Wave Life Sciences to Focus RNA Editing on Gene Upregulation

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

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

 

Mutations often scattered across genes

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

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

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

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

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

 

mRNA technology

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

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

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

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

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

 

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

 

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

Reconsidering RNA Editing for Alpha-1-Antitrypsin Disease

Alpha-1-antitrypsin (AAT) is the lead program in the RNA Editing pipeline with Wave Life Sciences recently licensing its candidate to GSK.  It is safe to assume that a number of other companies are working on similarly developing RNA Editing drugs in the AAT area as well.  Developments in the competitive environment using other platforms such as gene knockdown (RNAi, ASO) and genome editing are therefore of high interest.

Arrowhead Pharmaceuticals SEQUOIA data

This week, Arrowhead Pharmaceuticals and partner Takeda revealed that its RNAi candidate for alpha-1-antitrypsin-related liver disease worked as intended in the SEQUOIA phase II study and over time should translate into a measurable benefit in terms of developing severe liver disease.

After 52 weeks of treatment, mutant Z-AAT was knocked down by a solid -94%.  This in turn led to a 2/3 decrease in the more inert aggregated form of alpha-1-antitrypsin.  It is the aggregated AAT that is thought to cause cellular stress, followed by a cycle of apoptosis and hepatocyte regeneration, ultimately leading to inflammation and fibrosis, if not liver failure in ~20% of subjects homozygous for the Z-allele of alpha-1-antitrypin.  

Indeed, ARO-AAT/TAK-999 achieved a striking benefit of reducing portal inflammation (see above graph) which should be highly predictive of a benefit on liver fibrosis progression.  Although a high 50% of trial subjects on ARO-AAT experienced a reduction in fibrosis, this was not statistically significant due to small patient numbers.  A larger and longer (2-4 years) registrational study is therefore planned to confirm the benefit and gain regulatory approval.

RNAi unsurpassed in reducing Z-AAT, but RNA Editing may be more elegant

If the degree of reducing the insulting Z-AAT was all that mattered, RNAi would be difficult to beat. ADAR RNA Editing and genome editing for example would have to overcome the limitation that target sequences are limited to a very specific area around the mutation whereas RNAi has the entire mRNA to play with.

But looking more closely, the name of the game is actually inhibiting aggregation.  It is now that I increasingly appreciate the elegance and power of the RNA Editing approach   which fundamentally rests on the genetic observation that heterozygous carriers of alpha-1-antitrypsin disease have a much reduced risk of developing related liver disease, if an increased risk at all.  These people carry one 'Z' allele and one normal ‘M’ allele. 

I had not paid much attention to this, thinking that mechanistically this is simply due to a concentration effect of 50% less Z-AAT polymerizing less well and having a protective genotype that manifests after decades of life does not necessarily mean that converting a patient already suffering from liver disease to such a genotype will necessarily translate to the fastest possible treatment benefit.  RNAi with its highly potent Z-AAT reduction may be faster.

It is now that it dawns on me that there is likely more to the MZ genotype benefit.  Looking at the Wave Life Sciences mouse preclinical data on its RNA Editing candidate, it always irritated me that whereas the mRNA editing efficiency was ~50%, the increase in circulating AAT was 7x.  Since only ~15% of Z-AAT normally gets out of the liver, this basically means that the 50% of M-AAT allows essentially ALL of the AAT, including Z-AAT to get out of the liver.  This is consistent with the ratio of M-AAT/Z-AAT in circulation reflecting the 50% mRNA editing efficiency in hepatocytes.

It therefore appears that the M-AAT (and potentially non-wildtype variants generated by RNA Editing) functions to poison the Z-AAT aggregation chain reaction, keeps Z-AAT in solution and fit for secretion into circulation.  What is more, unlike RNAi, this has the additional benefit of also addressing the lung manifestations due to insufficient levels of circulating AAT and for which alpha-1-antitrypsin disease is better known.  RNAi by contrast may have to rely on other developments in improving the management of alpha-1 lung disease as it reduces circulating AAT even further.

In light of the above, as a shareholder I am now changing my mind and support ProQR with its industry-leading IP and know-how to enter the exciting alpha-1 race to generate a best-in-class RNA Editing drug.  We should soon find out when ProQR will reveal its line-up of initial pipeline candidates.

GSK Partners with Wave Life Sciences for Access to RNA Editing

This week, we have seen further confirmation of the increasingly recognized value within the pharmaceutical industry of Oligonucleotide Therapeutics in general, and RNA Editing in particular.

In a landmark deal, GSK obtained an exclusive license from Wave Life Sciences to the RNA Editing industry’s lead, albeit still preclinical WVE-006 development candidate for the treatment of alpha-1-antitrypsin disease.  In addition, GSK has the right to evaluate Wave’s oligonucleotide platform (editing, splice modulation, and RNAi/ASO silencing modalities) to then advance up to 8 programs into development.

In return, Wave will receive $120M in upfront cash, another $50M in an equity investment, and the potential to earn up to $3.3B in development and commercial milestones in addition to royalties on drug sales.  Because of its more advanced stage in development, WVE-006 stands to earn relatively more in milestones ($525M) and royalties (tiered double-digit, up to the high teens).

While I view Wave doing this deal largely to feed its voracious appetite for cash to feed what I consider to be less exciting clinical work in Huntington’s (ASO knockdown) and Duchenne muscular dystrophy (exon skipping), GSK will bring its genetics-based target insights to the collaboration table so that Wave could advance up to 3 related programs that it would wholly own.

Seeing the AATD program go to GSK was a disappointment to me at first.  Ultimately, I thought that this program would end up shouldering the weight of Wave’s market cap as the company’s lead program once the current clinical pipeline will meet its expected fate.  However, during the discussion of the deal the company’s CEO Paul Bolno made it clear that not only is GSK much better suited to advance ‘006 especially with regards to its lung-related endpoints, it is RNA Editing and gene upregulation that Wave considers the most valuable elements of its PRISM oligonucleotide platform and that it wants to maintain control over.

Gene upregulation can be achieved by either masking destabilizing sequences in an (m)RNA by antisense oligonucleotide, or by using RNA Editing to disrupt those or slightly change the protein to make it more stable.

After the 2021 deals between Shape Therapeutics andRoche (neuroscience, DNA-directed RNA Editing) and ProQR and Eli Lilly, this marks the third such Big Pharma deal in the ADAR sector.  It is reminiscent of the 2004-5 phase when Big Pharma started to take note of RNAi through a few measured investments.  

Expect the noise and excitement to grow over 2023 as RNA Editing approaches the clinic.  But unlike RNAi, a lot of delivery work has already been undertaken so that the trajectory of RNA Editing should be smoother from a technology point of view.  Only yesterday, Avidity Biosciences reported on the  expansion of the targetable tissue universe to the muscle and Arrowhead Pharmaceuticals is about to report important data on targeting RNAi to the lung.

HBV RNAi 2.0

Gene knockdown, in particular RNAi and RNaseH antisense, holds great promise in the treatment of hepatitis B viral infection.  It is currently the only practical means to potently inhibit all viral gene products*.  It therefore is poised to become a cornerstone of future treatment regimens aiming at functionally curing HBV, an infection predisposing more than 200 million patients worldwide currently to liver failure and cancer.

Arrowhead- lessons learned

Unfortunately, the field took a big hit last year when HBV RNAi trailblazer Arrowhead Pharmaceuticals had to abandon its efforts due to preclinical toxicity resulting from its particular approach to releasing the RNAi triggers into the target cell cytoplasm (monkey deaths due to the DPC).

Nonetheless, after more than a dozen trials in WoMan, the company had learned a great deal about HBV and how to best tackle it by RNAi.  Chief among those lessons were the observations that RNAi can suppress viral genes, most notably the surface antigens HBsAg by sometimes more than 2-3 logs.  Moreover, in HBe-antigen negative and those HBe-antigen positive patients previously exposed to polymerase inhibitors (‘nukes’), Arrowhead painfully found that most HBsAg is derived from host genomically integrated HBV.  Consequently, RNAi trigger target sites placed downstream of the HBsAg ORF may be lost and RNAi rendered futile (ARC520àARC521 transition). 

Finally, consistent with the experience with nukes and interferons, it appears that RNAi treatment success (functional cure) should follow complex viral and host immune dynamics and while intriguing changes were observed in the clinic with ARC520/1 (e.g. new lower baselines following treatment cessation), it remains unclear how long an RNAi agent would have to be given.  This has implications for whether intravenous routes of administrations are practical or not.

The competition

Arrowhead Pharmaceuticals, however, has not been the only RNAi game in town developing HBV therapeutics.  Its main competitor in terms of scientific prowess has been Arbutus Biopharma (renamed from Tekmira after biotech wonder boy Vivek Ramaswamy of Axovant fame spectacularly raided the company in 2015).  Its lead RNAi candidate ARB-1467 comprises of 3 RNAi triggers which are formulated in lipids (LNP) and is given intravenously alongside steroids.

While I like the 3-trigger strategy for pangenotypic coverage and for minimizing the risk of the virus developing drug resistance (including by genomic integration), the more cumbersome intravenous route of administration- now reduced to short 2-week intervals in an effort to increase potency- and the steroids makes ARB-1467 uncompetitive in a world of more potent and less frequent simple subcutaneous competition.  The use of immune suppressive steroids, of course, in HBV patients is a dicey proposition and would also seem to run counter to the ultimate aim of achieving immune control of the virus.

Unless it turns out to promote a functional cure along with other agents in short order, say less than 6-12 months, ARB-1467 will likely end up as a science project without much clinical impact.

RNAi powerhouse Alnylam Pharmaceuticals meanwhile is the third RNAi company that has begun clinical development of an HBV RNAi agent.  Importantly, it has been the first company using a simple subcutaneous GalNAc-conjugate format, therefore positioning it to be useful even when more prolonged treatment will turn out to be necessary.

After review of the program, however, it appears that the company prematurely rushed the single trigger ALN-HBV into the clinic without thinking too much about resistance issues.  To start with, ~2% of tested HBV genotypes have mismatches with the trigger that mitigate targeting efficiency.  As a single trigger candidate, ALN-HBV will also have to be given alongside highly potent replication inhibitors (nukes) as one can easily see how ALN-HBV resistant genotypes would otherwise eventually take over.

ALN-HBV moreover targets a site downstream of the HBsAg ORF, around the DR2 repeat element with marks the integration hotspot that has bedeviled ARC-520 before.  While Alnylam has been going around claiming ALN-HBV doesn’t suffer from ARC-520-type issues, I would challenge them with two points:

1)     under selection pressure by ALN-HBV to maintain HBsAg expression and thus evade host immune detection, the virus may ‘choose’ to break up upstream the ALN-HBV target site without affecting the HBsAg ORF; 

2)      more troublesome, closer inspection of the very paper Alnylam points to for making its claim (Jiang et al. Genome Research 22: 593) and which analyzes HBV integration hotspots, shows that ~40% of DNA break points appear to be upstream of the ALN-HBV target site (compare ‘position 1600’ below).      

One can therefore easily see why the project leader behind ALN-HBV, Laura Sepp-Lorenzino, has recently left the company to join Vertex Pharmaceuticals.  One has to get the impression that ALN-HBV, just like ALN-GO1 have only been rushed to the fore as a front in order to keep a lid on their competition by creating doubt about the ability of Arrowhead Pharmaceuticals and Dicerna, respectively, to compete with juggernaut Alnylam. 

Thrilled to join @VertexPharma as Head of Nucleic Acid Therapies!!!

— Laura Sepp-Lorenzino (@LauraSepplore) September 23, 2017

 

It is yet another lesson that in drug development, a detailed understanding of the disease is as important as the technology used to tackle it.  Half-hearted side projects typically lead nowhere.

Lastly, I would be remiss if I did not mention the RNaseH antisense efforts by Ionis along with partner GSK, and those of Roche.  Ionis/GSK are not only developing an unconjugated fully phosphorothioated antisense molecule, which I believe has little chance of competing in the market due to predictable safety and potency issues, but also a more interesting GalNAc-conjugate version (IONIS-HBV-LRx).  Although I currently see RNAi ahead of antisense in gene knockdown in hepatocytes (potency, frequency of administration, and safety), the GalNAc-conjugate version potentially has the advantage of also being able to access the pregenomic RNA directly, while direct pgRNA cleavage by RNAi of this non-mRNA remains to be shown.  My prediction is that  while RNAi can to some degree access pgRNA, this is not as effective compared to its cleaving mRNA.  What all of this means biologically remains to be seen.

Arrowhead HBV RNAi 2.0

Therefore, after all the drama and competitive noise, Arrowhead is poised to recapture the HBV RNAi lead with its new GalNAc-based candidate.  ARO-HBV is poised to enter the clinic in the first half of 2018.   It is subcutaneously administered and involves 2 RNAi triggers that are claimed to cover the viral resistance bases, including HBsAg derived from genomically integrated HBV.

The company expects the agent to be used once a month or less frequently, an attribute valuable should functional cures take longer to emerge.

Given knowledge leadership in HBV gene knockdown and prior practical experience, Arrowhead should also be able to navigate through the HBV clinical development maze faster than its competition.  As can be seen from its resurging stock price, this view is also shared by an increasing number of investors. 

Arrowhead has paid for taking some short-cuts when it was compelled to push ARC-520 into the clinic to give it a shot of becoming a serious player in RNAi before it ran out of capital.  It took some risks and failed, but that failure could well be the soil from which future success will emerge. 

Disclosure: long Arrowhead Pharmaceuticals

 

 

 

 

 

 

* it is unclear whether RNAi can directly cleave pregenomic RNA, whereas RNaseH antisense should be able to do that.

Ocular Applications Back in the Focus of Oligonucleotide Therapeutics

Following yesterday's disclosure that yet another one of GSK’s target picks for clinical development under their antisense options agreement with ISIS Pharmaceuticals is an ocular one, I thought it worth highlighting that ocular applications are regaining traction in oligonucleotide therapeutics in general.  This follows a temporary lull in the area due to setbacks with older generations of the technologies and funding issues for the industry.

Aptamers still in the lead

It may surprise you, but the eye is the one area in oligonucleotide therapeutics where aptamers, nucleic acids binding protein targets based on their shape not sequence (similar to antibodies), are most advanced.  Despite of the fact that the first approved aptamer, Macugen, is considered a great disappointment as it lost out to the monoclonal antibody competition in the VEGF market for wet AMD and DME, there are at least two new development candidates that are poised to become blockbusters in the same market: Fovista by Ophthotech targeting PDGF which has shown unprecedented activity in a phase II study in combination with anti-VEGF antibody Lucentis, and an earlier-stage, but potentially superior VEGF/PDGF bispecific aptamer approach by privately held SomaLogic.

It is now thought that the Macugen failure was due to it not targeting the relevant VEGF isoforms.  In other words, it was a failure of target selection/biological insight, not a failure of the technology.  Aptamers should work well for trapping extracellular proteins for ocular applications, because unlike their often rapid elimination following systemic administration, they can be maintained at elevated concentrations in the eye for sustained periods of time.  Their limitation, however, is in the number of targets available to them, similar to monoclonal antibodies.  Nevertheless, it should be kept in mind that with even just 2 or 3 commercial successes in a therapeutic area, a platform technology can be considered tremendously valuable there.

Gene-regulatory oligos catching up

Although ocular drug development has also been popular in both the antisense and especially RNAi fields, previous technology generations were inadequate to effect robust gene modulation, especially target gene knockdown.  This holds true for 1^st (à Vitravene) and 2^nd generation (cRaf inhibitor by iCo Therapeutics) antisense and the ‘naked’ RNAi trigger folly of the early days of RNAi Therapeutics (à Acuity Pharmaceuticals, Sylentis, Quark, and Sirna/Allergan to name just some of the worst offenders of sound science).

The reason why antisense and RNAi are both staging a comeback in ophthalmology is due to the use of higher affinity chemistries (e.g. cET by ISIS) and self-delivering RNAi triggers, both in the form of (partially) double-stranded (e.g. sd-rxRNAs by RXi Pharmaceuticals) and single-stranded RNAi triggers (à ISIS Pharmaceuticals).  The increased stability and lipophilicity combined with small molecular size should allow such an RNAi approach to efficiently penetrate the vitreous of the eye following needle injection and reach deep into the retina and other ocular structures.  Similarly, what used to be a mediocre 40% knockdown for ASOs could now be a genetically much more useful 70-80% knockdown with gen2.5 RNaseH.

It is too early to tell whether RNaseH gen2.5, ssRNAi, or sdrxRNAs will win out in the end.  At least in terms of timing, it will be as much a matter of investing in the technologies as it is about their potential.  In particular, I am disappointed by the failure of RXi Pharmaceuticals to recognize the need to further develop their sd-rxRNA chemistry.

So keep your eyes peeled as clinical results from the new wave of gene-modulating Oligo Therapeutics will start to emerge in 2016 and beyond.  It is possible that QPI-1007 by Quark Pharma for ocular neuroprotection for NAION may be earlier than that, although the chemical nature of this ‘2^nd generation’ non-AtuRNAi trigger remains unclear to me and therefore might be, or might not be a 'self-delivering' RNAi trigger.  If not this one, the upcoming clinical development of CTGF-targeting RXI-109 for retinal scarring by RXi Pharmaceuticals should be an interesting one to follow.

ISIS/GSK and Tekmira Come Out with HBV Knockdown Plans

If you did not appreciate the value the pharmaceutical industry has come to place on the HBsAg knockdown concept for achieving a functional cure for chronic Hepatitis B (HBV) infection, the last two days will have woken you up. 

Yesterday, ISIS Pharmaceuticals reported that it had received a $7M milestone payment related to the development of an antiviral RNaseH development candidate (ISIS-GSK3Rx, aka ISIS-HBVRx) which, although undisclosed for competitive reasons, hasgot to be for HBV.  And today, Tekmira publicly announced that they will file an IND for an HBV-RNAi candidate in 2014 while hinting at the partnering potential of such a treatment candidate.

Arrowhead Research is thus not alone in their efforts any more.  Coincidentally, Arrowhead reported today the completion of their enrollment of the phase I single-dose, healthy volunteer study with ARC520, their DPC-delivered candidate for chronic HBV.  Accordingly, the dose escalation was able to run through all the pre-planned 6 dose cohorts up to the top dose of 2.0mg/kg. 

Apparently, there were no signs of significant dose-related toxicities.  The only finding of concern among the 36 volunteers, 24 of which received drug, was 2 cases of lightheadedness of uncertain clinical relevance.  As these occurred at the highest dose, it seems that the company suspects that it could have been drug-related although the study remains blinded for follow-up.

A dose of 2mg/kg without any serious adverse events or dose-limiting toxicities is a great start for DPC delivery technology.  This is especially the case when one considers that the single-molecule subQ version of DPC that I hope will form the basis for the upcoming pipeline candidates, except for the next one perhaps, will be much more potent than the two-molecule version of intravenously delivered ARC520 based on the non-human primate data presented at last year's OTS meeting.

With 2mg/kg of ARC520, I further believe that HBsAg knockdowns of over 90% are likely.  The biggest challenge going forward with this program will be setting a knockdown goal and getting the dose and dose frequency right.

For more about increasingly lively HBsAg knockdown for the treatment of chronic HBV, please follow my HBV Knockdown Blog.

Also today: Tekmira and Arrowhead Research Rapidly Filling Pipeline

In addition to chronic HBV, Tekmira further disclosed development plans for candidates addressing Marburg infection, alcoholism, hypertriglyceridemia and severe orphan glycogen disorders.  The presentation made clear that the company has not sat on its hands since the settlement with Alnylam and is close to having at least 4 drug candidates in active clinical development by early 2015 (TKM-PLK1, TKM-EBOLA, TKM-ALDH2, TKM-HBV).

Meanwhile, previously $200M market cap Arrowhead Research has succeeded with a $60M private placement without having had to offer a discount.  In my opinion, this reflects the broadened investor interest in the space and makes Arrowhead an even better investment as it can now, freed from financial constraints, immediately pursue some of the attractive liver targets with best-in-class solutions and beyond.

Wondering whether we are in an RNA Therapeutics bubble?  Then I suggest you get my OTS 2013 meeting report.  Dare I say that oligonucleotide therapeutics is a more capable and exciting technology than monoclonal antibodies already?  At least if you believe orphan diseases are the way to go in today's pharmaceutical drug development, there is no doubt about it coming out of the meeting. 

RNAi and Antisense Targeting the Same Gene: Not a Zero-Sum Game

Transthyretin-mediated amyloidosis has become the single-most important factor for the 3-5 year valuation of certainly Alnylam, and possibly ISIS Pharmaceuticals as well. After TTR, Z-alpha-1-antitrypsin (Z-AAT) is gearing up as the next Alnylam(RNAi)-ISIS(antisense) battleground. Expect to hear competitive language from the two camps why their approach will prove superior over the other.  It is, however, worth keeping in mind that having two candidates race towards approval can also have significant pie-enlarging benefits.

Raising Awareness and Pressuring Regulators

A good competition will attract attention, from patient groups, physicians, regulators, and the investor community.  Patient groups and physicians will be primed that after years of lacking treatment options, the time for change has come.  We have seen this with the trifecta ofweight-loss drug candidates, two of which have recently been virtually willed into approval.  Given the enormous problem of obesity, it became politically untenable to further withhold treatment options.  Eventually, the regulators were forced to give in to the pressure despite their original objections over safety.

Closer to home, similar awareness and pressure is being brought to bear on getting exon-skipping oligonucleotides approved for the treatment of Duchenne Muscular Dystrophy, stoked by the competition between Prosensa/GSK and Sarepta .  If you caution about hurried, aka accelerated approval, then you are readily labeled as a heartless misanthrope.  Similarly, the homozygous familial hypercholesterolemia population has already been educated, targeted, and friended by both patient-access specialist Genzyme and competitor Aegerion and probably strongly expect the new drug approvals.  

Understand that these comments are not meant to be judgments on the respective drug candidates, but just the way I view current dynamics.  In fact, I agree that developing drugs for many of the rare and severe (orphan) diseases requires more risk-taking, usually biomarker-based approaches, and that not too long ago patient needs were not sufficiently considered in a highly risk-averse regulatory climate.

The TTR Amyloidosis Race

Given that Pfizer’s drug candidate tafamidis does not appear about to revolutionize the treatment of TTR amyloidosis, I see similar pressure building for the approvals of gene knockdown approaches ALN-TTR02 and ISIS-TTRRx in 2015-6.  The intense competition between ISIS and Alnylam will provide good fodder for the press (just as it does for this blog), engage the minds and pad the pockets of key opinion leaders and consequently raise the expectations of the public and hopes of those suffering from the disease.

While I have 2015-6 approvals baked into my projections of the RNAi Therapeutics story, I wouldn’t mind a year’s delay if the competitive dynamics would allow for it.  In fact, I expect the FDA to manipulate the process in a way that both drug candidates will come before an AdCom panel together, making the obsession with speed over good science even more so nonsensical.

My problem with ISIS and GSK speeding straight into a phase III study after a single phase I study is that they do not seem to have identified a suitable dosing regimen: 400mg/week good efficacy (-81% knockdown), but probably too high a dose from what we know about the safety of phosphorothioate oligonucleotides; 200mg/week could fare better in terms of safety, but has shown only modest knockdown efficacy (-44% knockdown).  In my opinion, another dose-finding study would be in order under normal circumstances.

[Update 29Nov2012: The Clinicaltrials.gov entry for the upcoming phaseII/III ISIS-TTRRx trial shows that the companies have chosen the stab-in-the-dark 300mg/week dose.  Although the January 2012 phase I results press release made no mention of such a cohort, the June 2012 Annual Shareholder Meeting TTR poster shows data from a 300mg cohort (~70% knockdown).  Unlike all the other cohorts were 90+ day data, the 300mg cohort data was less than 50 days.  It thus looks like a 300mg cohort was included post-hoc. Unfortunately, ISIS has chosen not to list the phase I trial on clinicaltrials.gov.]

This concern over uncertain dosing schedules also applies to Alnylam's ALN-TTR02 (-87% peak knockdown, -67% knockdown after 28 days following single dose) where the current ‘multi-dose’ phase II study only seems to cover two doses.  You would hope that Alnylam presses hard to amend the study to include further doses to gain more experience with the repeat-dosing pharmacology of ALN-TTR02 before betting their house on a single pivotal phase III trial.

Once approvals are obtained, however, the beneficial aspects of competition should disappear quickly especially when small orphan disease populations are involved.  But until then, the race towards approval should free energies allowing TTR amyloidosis to become the first real commercial success story of either RNAi Therapeutics or RNaseH antisense.

Who do you think will win the TTR race? Take part in the survey (upper right hand corner).   

The Mipomersen Briefing Docs: Gymnotic Delivery Revealed

After Alnylam’s CEO John Maraganore famously failed to deliver on his Big Pharma platform partnership promise in 2009 (aka ‘SRTL’), and ISIS Pharmaceuticals announced a preferred partnership with GSK in March 2010, it seemed that Dr. Maraganore salesmanship was outdone by one of the best biotech salesmen ever: Dr. Stanley Crooke. 

In addition to likely having deprived Alnylam of a deal, what is this salesmanship claim based on?  It is the fact that not all that long ago, antisense technology had fallen out of favor in Big Pharma.  This was because it required large amounts of oligonucleotides and, partly as a result of that, was associated with non-specific immunostimulatory effects.  And this is where the more elegant RNAi Therapeutics was supposed to take over. Despite this poor track record, the technology managed a comeback, ironically partly on the back of RNAi Therapeutics which was seen stumbling at the ‘delivery’ stage.  In some cases out of the frustration of making RNAi delivery work, the 'naked' delivery approach of antisense would be embraced by the likes of GSK, BiogenIdec, Pfizer, Genzyme, and VCs making the application of Santaris' LNA technology the basis of start-up biotechs targeting emerging classes of non-coding RNAs such as microRNAs or long non-coding RNAs.

The reason, however, why I had always favored RNAi as the more ‘natural’ gene silencing modality over antisense is that antisense, to this day, relies on achieving extremely high steady-state tissue concentrations (>100-300mg/kg of liver and kidney tissue e.g., see ISIS TTR patent application US2011/0294868) either by using reactive/sticky chemistries such as the phosphorotioate backbone or more gentle backbones such as the morpholino chemistry that, however, require even larger amounts of oligonucleotides.  

Mipomersen Briefing Docs Surprises

Unfortunately, the publication last week of the FDA Briefing Document related to the new drug application (NDA) for Mipomersen (tradename: KYNAMRO) by ISIS and partner Genzyme, should serve as a reminder that giving an old technology new names such as ‘gymnotic’ or saying that the algorithms now allow for the discovery of much more potent antisense sequences without any new chemistry (despite the fact that narrow tiling had always been practiced) are telltale signs that the biggest change that has occurred was in the marketing department.

The selective disclosure strategy by ISIS Pharmaceuticals on mipomersen has certainly contributed to the impression that antisense technology had indeed made important progress.  Dr. Crooke claimed in a recent interview on Mad Money with Jim Cramer that ISIS had been very transparent with mipomersen.  I could agree that there has been a lot of scientific data released on mipomersen by ISIS and their clinical investigators, but in hindsight this was arguably not for the claimed sake of transparency, but for providing the impression that mipomersen was a safer drug than it probably is. 

Here are a few examples of the ‘surprises’ that were revealed in the Briefing Docs:  

1) Liver Fat: More and Persistent

While the significance of the non-alcohol-related accumulation of fats in the liver in predisposing towards liver fibrosis is still hotly debated among experts, ISIS tried to dispel the still understandable fears about ApoB-knockdown-related fat accumulation in the liver by claiming a) that it was only modest, and b) that feedback mechanisms allowed the liver to adjust and that liver fat would decrease over time.  Supporting the impression that ISIS was concerned about transparency was the publication (Visser et al. 2010) of liver fat data from a 13-week study in a small, 21-patient study population which only showed a trend of such accumulation (median control-adjusted increase from baseline to day 99 of < 1%).  

Compare that to the actual results (as revealed in the Briefing Docs): In study ISIS301012-CS7 and CS12, 61.8% (63/102) in the mipomersen group had a > 5% increase in hepatic fat content.  This already shows that the median increase is definitely higher than 5%, not < 1%. Moreover, in study CS6, 16% had average liver fat of > 20% measured at least once.  30-40% liver fat contents also were not uncommon.  Such values were practically not observed at baseline or in control subjects despite ISIS’ repeated claims that such values are of no concern since NAFLD is so common anyway.

Furthermore, the data such as capture in Figure 10 of the Briefing Document show that there is no basis for claiming that the liver adapts and liver fat contents come down over time.  They only come down after you discontinue treatment.

Suggesting that the fat accumulations could be of clinical significance, a fibrosis-related biomarker score (ELF) was elevated in the mipomersen population.

To be clear, at least the liver fat accumulations (not sure about  the ELF score) is very likely a target-related side effect and cannot be attributed to the phosphorothioate antisense platform per se, but it is an important issue for mipomersen and raises suspicions about the way ISIS Pharmaceuticals has dealt publicly with platform-related safety issues.

2) Partial Clinical Hold: Vasculitis

Were you surprised by the fact that, according to the Briefing Doc, the FDA issued a Partial Clinical Hold for the non-severe LDL patient population in January 2008?  I certainly was taken by surprise and would also think that this should have been disclosed in an SEC filing as a Partial Clinical Hold for your lead program should be considered a material event.  Not only can shareholders be pitied, but also Genzyme which less than a month before invested more than $300M in mipomersen! I cannot imagine that they would have closed such a deal had it known that there was data that would trigger such a Hold (I do not exclude that the data was there under their nose, but in that case it was probably either buried under a pile of paper, or Genzyme just did not do their job well).

Apparently (à Briefing Doc), the ‘final interim report’ from the 52-week monkey tox study that was submitted in June 2007 showed vasculitis (inflammation of the vasculature which can impair blood supply and therefore organ function, possibly leading to organ failure) at clinically relevant doses of 3mg/kg and up, mainly, but not exclusively, in the gastrointestinal tract. Although the Hold was lifted after the actual clinical safety data did not indicate vasculitis to be an issue, subsequent skin biopsies around the injection sites showed an accumulation of inflammatory cells around the vasculature.

Although similar to the liver fat issue, the clinical significance and degree of systemic vasculitis due to phosphorothioate oligonucleotides remains unknown in Man, the potential for sustained inflammatory processes in the entire vasculature is a serious safety concern that needs to be studied in more detail before the technology can be applied to less than the most severe, 'orphan' patient populations- if at all.

3) Liver Enzyme Increases

That mipomersen increased liver enzymes in the serum, a measure of liver tox, has been widely known.  But ISIS used to claim that in a given individual these were one-off findings that could just as well have come after a night of heavy drinking.  By contrast, it seemed to me new news that there were cases where elevated (>3x ULN) liver enzymes were found on at least two subsequent investigations.  In fact, the FDA feels that the liver enzyme increases warrant them be tightly monitored as part of a REMS program should mipomersen be approved in the homoFH population.

4) Injection-Site Reactions and Discontinuations

One of the major marketing tools, and in fact actual draws, of ‘gymnotic delivery’ has been that it can be subcutaneously administered, while the leading RNAi Therapeutics delivery technologies, foremost Tekmira’s SNALP, still rely on intravenous infusion (for most applications at least).  Turns out that injection site reactions ranging from pain, redness, swelling, to skin discolorations and haematomas were a major factor for study drug discontinuations, especially in the open-label phases of the studies which should be indicative of what will happen in a real-world setting.  Overall, 61% of HoFH patients (23 of 38) discontinued study drug during the open-label phase, and 77 of 141 discontinued in the pooled Phase 3 population (including non-hoFH subjects).  Other adverse events attributable to the inflammatory potential of the technology, such as flu-like symptoms, contributed to the high discontinuation rate.

A curious, hitherto undisclosed finding, were injection site recall reactions with mipomersen (but not control).  This refers to an inflammatory reaction at an old injection site when the drug was administered at a new site.  The molecular basis and importance for such recall reactions seem unknown.  In fact, my literature searches suggest that they have almost exclusively been described for the TNF-alpha blocking antibody Enbrel.  However, to me, they once again indicate the systemic inflammatory potential of phosphorothioate antisense chemistry, likely involving adaptive immunity.  Accordingly, 30/50 patients (60%) in the CS5 and 6 studies exhibited mipomersen antibodies compared to none in the control population- another ‘surprise’ that should be applicable to the phosphorothioate platform in general.

5) Carcinogenicity

While there were ~2-3x more cases of tumors in the mipomersen-treated population compared to control, I agree with the reviewers that they are not the basis for concluding that mipomersen increases the risk of developing cancer.  This is because of the nature of the reported cases and the relatively short treatment duration after which many of them occurred.

However, what would worry me more is that in the preclinical carcinogenicity studies in rodents (mice and rats), mipomersen clearly increased cancer risk- despite ISIS’ claim that they have been ‘clean’.  Among the findings were hepatocellular adenoma and subcutaneous tumors.  Having a report by your CRO stating the findings are likely species-specific issues and not applicable to humans, does not mean that the findings are not material in a regulatory sense, especially as they were made during a period at the FDA when the agency was much more risk-averse.  I expect the clinical and preclinical findings to cause carcinogenicity to be included in a REMS program.    

6) Kidney Damage

The liver, spleen, and kidneys are the major sites of phosphorothioate accumulations.  Consequently, it is not surprising that kidney damage has been an important safety concern of such antisense technology.  Although the clinical safety findings did not demonstrate that mipomersen impaired kidney function, the study drug did increase the amount of protein found in the urine (proteinuria).  This finding supports that kidney function remains a safety concern and such monitoring should be included in a REMS.

In summary, the safety findings were not all that surprising given what we know about phosphorothioate safety in animals and study discontinuations by competitor Santaris, very likely due to safety.  However, if you indeed have taken the reassurances by ISIS CEO Dr. Crooke literally, you may be justified in feeling misled.  Even on that count, however, I am not all that surprised, and his salesmanship may indeed be one of ISIS' great assets.  

Disclosure: The author is 'long' the stock ($ISIS).

Clarification: The term 'gymnotic delivery' in the linked paper refers to a specific protocol for optimizing gene knockdown in tissue culture in the absence of assisted delivery.  However, the term has been more broadly used by Santaris to refer to unassisted delivery in general (including clinical applications), obviously in an effort to express that in contrast to phosphorothioate oligonucleotides, RNAi needed assisted delivery.  I found it suspect, however, that they used a new term to refer to an over decade-old industry practice.  In a further clarification, I have not seen ISIS Pharmaceuticals adopt the term either. But clearly, not using (in most cases) assisted delivery has been a greatly exploited marketing tool of the antisense industry built on the back of RNAi Therapeutics.

Simply SNALP: RNAi TTR Knockdown Data Impress

Clearly, RNAi Therapeutics is back in fashion.  Well, almost.

Alnylam’s announcement of profound gene knockdown in the ALN-TTR02 phase I study for the treatment of TTR amyloidosis was greeted with an over 50% jump in the stock price on very high volume.  Various observers pointed out that this might have been an over-reaction given the early nature of the data.  On the other hand, considering that the over 80% peak knockdowns achieved with only a single dose of 0.15mg/kg are representative of a widely applicable delivery strategy, namely Tekmira’s SNALP technology, and considering that an accelerated approval path  should be possible for this orphan disease, determining the fair value of a SNALP-based company like Alnylam becomes difficult.

Yes, this was a study that enrolled just 17 subjects, healthy volunteers at that.  But it may be this that makes the tight variability in the pharmacodynamic response (=TTR knockdown) even more remarkable so: less than 5% relative standard deviations in the 0.15mg/kg and 0.30 mg/kg 3-person dose cohorts, the relevant groups for this analysis.  

When I had mentally listed the technical risks of SNALP-mediated delivery several years ago when I did my fundamental research on SNALP delivery, intersubject knockdown variability was one of the major ones.  However, given the present results and those from the recent hypercholesterolemia study (ALN-PCS02), I am much less concerned about this now, also in patient groups where liver health may be affected.

Moreover, the dose-related TTR knockdown in humans was essentially what has been seen in non-human primates.  Although a number of analysts were apparently puzzled by the importance of this observation, its value to a scientist is obvious: you can essentially eliminate the risk that an SNALP program fails in phase I due to lack of knockdown efficacy (and if you are one of those scientists that also dabble in the stock markets, you can start investing in the stock based on the monkey data that you’ll see presented at an informal presentation).   

The safety profile with ALN-TTR02 in this study was good.  There was one infusion reaction seen in other SNALP studies before and that was managed by slowing the infusion rate.  This was said to be possibly related to the pre-treatment with corticosteroids, and not the liposomal formulation itself.  In any case, while corticosteroids and other transient immune suppressions will not be an issue for the initial patient populations in diseases like TTR amyloidosis, Tekmira and Alnylam should start to try and wean themselves of it.  The improved, more predictive immune stimulation assays may form a sound scientific basis for this.

The reason for why the study was limited to 17 healthy volunteers was mostly due to competitive concerns.  In particular, the ISIS/GSK TTR antisense compound is said to skip phase II studies altogether and go straight into a pivotal phase III.  In data presented in May of 2011, this antisense compound was reported to effect a 44% mean TTR reduction at the 200mg/week dose level when given for four weeks, and 81% knockdown at 400mg which was the next higher dose tested.  400mg/week, however, is a dose that I believe is too high for chronic phosphorothioate oligonucleotide administration.  Although ALN-TTR02 should thus be 100- to 200-fold more potent on a weight basis than the antisense drug candidate, being the first-to-market is seen by some as an important factor for the commercial success in this orphan market, especially since these compounds work by the same mechanism of action.

The value of SNALP delivery

Some readers of this blog may be tiring by now of reading me sing the praises of SNALP delivery technology and Tekmira.  Yes, I’m invested in the technology via my Tekmira shares.  In all fairness, however, it is the clinical data from the SNALP compounds, really starting last November with the ALN-TTR01 data (SNALP Works!) that are reviving the RNAi Therapeutics field: ALN-VSP, ALN-PCS, and ALN-TTR.  We have heard a lot about exciting new, revolutionary RNAi delivery technologies, often claiming to solve the putative problems with  SNALP technology.  However, in the end, it is SNALP that is set to unlock the first wave of RNAi Therapeutics value creation.

Even Arrowhead Research and Benitec which develop competing delivery and RNAi trigger technologies, respectively, admitted so much in congratulating Alnylam to the results.  Sadly, they missed the opportunity to also congratulate the real innovator behind SNALP technology, Tekmira.  It seems that since Alnylam is getting all the media attention, it was probably not a good PR strategy to do so.

RNAi delivery technologies are tough to develop and rare to find, but when they work, they open up a range of therapeutic opportunities.   

Preview: For my next post, I am planning to write something about last week’s MC3 paper and how it reveals that covering all the tracks is hopeless when a scheme is that elaborate (RE Tekmira-Alnylam litigation), and why Alnylam the stock may be a great short here. 

Impressions from the Abstracts of the 7th Annual Meeting of the Oligonucleotide Therapeutics Society (Part 1)

The Annual Meetings of the Oligonucleotide Therapeutics Society are among the best on the conference circus related to, well, oligonucleotide therapeutics drug development. One benefit of bringing together RNAi Therapeutics, traditional RNaseH and steric block antisense, aptamers, and a few other oligo-based approaches is that researchers can benefit from sharing lessons in safety, how pharmacology relates to chemistry and formulation, manufacturing etc. Remember, it is the experience with older oligonucleotide technologies that allowed RNAi Therapeutics to take 10, instead of 20 or 30 years, to get to where it is today: over a 1000 patients and healthy volunteers dosed with more than a dozen of RNAi candidates exhibiting a decent, and improving safety profile; the ongoing Atu027 and ALN-TTR01 trials having reached dose levels where, based on sound science, robust target gene knockdown, technologically the primary objective, can be expected. Moreover, data from hypercholesterolemia, solid cancer, ocular and respiratory disease studies have provided evidence of dose-related therapeutic efficacy.

Not able to attend the 7^th Annual Meeting to be held this week in Denmark myself, I eagerly went through the abstract book to learn of new developments and trends. Here are my thoughts on a few select abstracts that I thought might be of interest to the readers of this blog (presented in the order they appear in the book). Note that if you are a Tekmira investor, keep reading until the end. Part 2 of the discussion can be found here.

Oral presentation: Expanding the structural diversity repertoire of siRNAs (Dong-Ki Lee, Sunkyunkwan University, Korea)

This presentation highlights the realization that a number of non-Tuschl RNAi trigger structures are not just IP workarounds, but can be used to achieve novel biological outcomes such as targeting multiple genes with one RNAi trigger molecule (multipodal structures), inducing select innate immune stimulation while at the same time silencing genes (long siRNAs), and reducing off-targeting (asymmetric siRNAs and ‘wobbly’ siRNAs).

Oral presentation: Activation of RNA interference in animals with single-stranded oligonucleotides (Erice Swayze, ISIS Pharmaceuticals)

For some indications, the intravenous application of the nanoparticle RNAi formulations which are leading in terms of in vivo RNAi gene silencing potency may be a commercial drawback (for the purpose of long market exclusivities, I believe it is a widely underappreciated benefit). ISIS Pharmaceuticals, until recently in collaboration with Alnylam have been working on naked single-strand RNAi (ssRNAi) solutions that can be administered subcutaneously.

It has been long known that ssRNAs can induce RNAi gene silencing, just 100-1000 less efficiently, which is not surprising since RNAi has evolved as a dsRNA-induced mechanism. The abstract claims that using fully modified, partially phosphorothioated ssRNAs, they were able to come within 5-fold of the potency of corresponding double-stranded structures. The initial animal experiments, however, seem to have failed due to ssRNA instability, but after further modifications they have now achieved activity at ‘pharmacologically relevant doses with subcutaneous administration in saline formulations’.

Certainly an interesting abstract and it remains to be seen just how pharmacologically relevant these doses are and the related safety profile. Similar, or better to their current RNaseH antisense? ssRNAi...ISIS’ antisense 3.0? Another interesting question is at what point did Alnylam drop the ball on ssRNAi after considerable investments- before or after the initial animal experiment failures?

Oral presentation: Delivery of Nucleic Acids (Muthiah Manoharan, Alnylam)

Alnylam’s oral presentation will be, you already guessed, about RNAi delivery. By listing 25 papers on two pages without any meaningful comment or discrimination, the abstract obviously wants to make the point that Alnylam is the leader also in RNAi delivery. Somewhat reminiscent of Alnylam’s press releases that used to list seemingly all their RNAi trigger-related patents, no matter how relevant to their gate-keeping potential which was the reason for listing them in the first place. As such, the abstract carries the dubious distinction of being the longest one of the conference, but the one with arguably the least content.

It is not the amount of money spent, the numbers of patents (‘thousands’), or papers published that makes you a leader in RNAi Therapeutics.

Oral presentation: Non-covalent peptide-based delivery systems (Divita, CRBM-CNRS-UMR5237, Montpellier, France)

This abstract concerning a non-covalent cell penetrating peptide-siRNA systemic delivery technology to me has firstly sentimental value. This is not a specific criticism of the work to be presented, a body of work that is buttressed by some credible data, but the abstract still reminds me of the early days when RNAi Therapeutics was hot…hot, hot, and all kinds of, sometimes wild, delivery claims were made: oral, blood-brain, all organs to name a few keywords.

While I wished that there was more excitement around RNAi Therapeutics right now as the negativity, particularly in the commercial arena, threatens to choke deserving technologies, the one benefit of RNAi being less hyped and exploited for fund-raising purposes by the biotech promotion machinery is that the overall scientific credibility index has increased. This can also be seen from the abstracts at this year’s OTS meeting.

Oral presentation: Investigating the potential of therapeutic oligonucleotides for pulmonary diseases (Clark, GSK)

GSK and AstraZeneca are probably the two Big Pharma companies most interested in RNAi/oligonucleotide Therapeutics for pulmonary diseases. This is an area with high unmet medical needs and new therapeutic approaches are needed here more than anywhere else. There are fundamentally two different approaches to knocking down genes in the respiratory tract: local delivery by aerosol inhalation, or through systemic delivery. Based on the abstract, GSK seems to be primarily interested in inhalation methods.

Among the companies having explored inhalation are Alnylam, ISIS’ respiratory disease spin-off Altair, and most recently Tekmira. It has become obvious that Alnylam’s naked siRNA approach (e.g. in ALN-RSV01) is sub-optimal and conjugation plus chemical modifications need to be applied to give such ‘semi-naked’ routes a chance. Altair meanwhile has closed down following phase II results with their naked MOE gapmer antisense candidate for asthma. Based on Tekmira’s track record of publicizing only meaningful scientific progress, Tekmira's aerosolized LNP approach is to be considered a serious contender for the leadership position in gene knockdown in the respiratory epithelium. Tekmira this year has presented data that its aerosolized LNPs retain the ability to knockdown genes in tissue culture. It is quite possible that GSK was/is the undisclosed Big Pharma collaborator for this program.

Silence Therapeutics, probably more by necessity than choice, takes a systemic approach towards gene knockdown in the lung using their intravenously administered lipoplexes (DACC). Actually, since Silence’s and Tekmira’s technologies may be best suited for endothelial and epithelial cell knockdown, respectively, the two approaches are complementary. It would make sense if AstraZeneca had some familiarity with Silence’s DACC technology.

Abstract #9: [3H]-radiolabeling of siRNA (Christensen, Novartis)

Abstract #86: Characterization of side reactions during the annealing of siRNA (Noll, Roche)

I list the two abstracts from Novartis and Roche here together because I believe they illustrate the cultural differences between Big Pharma and pure-play RNAi companies. While pure-play companies emphasize biology and developing new RNAi trigger and delivery solutions, the established pharmaceutical companies are apparently more concerned about manufacturing and pharmacology methods. It is obvious that manufacturing and pharmacology is an essential part of the game, and such work is also happening at pure-play companies and their outsourcing partners, but such work obviously does not address the rate-limiting challenges and Big Pharma, perhaps with the exception of Merck, willfully relies on accessing that from the pure-play companies.

Abstract #16: Inhibition of complement C6 synthesis in the liver using antisense oligonucleotides affects neuro-regeneration (Fluiter, Academic Medical Center, Amsterdam, Netherlands)

This abstract highlights that by knocking down a gene in the liver, one can have therapeutic benefits for a wide range of non-liver diseases, such as neurodegenerative diseases. This is not really surprising given that all organs almost exclusively depend on their development and function on what they are provided for by the blood. Proteins made in the liver constitute the majority of free proteins in the blood and consequently impact all organs. Complement proteins which play a critical in immunity are one example of such proteins. As most diseases contain a complement-related immune/inflammatory component, RNAi Therapeutics could be a tool for modulating a wide range of autoimmune and other hypersensitivity disorders.

This principle of inhibiting a target in one organ to address disease in others (see e.g. transthyretin amyloidosis) is in contrast to other, post-translational therapeutic drug modalities that target the liver for which the therapeutic benefit is almost always restricted to the liver. As such, the medical and commercial potential of RNAi delivery technologies that work well for gene knockdown in the liver is larger than widely appreciated.

Abstract #27: Thirteen week non-clinical testing of miravirsen in cynomolgous monkeys (Hildebrandt-Eriksen, Santaris)

This abstract concerns the toxicological evaluation of Santaris’ exciting phase II LNA anti-miR122, a LNA-modified phosphorothioate steric block antisense, for the treatment of HCV infection. Despite the successes of the recently approved protease inhibitors for genotype 1 HCV, there is still considerable unmet medical need, including for those with less drug-responsive genotypes or those high-risk patients that have failed on established therapies.

Presenting on home soil, the reported toxicities were in line with expected class effects of phophorothioate oligonucleotides, including slight, but relatively persistent clotting abnormalities which was not judged an adverse side effect because of the apparently small extent of the increase; reversible kidney toxicities at doses above 10mg/kg (the effective dose of miravirsen is likely between 2 and 5mg/kg); and finally some enlargements in macrophages which does not appear to be of too much concern. Note that because miravirsen is not intended for chronic use, this safety profile may be adequate. In addition to liver toxicity, it appears however that the kidney toxicity will be something to watch out for in the development of miravirsen.

The first phase II study of miravirsen has just completed enrolment according to clinicaltrials.gov and I look forward to learning about the results in due course.

Abstract #30: Lipid nanoparticle formulations of minimal-length shRNAs show potent inhibition of HCV-driven, liver-specific gene expression in mice (Johnston, Somagenics- in collaboration with Tekmira)

This abstract concerns the evaluation of 40-50 nucleotide hairpin RNAs with Tekmira’s LNP delivery technology for liver gene knockdown (in this case using HCV as a model system). It is not a surprise that the abstract shows that Tekmira’s LNP technology works with various RNAi triggers. The real new insight for Tekmira investors, however, is that Tekmira did not go into this litigation in a way that its access to payloads would be threatened as a loss of access to Alnylam’s RNAi triggers may very well be one of the outcomes that could facilitate a settlement. Instead, Tekmira must have been evaluating various RNAi trigger structures and presumably other nucleic acid payloads as well, and when it chose to exclusively license Halo-Bios multivalent RNAi triggers one has to assume that this was after an extensive evaluation of their safety and potency.

Whether there will be a similar arrangement with SomaGenics remains to be seen. Synthetic shRNAs are credible RNAi triggers and may in fact have some advantages over two-stranded approaches, e.g. highly efficient unimolecular annealing. However, their development has been held back by increased cost of goods associated with such long oligonucleotides and concerns about clogging up the RNAi enzyme Dicer (probably not an insurmountable challenge). A licensing decision may also depend on how broad SomaGenics' intellectual property is with regard to shRNAs. It is highly unlikely that SomaGenics has any gate-keeping claim in this area, and partnering with them would have to be driven by their shRNA-related know-how.

To be continued...(for part 2 click here)