Oligonucleotide Strategies beyond the Liver

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

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

Chemical stability

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

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

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

PK enhancers

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

Receptors

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

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

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

 

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

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

Endosomal release

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

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

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

 

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

AstraZeneca Selects MicroRNA Development Candidate, Blazes Innovative Trail

When it is screaming into your face that your business model has failed you and the young competition is running circles around you, only then you might be compelled to change. 

This certainly is true for Big Pharma which have lost sight that their business is to make a buck while increasing the health of their customers instead of wasting time and energy on challenges like turning a twice-a-pill into a once daily therapy.  In its quest to optimize their business processes, it has thus thrown out of the window revolutionary, innovative technologies that just would not fit into those loved models.

Case in point, Merck writing off their multi-billion dollar investment in RNAi Therapeutics and selling it to Alnylam for $175 in largely equity and some cash. Alnylam then turned around and made at least a 10x return on the RNAi trigger stabilization chemistry by Merck in little more than a year.

This is a rough estimation of how much the Merck RNAi assets have approximately contributed to increasing Alnylam’s market cap.

AstraZeneca leading the way for Big Pharma in RNA Therapeutics

Not long ago, AstraZeneca was widely vilified for being the worst of the worst in terms of R&D productivity.  Their labs just would not produce new compounds that mattered to patients.

After a corporate shake-up, things certainly have changed on the innovation front.  AstraZeneca has fully emerged as a real risk-taker when it paid Moderna $240M in upfront monies alone for access to a comparatively early-stage mRNA Therapeutics platform in 2013. 

Before that, however, it already got active in the RNA Therapeutics in a less visible manner, notably with a much smaller, but possibly more profitable deal with microRNA Therapeutics platform company Regulus Therapeutics.

In the 2012 deal, AstraZeneca made a $25M equity investment in addition to a token $3M cash hand-out in the then privately-held Regulus Therapeutics.  In exchange, AstraZeneca received 3 microRNA target picks in the cardiovascular, metabolic, and/or oncology areas.

The best part of the deal for AstraZeneca (and the reason why I took money off the table today at what I considered an outsized reaction) was that it only has to pay $2.5M per target/candidate pick and Regulus Therapeutics has to pay for part of the work involved in generating the candidate at that.  There would, of course, be the milestones and royalties, but they should also be modest, in-line with the $2.5M payment due now. 

Oh, those were the good old times of abusive (because they could) Big Pharma biotech business development deal right on par with the steal that The Medicines Company got from Alnylam with regard to the PCSK9 target.

But still, you have got to credit AstraZeneca that unlike its brethren they not only sealed the deal, but actually advanced one of the first clinical candidates involving a fundamentally new molecular target class.  It will be interesting whether they will do the same in mRNA Therapeutics.

Anti-miR103/107 antagonism for improving liver health in diabetes

Initially, the focus  of the partnership had been on what looked like a very promising HDL-augmentation strategy by inhibiting miR33 in the liver, but this candidate has apparently taken a backseat in favor of the insulin-sensitizing strategy by inhibiting miR103/107.

It had been known that in type II diabetes, there is an inverse correlation between insulin sensitivity and miR103/107 expression.  Supporting a causal involvement, inhibiting miR103/107 in mouse models of diabetes with (unconjugated) antisense oligonucleotides increased not only insulin sensitivity, but also had positive effects on a couple of other diabetes-related parameters not only in the liver (e.g. triglyceride levels), but also body fat (adipocyte size/differentiation).

One puzzling aspect, somewhat akin to Regulus’ Alport’s program (--> miR-21), in exploiting anti-miR103/107 for pharmacological intervention is that it was initially uncertain what the target cells ought to be: adipocytes and/or hepatocytes?  A role for miR103/107 expression in adipocytes was particularly supported by the observation that its steady-state level there is higher than in the liver and the fact that single-strand phosphorothioate oligonucleotides also distribute to body fat.

However, with the adoption of GalNAc conjugation technology where most of the oligonucleotides now accumulate in hepatocytes it seems that AstraZeneca and Regulus have come to the conclusion that it is the liver that once again is calling the shots here as it usually does in diabetes.  You can deduce this from the fact that a GalNAc version was selected as the clinical candidate (AZD4076) slated to enter the clinic later this year.

Taking advantage of the observation that anti-miR103/107 has positive effects on liver triglyceride levels, the clinical development of AZD4076 will at least initially be geared towards treating non-alcoholic steatohepatitis (NASH) in diabetes patients.

Co-delivering Antisense and RNAi for Cancer

The upcoming phase I top-line data for ISIS-STAT3Rx in liver cancer (HCC) to be presented at the upcoming EORTC-NCI-AACR triple meeting in Barcelona (Nov 18-21) will be an important test of the potential utility of RNAseH antisense oligonucleotides (ASOs) incorporating high-affinity chemistry in oncology.  

Based on the body language by ISIS Pharmaceuticals* and last week's $7.5M milestone payment from partner AstraZeneca for progress on ISIS-STAT3Rx (aka AZD9150) , I am tempted to speculate on more than just ‘encouraging’ results.  On the other hand, Regulus Therapeutics partner Sanofi at the Canton Nucleic Acid Forum (CNAF) also last week, noted the need for formulating antisense oligonucleotides to get their anti-miR21 oncology candidate into liver cancer tissue. It is likely that they will be using liposomes for that (--> Tekmira?).

* I was surprised that at the CNAF in Guangzhou, China, Brett Monia from ISIS mentioned STAT3Rx and cancer right after gene silencing in the liver as the next interesting application for ASOs- that is ahead of even the exciting CNS opportunity.

The discrepancy in body language may be explained by just cultural differences (conservative, blasé Big Pharma versus risk-taking, enthusiastic biotech); it may also be a reflection of different requirements for effective tissue concentrations with RNaseH versus anti-microRNA modalities or different target requirements.  Whatever the reason, the Sanofi comments clearly support the notion that getting naked, even phosphorothioated oligonucleotides into cancer tissues is not as robust as with other tissues such as the liver and kidney.

I am therefore pleased that it is a Big Pharma, the last place where I had expected that from, that is connecting the dots and is considering delivery formulations, even the supposedly ‘toxic’ LNPs.  The concept is that the nanoparticle would facilitate a higher tumor concentration of the oligonucleotide, and once in the tumor interstitial space, cellular delivery may be facilitated via two routes.  Firstly, it may be traditional liposome-dependent cell uptake and cytosolic release.  Alternatively, those LNPs that get stuck in the interstitial space would spill the phosphorothioate oligo which may then diffuse further and get into the target cell by self-delivery. 

The same concept, of course, applies not only to phosphorothioate ASOs, but also to self-delivering RNAi triggers (+/- conjugation).

But why stop there? I propose that for cancer delivery, one should strongly consider and co-formulate RNAi triggers and ASOs into a shared nanoparticle.  They could target either the same gene, or they could target different genes thus taking into account the desire for a multi-pronged attack on cancer   (-> resistance).  In that scenario you would benefit from the superior gene silencing efficacy of RNAi triggers in those cells that they were able to reach, but then extent your reach with the help of the more agile, penetrative single-stranded antisense molecules. 

As such, PS-ASOs have an advantage in addressing intra-tumor heterogeneity of the EPR effect which is a well-recognized problem of nanoparticle delivery for cancer.

Another benefit of combining RNAi triggers with RNaseH ASOs is that you could achieve additive gene silencing activity when going after a shared target.  For example, if the RNaseH ASO and the RNAi trigger had both say a 70% knockdown activity on their own in the nucleus and cytoplasm, respectively, the combined activity would likely be ~90% which genetically could make a huge difference.

There is also a potency benefit, although more minor, when going after different targets because at least in RNAi, the best you can hope for when combining RNAi triggers against different targets is that they do not interfere/compete with each other.

With solid cancer data from both Tekmira (RNAi) and ISIS/AZ out over the coming weeks, we will soon get a sense of whether the field has moved forward in oncology and what the next steps ought to be.

Oligonucleotide Therapeutics Need to Embrace New Genomic Era in Cancer Medicine

With ASCO 2014 behind us, I am left with the impression that oligonucleotide therapeutics have failed to keep pace with important developments in cancer drug development.  Most importantly, cancer oligonucleotide therapeutics need to take advantage of the latest genomic tools and insights in cancer biology to select the patient populations most likely to respond treatment to both increase success rates and to save development dollars.  After all, this is what you would expect from a platform most suited to personalized medicine.

In some cases, drugs are being developed a priori with specific mutations in mind such as the selective EGFR-mutant inhibitors by Clovis Oncology and AstraZeneca which have caused quite a stir amongst clinicians and investors at ASCO.  In other cases, and this is something spearheaded by companies like Foundation Medicine and their pharmaceutical partners, comprehensive next-gen sequencing is employed as trials progress to identify often complex signatures that render a cancer susceptible to a treatment. 

And particularly relevant for delivery aspects, other companies such as Endocyte are using imaging diagnostics to identify those cancers that either express the right surface receptors for a ligand-targeted agent or those amenable to the EPR effect relevant for nanoparticle-based delivery.

Although I believe that Tekmira has also a delivery-related rationale in selecting adrenocortical carcinoma and neuroendocrine cancers for TKM-PLK1 based on their insights in liposomal delivery, the oligonucleotide field at large is failing to take advantage of delivery-related cancer selection strategies.

In terms of tumor response related to hitting the right targets and pathways, I would like to see the field embrace the personalized genetics approach.  Prospectively targeting KRAS mutant colorectal cancers with a PLK1-RNAi therapeutic would be one example.  Going one step further, a development strategy that looks at the genomic profiles of various cancers and then pull out the formulation with the right RNAi trigger or antisense oligo against critically activated pathways would play into the strength of the technology: every signature is ‘actionable’ as the technology leaves no target behind.

Especially for delivery approaches that can penetrate tumors well, innovative cancer development could unlock the considerable potential that oligonucleotide therapeutics have for oncology.  Beyond TKM-PLK1, the androgen receptor antisense compound by ISIS/AstraZeneca could be an important first test case for this concept in oligonucleotide therapeutics.

Pharmaceutical Mega-Deals Could Delay RNA Therapeutics Partnerships

Pharmaceutical mega-deals are en vogue again.  Though not official, rumor is that Pfizer intends to acquire AstraZeneca for $100B, and deal engine Valeant has gone hostile on Botox maker Allergan with a ~$45B bid.  As if that weren’t enough for what was supposed to be a quiet Easter weekend, double-digit billion figures are being moved across the table in an asset swap between Novartis and GSK.   

The motivations for all these deals are essentially the same: squeezing out short-term profits by slashing R&D.  Valeant is an interesting example as it never pretended to be in the R&D game in the first place.  Instead, it exists on the notion that R&D is inefficient and risky and financial engineering through M&A instead of drug development is the only way to Big Pharma bliss.  Considering its spectacular rise to a ~$50B market cap company and a relentless increase in its share price, it has the goods to show for it. 

Pfizer, on the other hand, like all Big Pharmas likes to tout its R&D prowess webcast after webcast, R&D day after R&D day, but in fact is the worst offender when it comes to squeezing profits from slashing R&D.  In just 5 years following its acquisition of Wyeth, the R&D budget of the combined companies has been cut in half.  AstraZeneca is partly a juicy target because it was forced to be more risk-taking in its R&D as it gained the reputation to be the Big Pharma with the least innovative and effective R&D. 

As a consequence of this, AstraZeneca has become one of the most active Big Pharma in RNA Therapeutics with deals in antisense (ISIS), microRNAs (Regulus), RNA modulation (PTC Therapeutics), and most famously the 2013 $240M upfront mRNA Therapeutics deal with Moderna. 

Unfortunately/fortunately, depending on whether you think Big Pharma involvement in RNA Therapeutics is a good thing or not, the other two deal protagonists from this weekend, Novartis and GSK are also amongst the Big 4 Pharmas in RNA Therapeutics (Sanofi/Genzyme being the 4^th).

In addition to cost savings by cutting R&D outright, RNA Therapeutics deals could also be affected by Big Pharmas becoming pre-occupied with re-organizing.  This is based on experience as the narrative is that when Pfizer acquired Wyeth in 2009, Wyeth had by far the superior RNAi development effort, but that this fell victim to the acquisition.  Similarly, when Roche acquired Genentech the same year, RNAi Therapeutics quickly fell down the priority list.

What deals may be canceled or at least delayed as a result of these developments? 

mRNA delivery is the first one that comes to mind as I believed AstraZeneca to be under pressure to do something in this area after having spent probably $300M on mRNA Therapeutics by now.  

We have already heard about Novartis which had been another top pick for a delivery deal to go with its target picks from Alnylam.

Fortunately, RNA(i) Therapeutics is in a different position from what it was in 2009.  Cashed up and with robust, clinically validated technologies, a number of companies do not depend on dilutive Big Pharma deals any more- at least for now.  Let the deals therefore happen.  They will only accelerate the demise of the old pharmaceutical model to be replaced by innovative biotech companies.  

Tekmira Grabs Leadership Position in Messenger RNA Therapeutics

Messenger RNA Therapeutics is the latest addition to the RNA Therapeutics tool box.  A simple concept, it has only been recently revived outside the therapeutic vaccine realm when it was shown (e.g. Kariko et al. 2012 and Kormann et al. 2011) that certain nucleic acid modifications can minimize, if not abolish innate immune responses triggered by these long RNAs.

Of course, in addition to representing an increased innate immune challenge compared to RNAi Therapeutics, like RNAi Therapeutics, the key technical challenge for mRNA Therapeutics is the effective delivery into the cytoplasm of target cells.  When AstraZeneca paid Moderna Therapeutics $240M in upfront alone earlier this year for a limited technology license, I half rolled my eyes telling myself ‘not again’.  

The ‘not again’ refers to Big Pharma getting distracted by supposedly gate-keeping IP claims regarding the RNA inducer all the while ignoring the critical importance of delivery.  As we know, this mis-attribution of value has caused much trouble in the history of RNAi Therapeutics.

Enter Tekmira

I further believed that the SNALP LNP technology by Tekmira is most readily adapted for mRNA delivery among the RNAi delivery technologies.  So it was pleasing when Tekmira disclosed last week at the 1^st International mRNA Health Conference in Tuebingen, Germany, that they indeed have been pursuing mRNA delivery and presented first data indicating that they are most advanced in this effort.

As it is difficult to compare the amounts of proteins expressed with the different technologies due to the unique half-life of each mRNA and protein, the leadership hypothesis is partly based on an assumption, namely that there is a close relationship between SNALP LNP delivery of siRNA and mRNA.  More importantly, however, due to their ample experience of commercially translating SNALP LNP delivery for RNAi Therapeutics with over half a dozen SNALP-enabled candidates that have entered clinical development and with the demonstration of scale-up, they should have a first-mover advantage.

Liver and solid cancers

In particular, Tekmira presented data on mRNA delivery in mice for the liver and solid cancers.  The liver is of interest both as a target organ itself and also as a factory for the production of proteins secreted into the circulation.  The data showed that robust levels of the luciferase marker protein could be made in the liver.  However, the data also reminded me of one (certainly not insurmountable) challenge with mRNA Therapeutics: the relatively short period of time (<24 a="" as="" been="" boundary="" data="" designed="" expression.="" expression="" gene="" half="" has="" have="" hours="" however="" indicate="" life="" lower="" luciferase="" nbsp="" of="" ote="" p="" platform.="" protein="" reporter="" robust="" short="" so="" specifically="" that="" the="" to="">

In that light, the tumor expression data were particularly intriguing.  Not only were the tumor peak expression levels comparable to the liver, but they also were maintained over a longer period of time (~2 days) and generally declined more gradually.  Since the same mRNA was delivered, this observation would be consistent with delivery to the tumor over time, as one would expect if the EPR effect plays an important role here.  The data also support a depot effect following extravasation meaning that intratumoral SNALP-mRNAs can be stable so that they can be taken up in a delayed fashion and yet be functional.  Such observations incidentally should also prove quite useful for the development of RNAi delivery for solid tumors and I look forward to more such cross-fertilizations between siRNA and mRNA delivery.   

Tekmira the logical partner for AstraZeneca

The new disclosures would seem to warrant AstraZeneca partner with Tekmira on mRNA delivery.  After all, having spent $240M for IP puts them under pressure to do something with their rights.  As I do not think much of Moderna’s own LNP delivery efforts (order my latest OTS 2013 Report to see why), AstraZeneca would be well advised to look outside of that partnership for delivery solutions.

It also so happens that AstraZeneca’s IP rights relate to protein expression for metabolic disorders (--> liver as a critical metabolic organ) and cancers, precisely the two areas where Tekmira’s technology should be most useful initially.    

And who knows, maybe Big Pharma is not always that dense as it sometimes seems and all this had been in the making months ago and the CEO of Tekmira is about to make good on his partnership ‘promise’ (in that case, sincere apologies to AstraZeneca).

A Sign That Big Pharma Could Recognize the Low-Hanging RNAi Therapeutics Fruits

It was with much amusement and head-shaking disbelief that I read the Li et al. paper oncancer RNAi Therapeutics  development from Abbott.   The amusement stemmed from the fact that in the paper, the authors had come to the obvious conclusion: current technologies should allow you to develop real-world therapeutics based on the RNAi platform if only you judiciously combine the delivery platform with the right target and indication.  Duh…

As such, Abbott is representative of the various Big Pharma companies that guttered in-house RNAi Therapeutics development as they chose to only see the challenges instead of realizing the obvious opportunities.  Even those still in the game like Merck have long liked to go around and teach everyone how super-diligent, but super-slow their RNAi Therapeutics game strategy was. 

As RNAi Therapeutics have created market values at lightning speed (Alnylam now at a market cap of $3.5 billion), not based on just hype, but based on paths well-trodden by orphan drug companies, I expect more and more Big Pharmas to re-think their strategies.  Maybe even listen to this blogger who has always advocated a pipeline strategy that is based on where your delivery technologies can go to, instead of the traditional cart-before-the-ox-I-want-the-next-blockbuster-pill wishful thinking by people who may have risen to the corporate tops in companies like Coca Cola.
  

C’mon scientists from Merck, Novartis, and Takeda.  Inside yourself is an innovator that finally wants to see how your technology performs in humans.  Instead of just focusing on what could go wrong, don’t you also have an obligation to address diseases of high unmet medical need? And if not you, then who is going to transform your organization into a dynamic science-driven one that you can identify with as a scientist?  On a more practical level, I cannot see how many of you will be with your present employer in 2-3 years if you don’t have the goods to show.  Look at what happened to your peers from AstraZeneca whose last job it was to look for partnership in the more innovative pure-play RNA Therapeutics space before they were given the boot. 

Whoa! AstraZeneca Pays RNA Therapeutics Start-Up Moderna $240M

In the age of rare/severe diseases in drug development and personalized medicine, RNA Therapeutics are enjoying broad interest like never before.  Following a series of RNaseH antisense, splice modulation, microRNAs, and RNAi Therapeutics deals with large pharmaceutical companies, the AstraZeneca-Moderna Therapeutics news today marks another high water mark in the deal-making.  According to their agreement, AstraZeneca will pay Moderna Therapeutics $240M in upfront alone for rights to Moderna’s technology in the cancer and cardiovascular/metabolic disease areas (40 targets).   

AstraZeneca’s Externalized RNA Therapeutics Efforts

The deal with an innovative biotechnology start-up while in the process of shedding yet another few thousand employees, particularly in internal R&D, illustrates AstraZeneca’s R&D externalization trend.  RNA Therapeutics here seem to play a key role as supported by additional recent multi-million dollar deals in the space with ISIS Pharmaceuticals (December 2012) and PTC Therapeutics (June 2011) in oncology.  Unfortunately for RNAi Therapeutics though, it has been left out so far from the AZ’s deal bonanza.  Only in 2011, it wrapped up a collaboration with UK-based Silence Therapeutics.  Still, as AstraZeneca faces the challenge of how to deliver messenger RNAs to the liver and cancer, I expect RNAi Therapeutics delivery companies to financially benefit from AZ's mRNA investment soon (not just for mRNA, but also for RNAi delivery).

Moderna’s Technology

Moderna’s approach is a gene therapy one.  However, while classical gene therapy involves the use of DNA vectors for expressing therapeutic proteins, Moderna aims to circumvent the need for DNA, which have certain regulatory and safety drawbacks, and deliver instead messenger RNAs encoding for the same proteins.  This, in fact, is not a new idea and particularly popular in the immunotherapy field (albeit delivered ex vivo here, by electroporation). Duke University for example had a clinical RNAi Therapeutics program that involved the transfection of mRNAs along with siRNAs (cancer vaccine).

A 2013 Nature Protocols paper by the company’s scientific co-founder Derrick Rossi also leaves me scratching my head as to why AstraZeneca concluded that Moderna’s IP was worth $240M to them.  According to the protocol, the mRNAs are generated by normal in vitro phage polymerase transcription as you would do in the lab using Life Technology’s MEGAscript kit.  The only difference from the standard protocol may be that modified CTPs and UTPs were included.  This is supposed to mitigate the immunostimulatory potential with RNAs just as in RNAi Therapeutics and also contribute to the stability of the long RNAs.  

Based on the fact that neither the concept of mRNA Therapeutics are novel nor the RNA modification strategy unexpected, I expect that Moderna has yet to come out with their secret sauce and that the Nature Protocol may be misleading.  I therefore look forward to studying the patent applications, two of which curiously just published today.  It must be the IP that explains why AZ took a $240M license, to get a sense of the secret sauce.  But still, given the hundreds of nucleotide modifications available, it seems hard to fathom that Moderna's a blocking IP position, and why pay $240M if not a blocking one?

Need for Delivery

In addition to AstraZeneca’s interest in cardiovascular/metabolic disease and cancer, the state of the RNA(i) delivery technologies explains their choice.  Compared to RNAi Therapeutics where small RNAs are involved, the longer mRNAs face an even steeper cytoplasmic delivery challenge.

The liver, of course, is a key target organ for metabolic and cardiovascular disease.  Among Arrowhead’s DPCs, Alnylam’s GalNAcs, and Tekmira’s SNALP, the most advanced RNAi delivery technologies for the liver, it is essentially only Tekmira’s SNALP technology which I regard to be readily applicable to mRNA delivery (also for cancer delivery).  Conjugate approaches such as DPCs and GalNAcs are disadvantaged for mRNA delivery because they would provide no extra protection to the long, fragile mRNAs.  Liposomes by contrast provide such protection by wrapping around the RNA.

I expect to hear more about Moderna’s and AstraZeneca’s mRNA delivery strategies soon.  I, for one, do not believe that Tekmira’s shares are trading up by 8% on strong volume on the back of a SeekingAlpha article.

Other evidence that RNA Therapeutics (and Gene Therapies) Are Hot

ISIS Pharmaceuticals yesterday presented phase I data for their spinal muscular atrophy (SMA) splice modulation drug candidate at the annual AAN meeting.  This compound is partnered with BiogenIdec.  The results from the single-dose PK-oriented study suggest that fully 2’-MOE phosphorothioate oligos are well tolerated in the CNS and that a once-a-year/once-every-half-year dosing regimen may be possible: phosphorothioate oligos sit like a rock in the CNS when intrathecally administered.  In addition to the hints of clinical efficacy at the highest dose level (9mg) presented at the conference, what makes me optimistic about this program is that relatively little (compared to RNaseH) phosphorothioate molecules seems to be required based on the pre-clinical results.

In other news, cancer drug developer Celgene will work with gene therapy company bluebird bio on cancer gene therapy.  Although financial details were not disclosed, they were probably substantial (wild guess: $20M) given the broad nature of the collaboration (multi-year, multiple targets). 

The $28M AstraZeneca-Regulus MicroRNA Therapeutics Deal

Yesterday, it was announced that AstraZeneca is paying microRNA Therapeutics company Regulus $28M for three preclinical-stage microRNA targets.  This is certainly good news not only for the field of microRNA Therapeutics, but also oligonucleotide therapeutics in general which is well on the day to be the third major drug development engine after small molecules and monoclonal antibodies.  

After GSK and Sanofi-Aventis, it is the third of its kind for Regulus and there were similar ones between Miragen and Servier late last year and Santaris and GSK before that.  The number of such deals, each usually involving a number of microRNAs, illustrates how far the field of microRNA biology has come in just 10 years from the discovery of microRNAs in humans to yield promising therapeutic targets that number in the dozens.  In fact, microRNA Therapeutics has been more successful than the more straight-forward RNAi Therapeutics approach in attracting the partnering interests of Big Pharma lately.

On the other hand, it’s been now five years since the founding of Regulus Therapeutics, and still no program has made it into the clinic.  Such a performance is certainly not good enough to support an IPO these days for which the company, based on job postings, appears to have had ambitions for for some time now.  With remaining ~25M in cash and an annual burn rate of around that, the revenues recognized from the AZ deal may well be the substitute for a public offering.  

As it was not disclosed how much of the $28M was for equity in Regulus, other than the cash added to Regulus’ balance sheet it is really difficult to tell whether the dealmakers at Regulus will be all smiles about it.  What’s more, the miR-33 atherosclerosis program which had yielded exciting data (including in non-human primates) in enhancing reverse cholesterol transport with a subsequent reduction in plaque size seems to be spoken for already at this early stage (before the magical phase II value-inflection point).

It is unclear what is causing the apparent delay of Regulus progressing programs into the clinic.  Is it the complexity of microRNA biology where each microRNAs often has dozens of targets, or has it something to do with the concern that the 2'-fluoro modification initially favored by the company may be genotoxic?

AstraZeneca’s Return to ‘Proper’ RNA Therapeutics

When AstraZeneca, in its farewell to Silence Therapeutics in January, said that the Silence effort was ‘part of its overallstrategy to explore this important therapeutic approach [i.e. RNA Therapeutics]’, I took it to mean that the Silence projects may not be their top priority in this regard and that it already had other oligonucleotide technologies and companies in mind.  

Confusingly, half a year before that AstraZeneca entered into a ‘small molecule RNA Therapeutics’alliance with PTC.  Beware of companies, particularly prevalent in the (cancer) stem cell field it seems, which claim to be pursuing new platforms and treatment paradigms, when the innovation actually rests on just tying pre-existing molecules to new biological rationalizations.  

Classic Big Pharma I thought then: advertising innovation, but really sticking to its old, rusty guns; and if AstraZeneca is widely thought to have the industry’s worst productivity, you have to look no further for its causes.

Yesterday’s news was therefore quite encouraging in that AstraZeneca has not given up on developing ‘proper’ RNA Therapeutics by which I mean that nucleic acids are the therapeutic agents.  Whether the stream of positive clinical results in oligonucleotide therapeutics (and vaccines) have provided AZ encouragement to go down this path is unclear, but they certainly did not hurt.  Maybe it will even make AZ re-energize its RNAi Therapeutics efforts (e.g. for oncology or respiratory disease).

Want to learn more about microRNAs?  Register for the 2012 Janssen Award Symposium in New York.

Next post: Tekmira's very busy quarter.

SNALP Delivery Keeps On Giving: Tekmira Receives OK for Ebola Clinical Studies

On Monday, Tekmira announced that it has received the Green Light from the FDA to go ahead with clinical studies of its SNALP-enabled biodefense candidate for the treatment of Ebola infection. Tekmira is developing TKM-EBOLA under a $140M contract from the US Department of Defense following spectacular results in non-human primates reported last year in The Lancet. Depending on whether you want to count in the stalled TKM-ApoB program or not, this marks the 5^th or 6^th SNALP-enabled candidate in clinical development, illustrating the strength of this systemic RNAi trigger delivery platform: TKM-ApoB, ALN-VSP02, TKM-PLK1, ALN-TTR01, ALN-PCS02, and TKM-EBOLA.

In other words, 6 of the last 7 systemic RNAi INDs or IND equivalents were for SNALP-enabled product candidates (period: 2008-2011). This plus the unparalleled, strong pre-clinical track record of this platform demonstrating efficient knockdown in the liver, solid tumors, and viral infections not only in rodents, but also a number of non-human primate models supports the notion that Tekmira’s SNALP is the industry’s most advanced and valuable RNAi delivery platform. There are thankfully other promising RNAi delivery technologies lining up behind SNALP, but this is not Lake Wobegon where everybody can be above average

Next Steps for TKM-EBOLA

Since TKM-EBOLA, as a treatment for a disease in which controlled human studies are ethically or practically impossible, is being developed under the Animal Rule, this phase I study will not only have to demonstrate adequate safety, but more importantly yield pharmacokinetic and potentially biomarker data that replicates what is seen in the successful treatment of the pre-clinical animal models of the infection. At the same time, it may be worth trying to test the limits of how long treatment can be delayed after symptom onset in the animal models as a common criticism of these studies is that in the real world it may take some time before Ebola victims are identified and treated. In The Lancet studies, rhesus monkeys received first treatments already 30 minutes after exposure to the virus which may model a needle stick scenario in an Ebola research laboratory, but not exposure of the civilian population e.g. in a subway system. Similarly, achieving similar pre-clinical efficacies with 1mg/kg as with the tested 2mg/kg dose in The Lancet studies may bring it more in line with the clinical SNALP safety experience so far. On the manufacturing front, it would be helpful if Tekmira succeeded in providing SNALP in lyophilized form which would increase its utility in the field.

On the other hand, the fact that the rhesus model seems to closely replicate, if not represent a particularly severe form of the human disease, and the absence of a (experimental) therapy for Ebola that has shown comparable promise, should position TKM-EBOLA well for stockpiling despite any lingering real-world concerns. From an Army point-of-view, as long as it has been shown to be safe and well tolerated in humans, having the most promising treatment as a stand-by for a virus as deadly as Ebola is better than nothing at all, a consideration that may result in stockpiling even before, or in the absence of FDA licensure.

In that regard, TKM-EBOLA will be mainly competing with AVI Biopharma’s morpholino antisense candidate AVI-6002 which is being developed under an essentially identical contract with the DoD. A Nature Medicine paper published last year reported that this candidate was successful in rescuing ~60% of infected rhesus macaques, although this represents a roughly 3-fold increase in risk of dying compared to the highly comparable SNALP studies in The Lancet. Nevertheless, AVI Biopharma still enjoys a slight time advantage as it has already begun phase I safety studies earlier this year. A late-October 2011 update by AVI stated that treatment in the first 5 of 6 dose-escalating cohorts was well tolerated and that the Data Safety Monitoring Board recommended further escalation to the last 9mg/kg cohort. Nevertheless, once years behind the AVI program, Tekmira has done well catching up with the competition.

Importance beyond TKM-EBOLA

Besides representing an invaluable strategic asset for Tekmira (it is earning the company significant hard cash now and revenues from stockpiling may come well ahead of the customary 5-10 years it usually takes a normal drug to navigate the FDA approval maze), the approval of the IND further demonstrates that SNALP is indeed the productive delivery platform that also I have long had hopes for it to be, with applications not only for knockdown in the liver and solid cancers, but also phagocytic cells (an important target cell population for the Ebola indication). It is also a stamp of approval by various regulatory agencies around the world that SNALP (including reliable manufacturing) is fit for clinical development. An IND for ALN-TTR02 and phase I results for ALN-PCS02 are next.

Comment on Roche Partnership with PTC

Roche disclosed today that it has signed a collaboration with PTC Therapeutics for the treatment of Spinal Muscular Atrophy, including a $30M upfront fee for pre-clinical assets. This follows a similar deal by AstraZeneca and PTC in oncology earlier this year. PTC develops a platform for the modulation of post-transcriptional processes using orally available small molecules.

What is disappointing to me is that these are examples of Big Pharma companies with an interest in RNA Therapeutics (note that AstraZeneca has a relationship with Silence Therapeutics for which a go/no-go is imminent), but which feel more comfortable risking their money on a technology based on phenotypic tissue culture screens with considerable uncertainty as to clinical relevance and the safety risks inherent in modulating very general gene regulatory mechanisms, instead of using the much more straight-forward oligonucleotide approaches. The reason? Oral bioavailability and coziness with small molecule chemistry. The fate of these collaborations will be an important test case of whether putting patient convenience and other marketing considerations ahead of what is the scientifically best approach will bring Big Pharma the desired outcome. Of note, only a few months ago, Genzyme handed back PTC a candidate for the treatment of Duchenne Muscular Dystrophy and Cystic Fibrosis after spending more than $100M on it.

My view: Technical success trumps patient convenience when it comes to diseases as severe as SMA, DMD, or cancer.

Interested in the Chinese market for RNAi Therapeutics, but language barriers exist? Get expert help from somebody that understands RNAi.

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)

AstraZeneca Shows Continued Commitment Towards RNAi Therapeutics through Partnership with Silence Therapeutics

One week after announcing the grant of a US patent covering RNAi triggers with enhanced guide strand loading, Silence Therapeutics today announced the one-year extension of its R&D collaboration with AstraZeneca for the ‘identification and optimization’ of five siRNAs targeting genes involved in cancer and respiratory disease. This relationship dates back to 2007 when AstraZeneca paid Silence a 7.5M pounds Sterling technology access fee plus the usual potential for future fees and milestones. After similarly extending and expanding relationships earlier this year with AstraZeneca and Dainippon Sumitomo in RNAi delivery, Silence Therapeutics has met important partnership goals earning it time to now prove to the investor and pharma worlds that its merger with Intradigm also brought with it technological synergies.

The way I read the AstraZeneca extensions is that Silence Therapeutics was certainly able to convince AstraZeneca that it is a RNAi Therapeutics company with scientific credibility, but that AstraZeneca still needed extra time before it could fully commit to Silence Therapeutics. It is interesting to speculate that AstraZeneca was a major force behind the Silence Therapeutics-Intradigm merger, as Intradigm had filed for a number of patents covering siRNA sequences against cancer-related as well as inflammatory targets also useful for respiratory diseases. Some of these have actually started to issue and may come in handy for this siRNA-specific collaboration [update: the company informed me that the Intradigm RNAi triggers would not be part of this collaboration, and re-confirmed that the RNAi trigger and delivery collaborations are separate. Sounds to me like they are holding back, possibly hoping to monetize some of these assets on even better terms to another potential licensee or as part of a larger collaboration with AZ eventually].

As to the targets, I could imagine that, based on the unmet need and AstraZeneca’s interests, RSV infection, asthma, COPD, and solid cancers esp. primary liver cancer are among them. Of note, mdRNA announced earlier this year an early collaborative effort with AstraZeneca China for studying that company's liposomal delivery technology for liver cancer, and today’s press release mentioned that AstraZeneca's Chinese R&D branch was part of the Silence collaboration, too. Since Silence Therapeutics appears to be keen to emphasize that the siRNA and delivery collaborations are separate- scientifically not necessarily the most sensible approach, but a deficiency that may open up the prospect of a more lucrative arrangement in the future- it is certainly an interesting thought if Silence’s siRNAs were to be formulated in mdRNA’s liposomes. Of course, it is more likely that mdRNA and Silence compete with each other for AstraZeneca China’s favor in liver cancer, but still...

As I said, it would eventually make a lot of sense to consolidate the two efforts, siRNA and delivery, into one larger collaboration. Based on Silence’s own data, I am fairly encouraged that its lipoplex-siRNA formulations could be generally useful for knocking down genes in the vascular endothelia, which not only would be of obvious relevance for cancer, but also lung disease which often involves vascular escape and infiltration of the lung by inflammatory cells. An alternative approach to siRNA delivery to the lung is, of course, by inhalation, desirable e.g. for RSV infection involving lung epithelial cells, although Silence hasn’t reported much progress in that regard. It remains to be seen what Intradigm's technology can add to that.

Ultimately, I consider today’s extension a major de-risking event for the near-term health of Silence Therapeutics. While much remains to be done, the stock at 5.5 pence and a market cap of little over $20M is simply too cheap, if not an insult to RNAi Therapeutics in general. It is especially encouraging that AstraZeneca shows continued interest in RNAi Therapeutics, a company that I honestly had at the bottom fifth of Big Pharma companies willing to embrace cutting-edge platform technologies to once again efficiently develop drugs that make a difference. Unless their goal is to buy back all their shares and eventually liquidate operations or become consumer healthcare companies selling diapers and skin lotions, realistically, it is their only way forward.

Can RNAi Therapeutics do a Better than Monoclonal Antibodies in RSV Infection?

Arguably, the next main proof point that looms for RNAi Therapeutics is proof-of-concept that RNAi can be made to work in humans. Alnylam takes its first shot at this in their randomised, placebo-controlled, double-blinded phase II study for the treatment of respiratory syncytial virus (RSV) infection. In this study, volunteers infected with an attenuated form of RSV will be treated with ALN-RSV01, an siRNA targeting a conserved region of the nucleocapsid (N) protein mRNA, and its effect on viral load and symptoms evaluated. Results from this trial, initiated last month (see Blog from 25 June 07: “Alnylam Progresses RSV RNAi Program into Phase II Clinical Studies”) are expected by the end of this year.

Currently, the only effective drugs in addressing RSV are neutralising antibodies that were developed by MedImmune (now AstraZeneca). These monoclonal antibodies (MAb) are directed against the F-protein on the surface of RSV and block cellular entry of the virus. Importantly, whereas these MAbs are used for the prevention of RSV infection in a small at-risk population, premature infants, ALN-RSV01 is geared towards the treatment of RSV.

Numerous studies have shown that the effect of RNAi, and probably any type of drug, on viral replication is most potent when given around the time of infection. I therefore wondered why ALN-RSV01 should succeed in the treatment of RSV when other drug classes such as MAbs have failed. Indeed, my own literature research confirms that MAbs have been tested in animal models for the treatment of RSV, but were found to lack sufficient therapeutic activity.

A study by Mejia et al. [Antimicrobial Agents and Chemotherapy 49: 4700 (2005)] compares 50mg/kg of the latest generation of anti-RSV MAbs when given either before or after viral infection in mice, and finds that on almost all accounts (viral load, inflammation, lung pathology) MAbs were only effective when given shortly (24 hours) before infection. The only assay that showed an effect when MAbs were given 48 hours after infection was a viral plaque forming assay which may reflect the presence of neutralising antibodies in the assay.

Bitko et al. [Nature Medicine 11:50 (2005)] on the other hand showed in an almost identical mouse model that intranasally delivered siRNAs had a profound effect on RSV replication even when given after viral infection. Moreover, 3.5mg/kg doses already proved very effective. Importantly, siRNAs were able to limit viral replication even when given up to 5 days after viral infection, the time when the acute phase of RSV peaks in this particular model. This is crucial in the clinical setting where the treatment benefit will likely be optimal if RNAi therapy can be initiated before acute infection has peaked. The authors then go on to show that on a number of counts (respiratory rate, pathology score, leukotriene production), anti-RSV siRNAs almost abolished any pathological signs of the disease.

These results suggest that while current MAbs are potent in reducing the initial infection by neutralising the interaction of the virus with the host cell, they are ineffective in preventing the subsequent spread of the virus. This could be due to the kinetics of viral re-infection in close proximity to the next host cell. By contrast, unless they target host surface receptors, siRNAs will not be able to prevent viral infection. The can, however, prevent and limit the ability of the viral genomic RNA to replicate and/or inhibit virion formation. Although Bitko et al. have not measured viral RNA levels directly, it is very likely that these were also reduced, and treatment with siRNAs even after the acute phase of infection may have a clinical benefit on RSV co-morbidities such as asthma/wheezing later in life.

Silence Therapeutics in RNAi Therapeutics Deal with AstraZeneca

Silence Therapeutics, formerly known as SR Pharma/Atugen, disclosed today the signing of a significant collaboration agreement with AstraZeneca. In addition to initial upfront investments of £7.5M (~$15M), Silence stands to receive up to £200M in future developmental milestone payments plus royalties on any resulting drug sales. For this, AstraZeneca may target up to 5 genes for therapeutic purposes using Silence Therapeutics’ AtuRNAi platform with a focus on respiratory diseases.

The AtuRNAi platform is at the heart of Silence Therapeutics. AtusiRNAs are blunt-ended, double-strand oligos with a particular 2’O-methylation pattern that induce post-transcriptional gene silencing. Silence believes that its AtuRNAi platform sufficiently differentiates it from competing RNAi platforms, most notably Tuschl siRNAs, to be considered proprietary. Indeed, the European Patent Office has been a good ally of the company by granting them a core patent relating to this technology earlier this year and restricting the scope of the competing Kreutzer-Limmer patents before that. This has allowed Silence to attract a number of reputable collaborators even before today’s deal with AstraZeneca.

It will be interesting, however, to see whether the value of the AtuRNAi patents are as significant as is claimed, since the seminal Tuschl II patent series demonstrates the use not only of 3’ overhang siRNAs, but also those without such overhangs. It should be noted that while retaining RNAi activity, blunt-ended siRNAs have been shown to be less potent than 3’ overhang siRNAs. Moreover, Tuschl II, to which Alnylam Pharmaceuticals holds exclusive rights, covers siRNA modifications in general and 2’O-methylations in particular. Only last week, Alnylam has strengthened this position by securing exclusive rights to ISIS’ fundamental nucleic acid modification patents for the use in RNAi Therapeutics.

Indeed, at least one licensee appears to have started to doubt the strength of the AtuRNAi IP position. After it had licensed two AtuRNAi agents from Silence that have entered phase I clinical studies this year, Quark subsequently decided to be covered through Alnylam’s InterfeRx siRNA licensing program as well.

However, unless Alnylam feels threatened in its ability to negotiate high-value collaborations due to secondary RNAi IPs such as the AtuRNAi platform, I do not expect them to resort to legal measures at this point. Today’s deal should be seen as yet another validation for the RNAi Therapeutics platform and the considerable funding stream flowing in should strengthen the whole field. Silence has certainly proven its business savvy, now is the time for them to show that they can also execute on their own cancer focussed clinical programs.