQ administration of 1.0mg/kg SNALP in rodents.  This compares to a much more modest ~55% knockdown with ALN-TTRsc at 1.0mg/kg given on 5 consecutive days, the subQ GalNAc-siRNA candidate by Alnylam which just entered clinical development for the FAC form of transthyretin amyloidosis.

Of course, we need to learn more about the safety of subQ SNALP to know whether it really is a clinically viable strategy and superior to the competition.  GalNAc-siRNAs in particular look quite benign, albeit not very potent.  However, since peak plasma concentrations seem limiting for SNALP in terms of safety (infusion reactions can be managed by slowing the infusion), subQ, due to its slower systemic release might actually turn out to be safer, not just more convenient.  On the other hand, my impression is that subQ SNALP could cause the type of ‘nuisance side effect’ that is frequently seen with phosphorothioate antisense.

The GalNAc comparison is obvious since Alnylam developings both a SNALP- and a GalNAc-based TTR RNAi Therapeutics in parallel.  In terms of competition for potential partnering, however, the DPCs by Arrowhead Research would be the more appropriate and challenging comparison.  I look forward to watching the race unfold as healthy competition is propelling the RNAi Therapeutics field into a highly competitive industry.

Antisense Therapeutics Dressing Up with RNAi Delivery

The boundaries between RNAi Therapeutics and Antisense Therapeutics have become blurrier.  Originally, the basic practical difference between RNAi and antisense was as follows: on the one hand, RNAi is very powerful, but it requires special formulation chemistry which often is the tox-limiting factor and necessitates that the drug be administered intravenously.   On the other hand, antisense can be utilized in unformulated/naked form and be administered subcutaneously, but it is less potent and involves the accumulation of large, problematic levels of phosphorothioate molecules in a range of tissues, primarily the kidney, liver, spleen, and probably the vasulature.

   

Recently, however, some RNAi approaches have become more antisense-like in terms of patient convenience in that conjugate technologies and ‘self-delivering’ RNAi triggers that can be administered subcutaneously have advanced into the clinic.  Over the last two weeks then, two pieces of evidence emerged of the reverse trend of antisense technologies adopting delivery technologies originally developed for RNAi Therapeutics.  The is done for improved potency and could also bring critical safety advantages.

Evidence 1: ISIS and Alnylam Find SNALP Delivery to Increase RNaseH Potency by ~20-fold

In a paper by Prakash et al. in ACS Chemical Biology, scientists from ISIS Pharmaceuticals and Alnylam demonstrate significant potency enhancements for RNaseH antisense and single-strand RNAi to be gained from the use of Tekmira’s SNALP technology.  As a reminder, the two companies once collaborated on single-strand RNAi before Alnylam terminated that relationship with ISIS continuing work on the subject, so the present publication relates to historical work during their partnership, but is relevant to ISIS' continued efforts.

The study simply compared the knockdown potencies of RNase H gapmers (phosphorothioate 2’ MOE chemistry), single-strand RNAi triggers (heavily modified), and dsRNAi triggers (ssRNAi molecule as guide together with complementary unmodified passenger strand), either as unformulated oligonucleotides or when formulated with SNALP containing the DLin-KC2-DMA lipid.  This lipid, developed by Old Tekmira (see Semple et al. 2010, Nature Biotech), was a critical ingredient of the first ‘2^nd-generation’ SNALP showing significantly improved liver knockdown potency over the original DLin-DMA SNALPs. 

When targeting PTEN in mouse livers, none of the oligonucleotides alone exhibited knockdown activity at the 4.5mg/kg dose tested.  Only when increasing the dose up to 100-200mg/kg did the RNaseH gapmer and ssRNAi trigger exhibit marked knockdown.

   

In stark contrast, profound knockdowns were achieved with SNALP-formulated RNaseH gapmer and the ssRNAi trigger at the 4.5mg/kg dose.  Remarkably, for the RNaseH gapmer, the activity was obtained at liver oligonucleotide levels that did not differ much from that of the unformulated oligonucleotide (which did not exhibit PTEN knockdown).  This means that SNALPs not so much increases the delivery to the liver per se, but the functional delivery of RNaseH gapmer, possibly by liberating phosphorothioate oligos along the endosomal cellular uptake pathway.  This finding also makes it difficult to believe some reports that phosphorothioate oligos administered without special formulation accumulate in the nuclei as this is the site where they are supposed to be mostly active.  Where the non-functional oligos actually end up is an important question as this also relates to the safety of these molecules.

Overall, the study shows that formulation with RNAi delivery technologies can greatly enhance knockdown potency.  It should be added here, however, that this single-dose study somewhat underestimated the potency of RNaseH antisense as normally a loading dose schedule would be performed to reach threshold tissue levels required for knockdown activity of unformulated PS-RNaseH gapmers.

The lower dosages and more targeted delivery with RNAi delivery may also reduce the typical toxicities associated with phosphorothioate antisense.  Indeed, one may even be able to do away with the phosphorothioate modification altogether.  Of course, toxicity related to the delivery vehicle also needs to be considered, but the authors noted in their paper that SNALP was ‘well tolerated’ and that no changes in liver enzymes or in body weight loss were seen.

To further gauge the value of SNALP and RNAi delivery in general for gapmer antisense technology, a more detailed time-course analysis of the knockdown and the tissue concentrations would be useful.

Evidence 2: Regulus Therapeutics to Employ Alnylam GalNAc-Conjugation for Mir-122 HCV Program

In another example of the value of delivery technology originally developed for RNAi Therapeutics for single-strand oligonucleotide Therapeutics, Regulus Therapeutics disclosed at last week’s Needham conference that it would use Alnylam’s GalNAc conjugation for the delivery of anti-miR122 in its HCV program which is to enter clinical development in 2014.

With this, Regulus expects to achieve once-monthly-dosing with their single-stranded anti-miR at lower doses (0.3mg/kg) than the weekly LNA-based phase II compound by competitor Santaris (>1mg/kg).  Importantly, Regulus claims that HCV patients go to see their doctors to check their antiviral responses on a monthly basis anyway.  Consequently, these routine visits for patients on anti-HCV therapies could then be combined with the subcutaneous administration of the anti-miR.  This obviously would increase compliance in an era in which oral HCV meds reach high cure rates in clinical trials, but the actual cure rates in the field are significantly lower due to rate-limiting compliance issues (h/t to John Alan Tucker from Zacks Investment Research).   

Without showing the data, it remains to be seen whether and by how much GalNAc conjugation improves anti-miR potency.  The once-monthly dosing, however, indicates that once the single-stranded oligonucleotide is in the cytoplasm (where the target is), it is trapped and stays there for quite some time instead of being washed out again into the interstitial space (outside the cells) which would be the case if extracellular and cellular oligonucleotides were in dynamic equilibrium.     

It will be interesting whether the GalNAc approach will utilize the phosphorothioate backbone which ISIS Pharmaceuticals is using at all.  If not, the decision would indicate that ISIS satellite company Regulus is concerned about the safety implications of that chemistry, especially in patients where the liver is already diseased.