RNAi Also Conquers the Central Nervous System

In the span of a day, RNAi Therapeutics have gone from a mechanism widely viewed as being constrained to the liver only, to a major therapeutic modality for many targets and indications in a variety of tissues.  Due to the demonstrations in the liver, lung (yesterday), and today the central nervous system to potently and specifically knock down genes with infrequent dosing, RNAi will play a prominent role in today’s precision medicine-oriented drug development.

Employing C16 lipid-conjugated, chemically stabilized RNAi triggers, Alnylam and their partner in CNS drug development Regeneron achieved 84-90% maximal target gene knockdown with knockdown persisting at >70% for at least 3 months after a single dose.

Since chemical stability has been key to the successes in the lung, CNS, and also liver, it seems very likely that similar breakthroughs will be achievable for muscle, kidney, adipose tissues, and (in the words of Alnylam's President) 'even tumors' that Alnylam and Arrowhead are working on.

The initial target in the phase I study of ALN-APP was amyloid beta precursor protein (APP). Unlike the armada of antibodies that have targeted every known aggregation form of abeta for the treatment of Alzheimer’s, ALN-APP reduces them all and before they are even made thereby offering a unique angle to this important target.  An even more exciting near-term application of ALN-APP in my opinion is for cerebral amyloid angiopathy (CAA) where abeta accumulation near blood vessels can lead to intracerebral hemorrhage.  Studies with antibodies in Alzheimer’s have actually led to fatal damage to those very intracerebral blood vessels by causing local inflammation, and thereby make them a bad choice for CAA.

Beyond abeta and tau for Alzheimer’s, the CNS in particular abounds with otherwise difficult-to-drug important targets for diseases like Parkinson’s, Huntington’s, ALS, spinocerebellar ataxias for which RNAi is ideally suited.

The prolonged and robust knockdown observed is significantly better than what has been observed for previous RNaseH antisense candidates such as against SOD1 and tau (~50% target gene lowerings).  Safety also appears to be superior to the broadly phosphorothioated antisense molecules with no changes in neuronal markers of damage and inflammation seen with ALN-APP compared to placebo.

The US FDA though slapped a clinical hold on the multi-dose part of the trial based on findings in standard preclinical animal tox studies at doses well above what will be needed in the clinic.  The single dose exploration study, however, has been allowed to continue, and Canada has already allowed the multi-dose part to go ahead.  It therefore seems highly unlikely that the findings could derail ALN-APP or even this technology approach at this point.

With the recent news, the pharmaceutical landscape has changed and Big Pharmaceutical companies will have to think hard whether not having a stake in RNAi as a platform is viable.  The achievement is also one of delivery and stabilization chemistry which can be more broadly applied to other oligonucleotide therapeutics modalities in the CNS.

Korro Bio Opts for LNP in Liver-Directed Lead Program

Korro Bio yesterday announced that it would collaborate with Genevant to develop liposomally formulated oligonucleotides for the ADAR editing of alpha-1-antitrypsin in the liver.

This is a big surprise for the field since based on the successes in the oligonucleotide therapeutics industry in general and data from competitors Wave Life Sciences and ProQR in particular, it would have seemed obvious to employ GalNAc-conjugated editing oligonucleotides for alpha-1 antitrypsin-related liver disease.

Korro Bio is a privately held pure-play ADAR editing company that has raised more than $200M since 2020 and is developing oligonucleotides, as opposed to DNA-directed small editing RNAs, for mediating AàI conversion.  Given this substantial funding and what appears to be the ready availability of GalNAc, it is a big mystery to me why Korro has chosen intravenously administered LNPs and in the process is giving up substantial ownership in this program through the collaboration.

Just last month, Korro Bio and scientific founder Joshua Rosenthal published a selection strategy for efficient editing oligonucleotides.  The paper (Quiroz et al, 2023) finished off with experiments illustrating the need for extensive oligonucleotide modification, reminiscent of what ProQR and Wave Life Sciences have practiced, for effective ADAR editing.

 

Learnings from RNAi

One explanation for why Korro may have favored an intravenously over a subcutanously administered technology may be potency.  In yesterday’s press release and a recent Nature Biotechnology RNA editing industry article, the company is boasting that it wants to return serum alpha-1-antitrypsin levels to within the normal range.

A lofty goal and perhaps most readily achieved without having to balance the demands of chemical modification for stabilization purposes and inherent ADAR activation potency. 

In the earlier days of RNAi, Alnylam’s LNP-formulated Patisiran actually won out over an internal GalNAc competitor that didn’t quite have the potency and was also associated with toxicity.  Patisiran has also won the commercial race against a subcutaneously administered antisense oligonucleotide by Ionis due to superior clinical data.

Clearly, depending on the stage of chemical modification know-how with regard to a specific oligonucleotide modality, LNPs may be preferable even for the targeting of genes in hepatocytes.

Maybe Korro Bio does believe it still has a potency edge over the competition, and combining its oligonucleotides with LNPs may also get them faster into the clinic.

 

The Vivek Factor  

There is no reason to believe that in AATD an LDL receptor-targeted delivery strategy may be beneficial over an ASGPR-targeted one because of changes in receptor expression levels.

I would, however, not rule out that Korro Bio succumbed to the magic of the bewilderingly fast-talking executives from the Roivant universe (Genevant is a Roivant subsidiary).  I still cannot get over the fact that Tekmira handed over half the company plus LNPs to (now US Presidential candidate) Vivek Ramaswamy for some toxic small molecules scribbled on the back of an envelope.

When you hear Vivek on the campaign trail these days and his sharp fast talk full with twisted arguments that make your head spin, then I understand why people that for whatever reason like this energetic person may throw out reason and just want to trust this guy.  But beware: while making billions for himself and his family, he has lost many more of shareholders’ money for projects like the Alzheimer’s drug that he dug out from a dumpster and IPO’d at a valuation of over a billion USD.  I digress…

 

Until we see non-human primate data from Korro Bio and Genevant, I will count this candidate out of the race in AATD.  Whether ProQR will fill the void and throw down the gauntlet to Wave Life Science will be seen by its pipeline reveal at the end of this month.

Gentle Endosomal Escape to Expand Reach of Oligonucleotide Therapeutics

One of the attractive aspects of oligo-mediated ADAR RNA Editing is that it can piggyback on decades of oligo delivery R&D.  New breakthroughs in oligonucleotide delivery are therefore instantly applicable to and greatly enhance the value of the platform. 

In order to make extrahepatic target engagement of oligonucleotide therapeutics as robust as in the liver, various cell-targeting ligand conjugates are being attempted. This includes transferrin receptor-1 in the muscle or various lipid conjugates for the CNS.  The goal is to get highly stabilized oligonucleotides being taken up into the endolysosomal compartments of target cells from which they would somehow escape into the cytoplasm.

Increasing their release through the addition of endolysosomal disrupting agents is highly effective, but it may not be surprising that spilling the insides of endosomes and particularly lysosomes into the cytoplasm in addition to the intended oligonucleotides is a dangerous proposition.  This is illustrated by (not just) the experience with Arrowhead’s DPCs, but also various cationic polymers before it. 

Certain liposomal nanoparticle (LNPs) chemistries are better tolerated.  This may be related to how they release their payloads into the cytoplasm and which may involve complex lipid bilayer dynamics that provide oligonucleotides the opportunity to slip through.

Trapping Oligonucleotides in Metastable Endosomal Compartments

Trapping stabilized oligonucleotides in a similar metastable endosomal membrane environment is what Finicle and colleagues (in a collaboration with Ionis Pharmaceuticals) sought when inhibiting both endosomal recycling and lysosomal fusion with the synthetic sphingosine analog SH-BC-893.

Indeed, ‘893-treated tissue culture cells accumulated ~4 times the amount of a model phosphorothioate gapmer and there was a 9x oligonucleotide increase in the desired cytoplasmic-nuclear compartment.  This in turn lowered the IC50, the concentration of gapmer oligo necessary to inactivate 50% of target RNAs, by over 100 fold.  The IC50 of a model RNAi trigger was similarly lowered 20 fold.

As expected, crude endosomolytic agents proved extremely effective when added to tissue culture cells after the addition of oligonucleotides, but highly toxic.  And when compared to gentler oligo delivery enhancers reported before (AZD8055, 6BIO), ‘893 turned out to be significantly more effective.

Importantly, the sensitization benefit translated to the in vivo setting in a mouse model. Here, ‘893 was given orally around the time of subcutaneous oligonucleotide administration. 

While no gene silencing was really observed when a gapmer antisense oligo was hybridized to a cholesterol-conjugated complementary oligo (which was supposed to aid CNS delivery) and then subcutaneously administered, ‘893 led to noticeable, albeit still quite modest target engagement in some areas of the CNS.

This means that either the approach is optimized and ‘893 can be used in a subcutaneous oligo delivery setting when invasive intrathecal administration is prohibitive, or that ‘893 may be better suited for use along with intrathecal administration of oligos with the goal of achieving liver-like robust target engagements.  For highly severe and relatively rare diseases, the latter would be my preference.

To generalize the in vivo benefit of ‘893 following systemic oligo administration, greatly enhanced RNA silencing was seen in the mouse lung.  Again, I would have liked to see it compared to the benefit that ‘893 might bring to local, in this case aerosolized delivery.

 

Safety of Approach

That this endosomal kinetic entrapment strategy may have clinical legs is supported by the apparent safety of ‘893.  Indeed, this compound is already being considered for cancer and obesity-related indications.  So who knows, you may end up taking your oligos along with ‘893 and similarly acting agents that have anti-ageing activity.  And even if ‘893 should reveal negative side effects following chronic dosing, as a PK enhancer of oligonucleotides it may only have to be given infrequently, depending on how often the oligonucleotides need to be administered.

 

It is nice to see oligonucleotide deliveries making continued inroads throughout the body.  After the liver and CNS, the lung and muscle are becoming fertile hunting grounds for Oligonucleotide Therapeutics developers.  Because of its novelty and differentiation, RNA Editing will be busy with liver- and CNS-related applications alone for some time to come, but when there is robust clinical target engagement by oligos in other organs, RNA Editing can instantly jump on those opportunities.  Next up in terms of extrahepatic delivery is Arrowhead Pharmaceuticals with a number of early clinical readouts for RNAi gene silencing indications of the lung.

One Pitfall to Look Out for in RNAi Therapeutics

RNAi Therapeutics have turned the corner to being a generally accepted drug modality.  Following years of ridicule and benign neglect, the industry just celebrated the FDA approval of a 2nd drug, Givlaari for the treatment of acute hepatic porphyria. This was followed in short order by the $9.7B acquisition of The Medicines Company by Novartis for its cholesterol-lowering RNAi drug candidate, Inclisiran, a harbinger for how RNAi medicines will change the game in cardiovascular risk management.

Amid all the understandable investor excitement, it is probably a good time to reflect on the potential pitfalls as we move into a world where RNAi Therapeutics dominate liver-related diseases and advance into new big areas such as the CNS and ocular disease.  Remember, investor, it takes one safety event for the most highly valued programs like HBV RNAi candidate AB-729 by Arbutus Biopharma, Arrowhead Pharmaceutical’s ARO-AAT, or the Lpa program by Silence Therapeutics to decimate your investment by half or more.

Off-targeting safety, off-targeting safety, off-targeting safety

When the discovery of RNAi was rewarded with the Nobel Prize in 2006, the Andy Fire and Craig Mello hammered home a message that would resonate for the years to come: to make RNAi Therapeutics, it’s all about the delivery!

Delivery, delivery, delivery

But as the liver has been conquered and the industry is chewing on a number of liver-related therapeutic opportunities and opening up opportunities in new areas, we still have to fully understand the odds of seeing program failures due to the difficult to predict consequences of off-targeting.

Off-targeting is the moderate knockdown of sometimes dozens of genes sharing sequence homology to the target gene.  This was shown over a decade ago and soon thereafter shown to be addressed by using simple modification strategies.

This, however, was at a time when the delivery mantra was practiced and nobody wanted to risk reducing knockdown efficacy by simultaneously considering off-targeting in their RNAi trigger designs.  I was therefore upset, as it turned out rightfully so, that Alnylam let Nastech essentially go bankrupt a decade ago when it should have acquired rights to Nastech's then industry-leading off-target-mitigation technology.

Alpha-1 Antitrypsin as testbed

Alnylam would have to pay dearly for its pride in never treating a smaller competitor well, even if it served its own interest, with off-targeting manifesting itself in markers of liver toxicity in first-generation clinical programs like chronic HBV and alpha-1 antitrypsin-related liver disease.

These two indications, unlike the now approved AHP indication for Givlaari which addresses symptoms outside of the liver, represent the most exacting off-targeting challenge as they affect liver health directly and any further exacerbation of liver stress should be avoided.  

As a result, Alnylam dug out the old off-targeting literature and in their incarnation added a GNA modification at position 7 of the guide strand while, for experimental purposes, leaving the rest of the first-generation molecule unchanged. Translational science at its best (!) and the industry can be thankful for it.

Indeed, for both the ESC+ chemistry ALN-AAT02 and ALN-HBV02 no liver enzyme elevations (above 3x ULN) were observed in 49 subjects tested, whereas 3 of 33 had such elevations with the ESC-only versions.

In Arrowhead’s case, there also were no ALT elevations >3x ULN.  However, 3 treatment-emergent grade 1 ALT elevations not exceeding 2x ULN were reported in the 28 normal healthy volunteers receiving ARO-AAT.  I am curious to see the individual time curves and the doses at which they occurred.  It is certainly an area to watch for that program as it only now moves into patients. 

Add to this considerable uncertainty about patient selection, dose and duration required to have an impact on AAT liver disease, increasingly analysts topping their price targets by the day may have to re-think how they value this program (see disclosure below).

Further behind, we have to see how Silence Therapeutics and Arbutus have addressed off-targeting with first data expected in 2020.  While Silence has avoided indications directly affecting liver health (iron overload, cardiovascular disease), Arbutus with its HBV indication will have a steeper hill to climb, although the pay-off for investors in this $80 market cap company should be considerable should it climb it come Q1 2020.

Personally, my guesstimate for non-optimized GalNAc designs would be a 50% chance of off-targeting success, also depending on preclinical vetting (after all, the very clean $9.7B Inclisiran is such a design); a 85% off-targeting success for optimized designs. 

Disclosures: long Silence Therapeutics, Arbutus Biopharma; short Arrowhead Pharmaceuticals; no position in Alnylam.

Leveraging New RNAi Trigger Chemistries for Gene Knockdown in Phagocytic (and Other) Cells

Much of the achievement of solid RNAi gene knockdowns in hepatocytes (liver) by non-LNP means (Arrowhead DPCs and Alnylam’s 2^nd gen GalNAc-siRNAs) has involved the use of heavy modifications that render the RNAi triggers highly stable. This is because nucleic acids that are not protected by the delivery chemistry itself would otherwise be subject to rapid degradation in extracellular body fluids.

In the case of Alnylam’s GalNAcs, they can even function in the absence of an explicit endosomal release chemistry.  Moreover, GalNAcs and DPCs have shown more sustained gene knockdowns than is achieved with LNP delivery which historically has relied on minimally modified RNAi triggers. 

All that is required is receptor-mediated uptake into the endosomal-lysosomal pathway which is an immensely degradative environment (esp. the lysosomes).

Considering the extended duration of silencing and degradative environment, it seems as if the RNAi triggers have to be able to survive for long enough in late endosomes/lysosomes so that when they get an opportunity to escape by as yet undefined mechanism(s), they are still there ready for gene silencing action.

Such chemistry progress may also be particularly useful for RNAi gene silencing in phagocytic cells of the immune system.  This is because there have been multiple reports, especially concerning the use of LNPs (e.g. Novobrantseva et al.)  where some, but not the very robust, hepatocyte-type of gene knockdown have been obtained.  It is the ability to confidently achieve robust knockdowns that opens the gate to a flood of therapeutic applications, and this is why pushing borderline-technologies over the edge is so tremendously valuable.

Uptake into phacocytes is usually not the problem as these have evolved to scavenge for foreign particles and macromolecules.  In fact, phagocytic uptake is often a nuisance in RNAi delivery both because it may cause off-target toxicity and because it can make pharmacology less predictable.

In the case of untargeted nanoparticles such as LNPs, phagocytosis is the likely uptake mechanism.  Similar to endosomal uptake, phagosomal uptake involves the fusion with lysosomal compartments meaning that phagosomal contents are also exposed to a highly hostile environment.  Since the normal endosomal escape chemistries and mechanisms do not appear to be very effective in phagosomes, one strategy besides of possibly tailoring existing mechanisms to the phagosomal environment (e.g. lipid pKas) is to simply use the same ultra-stable RNAi trigger chemistries that are showing promise in the liver.

It is also possible that ligand-targeted conjugate approaches will be useful here, whether they enter the cells via phagocytic mechanisms or not.  In fact, Arrowhead Research (then Mirus Bio) in their seminal publication on DPCs (Rozema et al.) have demonstrated efficient uptake of DPC-conjugates into liver phagocytes (Kupffer cells) by using mannose as the targeting ligand (they haven’t tested and/or shown the corresponding RNAi knockdown though and this may relate to their using much less nucleic acid modification back in 2007). 

Since the mannose receptor is expressed on phagocytes throughout the body and not just in the liver, more extended circulation times promises the application of this conjugate-targeting strategy more generally.

Similar principles may apply to the targeting of other ‘frontier tissues’ for RNAi delivery.  However, given the degradative and differing nature of phagocytosis which means that other release mechanisms are not readily applied to this process, RNAi in phagocytes should particularly benefit from the new RNAi trigger chemistry developments.  I look forward to seeing the results.

Tekmira- A Liposomal Delivery Company No More?

Recently, I have begun to wonder whether being known as a liposomal ‘delivery company’, a company that delivers the payloads of others, is starting to hold back Tekmira’s valuation all the while high-quality RNAi Therapeutics advance towards marketing approvals in the field.  Similarly, as SNALP potency has dramatically improved over the years, particularly for gene knockdown in the liver, the effort of improving its potency may have reached a point of limited returns (~1000-fold improvement from 2004-2009, ~3-fold improvement from 2009-now) when aggressively competing with Alnylam, Arrowhead, and ISIS on the already available targets may be more important.

Consistent with such thinking, Tekmira this week announced a re-organization that goes to the core of the company.  Most importantly, former CSO Dr. Ian MacLachlan will cease to be in charge of the overall scientific direction of the company.  It has been his work on developing the various applications of liposomal delivery that defined the company.  Instead, he will now serve as the Chief Technology Officer with a particular focus on the development of medical countermeasures as exemplified by Tekmira’s Ebola program which has just moved into pivotal human safety trials.  I could imagine that with flu being an obvious target in addition to Marburg, some of his liposomal development efforts will be spent on SNALP knockdown in phagocytic cells and aerosolized LNPs for the respiratory epithelium.

Unclear to me is where this move leaves SNALP technology for oncology applications where Tekmira had indicated that they made progress in over TKM-PLK1 which is based on ~6 year-old technology.  This would offer obvious TKM-PLK1 life-cycle management and new target opportunities.  Perhaps MacLachlan will be responsible for oncology platform development as well in the form of business development initiatives just as in mRNA and AgBio that I believe represent exciting partnering opportunities.

The other half of the former CSO position will be filled by former Director Mike Abrams as the Chief Discovery Officer.  This move is clearly consistent with the company’s focus on rapidly expanding its product development pipeline with 2 INDs for new candidates planned in 2014 (HBV, alcohol use disorder) on top of TKM-PLK1 and TKM-EBOLA already in clinical development, and a series of opportunities in the rare and orphan disease space being actively pursued.

Congratulations to Ian MacLachlan for making himself redundant as the CSO and having the integrity to continue to serve in the best interest of the company and shareholders.  

Delivery versus Product Candidates? RNAi Therapeutics Business Development Strategies Need to Adapt

Since the beginnings of the RNAi Therapeutics industry, it has been a mantra that platform/enabling technology licenses are the business development way to go.  Indeed, broad and financially attractive platform deals by Alnylam and Sirna Therapeutics supported that notion.

The time has come, however, for companies focused on developing RNAi enabling technologies to carefully reconsider their business development strategies.  The biggest warning sign is that despite of the clear technological value of delivery, in my mind there has not been a financially attractive transaction in this area for over 4 years now despite the intense and also public focus on this bottleneck.

On the other hand, unlike you have carried a halo since birth similar to Regulus and Moderna Therapeutics, it is product-specific deals and attractive capital raisings based on interest in certain product candidates (e.g. TTR amyloidosis) that gives you the most attractive cost of capital right now.  Also witness the ~1 billion dollar market capitalizations of RNA Therapeutics companies Prosensa and Sarepta Therapeutics where single ultra-orphan product candidates have captured the imaginations of investors willing to pay up for a slice of the dream.  Contrast this to anemic $100M and less market capitalizations of companies with much stronger technology engines such as Arrowhead Research and Tekmira.

Misled by Big Pharma

I have intensely followed the Tekmira story and how they invested in developing SNALP and their derivatives for not just knockdown in the liver, but cancer, respiratory disease and beyond.  The apparent motivation has been that Big Pharma companies are interested in respiratory disease and especially oncology, so work on something this constituency, once believed to be the only source of capital, wants.  But is it really the right strategy to invest in something where you may get 50-60% knockdowns rather than genetically much safer 80% knockdowns and more in other tissues, all in the hope of maybe fetching low double-digit upfronts in the end?  Wouldn’t be the capital better invested exploiting what you already have and drive forward specific development candidates for a few million dollars and which the public markets may value in the hundreds of million dollars in the not-so-distant future?

Arrowhead e.g. recently decided to double its share count to raise $30-40M when it probably easily could have sold its then preclinical HepB program for that amount (see also blog entry yesterday on HepB area getting hot).  It would have gotten even less for monetizing its DPC delivery technology. the key element in the HepB program.  In the end, also as a shareholder, I agree that a 50% dilution, but retaining full control of this product was the far more attractive option.

My advice to these companies: don’t build your future on the notion that Big Pharma makes rational decisions.  Big Pharma would rather pay $100M for a bundle of RNAi trigger and delivery (=product candidate) with some type of early clinical knockdown result than $5M each to essentially get the same thing.  And in the end, you have to realize that not many companies in Big Pharma land, maybe Merck, Novartis, Takeda, GSK and AstraZeneca, would consider a platform-type RNAi Therapeutics deal these days in the first place.  So why try to please a handful of Big Pharma players which have proven to be penny wise but pound foolish when it comes to RNAi Therapeutics?

In the end, I am still hopeful that the RNAissance will lead to large technology transactions and acquisitions.  I do not believe, however, that most of the companies developing enabling RNAi technologies will benefit from such deals.  Instead, you are most likely better off focussing your attention and capital where it can create the most promising and differentiated product candidates.

The times have changed.  If Big Pharma wants a piece of RNAi Therapeutics, they have to pay up to compete with the public markets.

The RNAi Therapeutics Money is in the Product Candidates

If you believe, like I do, that the RNAi Therapeutics money ought to be in delivery, the track record suggests that you are mistaken: with the exception of what was euphemistically referred to as a $65+10M 'restructuring' of the agreement between Tekmira and Alnylam, over the last 4 years there has not been one deal where an RNAi delivery company reaped substantially non-dilutive funding for their delivery technology.  By contrast, the money was to be made in RNAi triggers and especially in partnering/licensing out RNAi Therapeutics candidates (for more details, see the RNAi Therapeutics in 2013 Report).

The failure of companies like Tekmira, Arrowhead Research, Silence Therapeutics, and Marina Biotech (plus many others not so much in the spotlight), to capitalize on the uncontested value of delivery may be due to one of the following factors:

1) their respective delivery technologies do not meet the expectations of the pharmaceutical industry;

2) RNAi Therapeutics has lost its attraction as a platform technology. Consequently, an investment in platform technologies such as delivery is not compelling;

3) Potential partners want a One-Stop Shop. Licensing in delivery AND the RNAi triggers separately can be burdensome and may mean decreased potential profits down the line (royalty stacking);

4) intellectual property issues around the payload when monetizing delivery; Alnylam and ISIS have famously sued Tekmira for their delivery deal with BMS (litigation terminated now) claiming that Tekmira was indirectly selling RNAi triggers it did not own.

I believe that all the above factors have contributed to some degree with the relative weight of the issues varying depending on the specific technology, geographies, and other company-specific factors such as the trade secret litigation between Tekmira and Alnylam that has delayed deal flow related to the most advanced RNAi delivery technology.

However, RNAi Therapeutics is regaining favor as a platform technology and I am hopeful that this will translate into the long overdue transactions, also involving the SNALP and DPC technologies by Tekmira and Arrowhead Research, respectively, which have technologies of clinical maturity (disclosure: I am a shareholder of both companies).  The two most likely customers here are Novartis and Merck.  Novartis has an active interest in RNAi Therapeutics, but the scientific and patent literature suggest that they have no tangible delivery technologies and their internal efforts are not even half-hearted so that I expect them to go out and buy in the technology instead of replicating Merck's inreasingly costly mistake of investing hundreds of millions in internal delivery research, including SNALP and DPC lookalikes, when the combined market caps of the originator companies is a fraction of that (please somebody explain that logic to me).

Nevertheless, RNAi delivery companies cannot rely on such platform partnering and need to push ahead with creating RNAi Therapeutics candidates.  This last mile is currently the most rewarding way to monetize on their delivery technologies.  Alnylam has long understood this (see recent partnering around TTR, PCSK9 and VSP), and is now reaping the rewards. 

What Alnylam has done, however, is no magic, far from it: identify genetically attractive gene targets, formulate into established delivery tech (e.g. SNALP, GalNAc), and do some biology around them to build a scientific story to be sold to the pharmaceutical and investor world.  With an established delivery technology, the hard work, or maybe better, the most uncertain factor has been removed, so it's pretty smooth sailing from there as long as the gene target is good and desired by the industry.

It is thus ironic that platform companies get loved for their specific product candidates (not just in RNAi Therapeutics it seems), and not for the technologies that critically enabled them. For RNAi delivery companies, it is important to swallow this scientific toad and invest the additional $5M or so it takes to build that IND-enabled package to reap disproportional rewards.

Sifting Through the Noise in RNAi Therapeutics Delivery

Hardly a day goes by without news of delivery deals, or universities issuing a press release that their scientists have just published on a revolutionary and ‘novel’ delivery technology that, by the way, is available for licensing. To be fair, public relations is a necessary evil of drug development and even academia these days, and don’t expect any company to blink and admit to their weaknesses.

This, however, poses a real problem for RNAi Therapeutics as the noise causes investments to be diverted away from the most deserving technologies, technologies that are more than just wishful theoretical thinking and with reasonable paths towards the clinic. There is, of course, also a place for the more audacious technologies, but this is what universities are for.

When reading about a new delivery breakthrough or deal announcements from sources you have hardly heard of before, it pays to browse through the peer-reviewed and patent literature to get a first impression of whether there is anything of substance to start with. Rest assured, a new delivery technology, especially if ground-breaking, won’t successfully go from scratch into the clinic within the next 10 years.

I don’t want to name any deal or company in particular, but I would also caution you not to put too much faith into the delivery claims of those biotech companies that are run by management and Directors that constantly chase the latest and hottest in science in order to generate constant buzz around their companies so that more shares and options can be issued and dumped on retail investors. Be wary of those companies that get regularly promoted in cheap analyst reports listed on Yahoo! and Google Finance pages, companies that select academic collaborators not based on how they can contribute technically, but based on politics and fame, and where companies are happy to have rumors spread on message boards.

When asked by a newspaper reporter what, in my mind, were the key publications in RNAi Therapeutics delivery over the last 3 years, I answered that it’s not been one or two, but the series of papers describing the transitioning and refinement of SNALP delivery technology from the first non-human primate proof-of-concept data reported in 2006 in Nature into the clinic today. Needless to say, despite the criticality of these de-risking events, also as it relates to the overall investment climate in RNAi Therapeutics for the coming years, this wasn’t picked up as being quotable.

There is no easy solution to this capital allocation problem. All one can hope for is that some of the more deserving technologies can build a network of supporters that, among the boom and bust cycles of the industry, will see them through to at least early clinical studies where they will have to prove their mettle.

Targeted Delivery Strategies Coming to the Fore

Hand in hand with a rapidly expanding understanding of the biological mechanisms of RNAi Therapeutics delivery, we are hearing more and more about ligand-guided targeted delivery. Just today, mdRNA announced notice of allowance for a patent application on the identification of a peptide specifically binding to the cell surface of hepatocellular carcinoma cells, and Alnylam has recently started to talk about their discoveries on ApoE-dependent and Apo-E independent SNALP cellular uptake pathways paving the way towards new targeted SNALP delivery strategies (Systemic RNAi Delivery Roundtable). As it increasingly looks like Calando’s transferrin-targeted CALAA-01 will not remain the only targeted RNAi Therapeutics formulation in the clinic for long as targeted delivery is set to provide the next push towards more potent and safer RNAi Therapeutics delivery, I will try and briefly explain the rationale behind targeted delivery and some of the challenges that need to be overcome.

Success in targeted delivery is measured by either preferential uptake of the siRNA in the target tissue compared to non-target tissues or achieving lower efficacious dosages by taking advantage of particularly productive receptor-mediated uptake pathways, preferably both. The detailed pharmacological effects are not only determined by the ligand, but also by where it is attached to. Steps that may be affected can include biodistribution, cellular uptake once at the target tissue, or the avoidance of certain cells and tissues.

SNALP delivery for example may benefit from targeting ligands by reducing uptake by macrophages or relying on non-specific charge-charge interactions for cellular uptake, both of which can be safety liabilities. For the liver, de-targeting from macrophages may be even more important than active targeting. The DPCs by Mirus (now Roche) were particularly exciting here in that the data suggested that the right presentation of ligands (simple sugars in this case) on the particle surface may eliminate the unspecific uptake of a delivery platform and instead re-target it to new cell types. Equally exciting data by Alnylam suggests that it should be possible to greatly limit the ‘non-specific’, mainly ApoE-mediated uptake of ionizable SNALPs by shielding their surface and then re-direct them by adding new ligands on their surface. By then further increasing their circulation times through creating very stable particles (e.g. by increasing the stability of the stealth shield), a delivery platform may then also be applicable to new therapeutic application fields by increasing the chances that a ligand recognizes receptors in distal tissues.

Lipoplexes such as Silence Therapeutics’ Atuplexes may also benefit from targeting ligands. Since the interaction of immune cells with blood endothelia is very well studied this may e.g. allow it to be targeted to specific endothelia such as the blood-brain-barrier.

Targeting ligands are already part of many siRNA-conjugate approaches. Achieving endosomal release in addition to cellular uptake is a big challenge for this area of delivery, and it will be interesting to see whether in fact those receptors that prove effective for nanoparticle delivery may be the types of receptors to be avoided for siRNA-conjugates in favor of channeling them into more non-specific pathways.

There are, of course, also challenges associated with targeted delivery. One is to identify suitable ligand-receptor interactions as endosomal maturation processes can differ greatly, e.g. in the degree and rate of acidification and receptor recycling, which imposes new types of pharmacokinetic demands on a delivery system.

A systematic effort to discover the best receptors may be to screen a panel of siRNA-nanoparticles containing (single-chain/nanobody-type) antibodies on their surface and that are targeted to a wide array of cell surface receptors and then select those with the best silencing results. The most promising receptors, hopefully patentable, may be pursued then either with the antibodies themselves or alternative, smaller ligands. New ligands to given receptors may be discovered through panning peptide display libraries against that receptor, something e.g. that mdRNA does with their trp-cage peptide libraries.

Avoiding adaptive immunity, especially to novel designer ligands is another added challenge for targeted delivery. And finally, when all these questions have been answered, the not-so-trivial task is to find formulation methods that allow for clinical and commercial scale-up of the more complex particles. At the end of the day, however, it is those platforms for which a detailed mechanistic basis has been established and those teams that have turned formulation into an art that will succeed. It can be done.

Money from the Sidelines Moving into RNAi Therapeutics Again

The recent signs that the appetite for RNAi Therapeutics by Big Pharma is returning coupled with accumulating evidence that RNAi can be triggered in Man with some of today’s delivery technologies, is finally translating into investors buying into the promise of RNAi Therapeutics again. Today’s almost doubling in the shares of Arrowhead Research (ticker: ARWR) on volume of over 40% of outstanding on the heels of a Nature publication showing that their RONDEL delivery system was able to induce an RNAi mechanism of action in solid tumor tissues in real patients, is quite impressive evidence for this. The incredible performance of RXi Pharmaceuticals (ticker: RXII) which has made much out of their ‘self-delivering siRNAs’ and who are also positioning themselves as an RNAi trigger alternative to Alnylam another one.

If the trend were to indeed continue, this time around I would expect much of the action to be in the limited RNAi delivery technologies that have made it into the clinic. Except for the $125M acquisition of Mirus Bio by Roche, I have long felt that the RNAi Therapeutics marketplace and investment community has never really reflected the importance of delivery in making RNAi Therapeutics a reality. In addition to being the critically enabling factor for RNAi Therapeutics, such IP should also be the most valuable kind.

The argument for delivery-related investments is based on the expectation that any Big Pharma that is seriously considering RNAi Therapeutics would first want to secure access to technologies that have shown potential as clinically viable platforms. This would allow them to gain familiarity with RNAi Therapeutics and, by being on its cutting edge, assemble know-how and lay down further IP to help position them as leaders in the field, an opportunity that was missed in the case of recombinant proteins and monoclonal antibodies.

A scarcity of technologies that have made it into the clinic providing some evidence of RNAi efficacy in the absence of show-stopping toxicities should further increase their value. At the moment these are SNALPs, RONDEL, Atuplexes, a lentivirus and tkRNAi bacteria. You will not be surprised to hear that based on the strong and abundant pre-clinical, including non-human primate data, highest quality science, modularity as a platform, well-defined structures and scalable manufacturing, and the proven ability to assist partners in bringing their RNAi triggers into the clinic, I am particularly fond of SNALPs and Tekmira. I cannot believe that companies like Novartis or Pfizer do not consider it a risk that access to Tekmira could be lost due to an outright acquisition of the company.

In times of cost-cutting, it is important for companies that want to partner or sell their technologies to keep the necessary talent to make technology transfer possible. Such a group of people would also enhance the attraction to acquire a company as an important pillar of the broader RNAi Therapeutics efforts of a Big Pharma, similar to what Alnylam Europe was for Roche or Coley for Pfizer. Losing such capabilities could quickly render a technology stale as Targeted Genetics seems to be running the risk.

All the ingredients of a small acquisition wave of RNAi Therapeutics technologies and companies are therefore in place: a limited supply of enabling technologies and scientific talent, capital markets that make it difficult, if not impossible for small RNAi Therapeutics companies to realize the full value of these technologies on their own, and big pharmaceuticals that will consider the recent results by Tekmira and Calando/Arrowhead as important de-risking events and feel the urgency to act now.

RNAi Therapeutics Delivery Wave Continues to Build

After listening to the presentations of a number of RNAi Therapeutics companies at the BIO CEO & Investor Conference, I came away thinking that while we are rightfully focused on discussing the challenges of RNAi Therapeutics, underneath of it there is a powerful wave of RNAi delivery solutions building and that is making its way into the clinic.

As a number of the presenters rightfully pointed out, there is not one universal delivery solution, such that this wave consists of various approaches addressing one cell and tissue type after another. Given that a cell/tissue is relevant for many diseases and given that there may be more than one suitable cell/tissue type for most diseases, this means that RNAi Therapeutics is quickly becoming broadly relevant for drug development. It is also true that while 21-23bp siRNAs will be the sweet spot for most applications, different siRNA lengths may work best for different delivery solutions, and which is facilitated by the remarkable robustness of RNAi in our cells that efficiently recognizes many forms of dsRNAs as substrates for RNAi.

To mark the occasion, I have compiled below a list of organs together with what I consider the leading respective delivery technologies. The list does not seek to exclude other technologies that could also be used for a given organ, but which are somewhat behind in development. The list is in order of clinical maturity with green highlighting those that I consider ready for prime-time, orange for those that are just on the verge, and red those that look very promising, but will require more research before I would feel confident about their clinical success.

The orange category is particularly exciting, because RNAi in immune cells looks like a low-hanging fruit waiting to be picked. Particularly when it comes to phagocytic cells such as dendritic cells that are important for vaccines, the opportunity lies in taking advantage of the natural tendency of siRNA nanoparticles to be taken up by such cells while there is increasing evidence that with the right chemistry you can get functional release of siRNAs into the cytoplasm instead of their degradation in phagosomes. I similarly feel that a renaissance of RNAi for the eye is in order given that DNA vectors and siRNA-conjugates are just begging to be applied as the unmet need grows in the exponentially ageing population.

Liver: cationic liposomes

Solid cancer proper: cationic and targeted liposomes

Endothelial cells: lipoplex/Atuplex

Phagocytic cells: liposomes

Other immune cells (incl. cancer of blood): targeted immunoliposomes

Eye: lipophilic-siRNA conjugates and ddRNAi (AAV and lenti)

Respiratory epithelia (incl. lung): modified siRNAs (inhalation; topical)

Brain (instillation): lipophilic-siRNA conjugates and ddRNAi (AAV and lenti; instillation)

Injured skin: lipophilic-siRNA conjugates (topical)

Heart and intestine: lipophilic-siRNA conjugates in reconstituted lipoproteins (intravenous)

Kidney: modified siRNAs (intravenous)

RNAi Therapeutics: A Spoilt Market, or Waiting for Clinical Progress?

The market reaction to Alnylam’s deal with Takeda was, well, a bit disappointing. Although the argument can be made that next to the Sirna acquisition by Merck this has been the most favorable deal for the RNAi Therapeutics field yet, shares of Alnylam not only did not advance, but even declined on the news. Can the muted reaction be explained by a market spoilt by Alnylam’s RNAi mega-deals and that has come to expect an even larger cash component, or has Alnylam’s hinting at more deals given the shorts plenty of time to prepare for such an event by aggressively shorting the stock on the news as the considerable volume may have suggested? Possible. However, here I would like to consider the third possibility that for Alnylam’s stock to move to new highs, the market would like to see the deal making being complemented by scientific and clinical progress.

Actually, these platform licensing agreements are evidence for just that as the therapeutic areas covered by the non-exclusive license, namely metabolic disease and cancer, clearly indicate that the deal was driven by the maturation of liposomal RNAi delivery technology. Moreover, an important component of the Alnylam-Takeda relationship will be the exchange of RNAi delivery capabilities, suggesting that Takeda had also made progress in this area.

However, the bears would argue that what Alnylam, and the wider field, needs is further demonstrating progress with their development programs. Of course, we had the phase II ALN-RSV01 proof-of-concept data which was a great relief for everybody and should strengthen Alnylam’s position at the deal table if not on a PPS basis, but we should also remember that Alnylam had previously guided that it would advance one or two SNALP-related programs into the clinic in 2007. This has not happened and I can understand that some may have well been disappointed by that. Paradoxically, rather than reflecting science that has stalled, the delay can be explained at least partly by ongoing progress in SNALP RNAi delivery and it may not be wise to commit to a development candidate when improvements in both safety and efficacy can be made so readily as supported by recent conference presentations and publications.

Having followed biotechs for a while now, one major question I ask myself before investing in a biotech company is whether programs have been rushed into the clinic well before they have been properly validated pre-clinically. That often happens with small biotechs with acute fund-raising needs or when existing investors would like to exit and an IND is the goal, not final approval by the FDA. How otherwise could one explain e.g. Nucleonics’ decision to advance a plasmid-based, DNA-directed shRNA program for Hepatitis B into phase I when all the in vivo data consisted of some co-transfection studies (= glorified in vitro data) and when current non-viral plasmid delivery methods are unlikely to achieve the transfection rates sufficient for curing such a viral infection through direct gene knockdown. Next to Nucleonics, other early RNAi pipeline candidates, while somewhat more promising scientifically, may also serve the role of flag-waving programs demonstrating to investors that RNAi is not just a fascinating science, but also of clinical relevance.

Much has been learnt since the first candidates entered the clinic in 2004 and, like for SNALP RNAi in particular, it makes a lot of sense, also economically, to take advantage of the steep learning curve in general to increase the odds of late-stage pipeline success at the cost of slight early delays. Meanwhile, the irresistible expansion of RNAi into in vivo applications and the increasing number of RNAi-related conferences (another one, Beyond Genome, is just about to take place a little bit North of where I live) demonstrate that the field is vibrant and the science is making rapid progress.

If that weren’t enough, the coming months should bring more than enough RNAi clinical results to contemplate. Calando just reported this past week initial results from their phase I studies on CALAA-01, an unmodified siRNA in a targeted nanoparticle formulation for the treatment of solid tumours. According to their press release, the first patient has now completed a course of 4 intravenous doses over two weeks without any show-stopping adverse events. As this is the first clinical experience of systemically administered siRNAs, unmodified at that, it therefore also marks an important and reassuring milestone for the synthetic siRNA field in general. It is easy to envision scenarios how an adverse event could have had disastrous repercussions for RNAi Therapeutics. Well done, Calando!

Then there are the SNALP RNAi programs that, once entered, could yield relatively soon clear in vivo efficacy data on systemically administered RNAi Therapeutics. Finally, Benitec’s HIV program is another program that deserves some attention as quantitative patient data may emerge relatively soon; for example, an expansion of T-cells derived from the stem cells transduced with the lentiviral vector encoding for a triple-RNA antiviral relative to unmodified cells would be very promising.

RNAi Therapeutics versus Antisense- Where Delivery Makes a Difference

Regulus, the microRNA joint venture between Alnylam and ISIS Pharmaceuticals, is the most visible manifestation of the scientific overlap that exists between antisense and RNAi. The overlap, however, is not just limited to the science, but also extends into the capital markets. To better help the investor differentiate between the two technologies, I’d like to use this blog to provide an overview of some of the fundamental differences underlying the development of RNAi and antisense into therapeutics and their long-term prospects, with an emphasis on delivery. Needless to say, beware this discussion will be heavily biased in favor RNAi, but then again these are the reasons why I’ve been attracted to RNAi in the first place.

Certainly, progress in both areas in the last 3-5 years has mutually benefitted the investment climate for both technologies as it has heightened interest and increased confidence in RNA therapeutics in general. However, the two technologies also compete for investment dollars with many of the same investors, which are typically upbeat about the future of gene-based medicines but unsure where to place their bets, allocating their investments based on where they see most promise. One issue that often comes up in making this decision is the observation that while for systemic applications antisense, as practiced in the most advanced programs today, is typically administered without a particular delivery formulation, the development of specialized delivery technology is frequently cited as the key challenge for RNAi to realize its ultimate therapeutic potential.

Antisense for gene knockdown works largely by two mechanisms: interfering with translational initiation (e.g. AVI Biopharma's morpholinos) or through an RNase H-type mechanism (e.g. Santaris and ISIS Pharmaceuticals). For this, the key factor is to achieve efficient hybridization of a single-stranded oligonucleotide antisense with its target mRNA which either prevents productive ribosome association to the mRNA (inhibition of translation initiation) or may be recognized as a substrate for the RNase H enzyme which may degrade the RNA portion of the mRNA-DNA duplex, but normally functions in the degradation of the RNA primer during DNA replication.

Various oligonucleotide chemistries have been developed to optimize these processes for in vivo applications. Essentially all of these are single-stranded oligos of which the sugar phosphate backbone is heavily modified to a) increase their in vivo stability; b) improve their pharmacokinetics and avoid rapid renal excretion by promoting their association with components of the blood; c) similarly allows them to be retained in tissues; d) facilitate crossing of cell membranes; and finally e) increase their target mRNA binding. By contrast siRNAs, because of their charge and more rigid double-stranded nature and with apparently some exceptions that include mucosal epithelia, do not cross cell membranes efficiently on their own and therefore need to be specially formulated for most applications.

The use of unformulated antisense is consistent with their mechanism of action. Since antisense does not harness a naturally existing endogenous gene silencing pathway, it relies on achieving concentrations of oligonucleotides in the target tissue over a prolonged period of time that are high enough such that, as a result of the rules of thermodynamics, a sufficient fraction of target mRNA will be recognized. Similarly, unlike RNAi, the specificity of antisense is largely governed by biophysics and benefits only relatively little from biological proof-reading.

In practice, to achieve the necessary tissue concentrations, patients are typically dosed frequently at the initiation of therapy so that the tissue concentrations reach steady-state therapeutic levels. Targeted delivery of antisense into cells of interest may allow one to achieve a knockdown earlier, but any benefit would only be short-lived as antisense is not retained in specific gene silencing complexes but will soon redistribute according to their partition coefficient throughout the entire tissue so that ultimately similar amounts have to be administered and a formulation would only be a nuisance with little benefit.

By contrast, RNAi harnesses an endogenous and catalytic gene silencing mechanism, which means that once it has been delivered, either by conjugation or in nanoparticles into the cytosol, they are efficiently recognized and stably incorporated into the RiSC silencing complex to achieve prolonged gene silencing. In fact, measurable RNAi-mediated gene silencing can be observed at siRNA concentrations so low that it becomes difficult to detect them (e.g. fluorescently-tagged siRNAs by microscopy). This means that as the majority of siRNAs that do not reach the cytoplasm may disappear quite rapidly, the total exposure of the body to the nucleic acid can be much lower compared to antisense which should be beneficial both in terms of safety and pharmacodynamics (activity profile of drug over time).

This is not to say that chemical modification is not practiced in RNAi. However, unlike in antisense, the purpose of modification in RNAi is mainly to avoid triggering innate immune responses, making the siRNA sufficiently stable so that they survive their journey into their target cells, and also to stabilize them as part of RiSC (Merck has been talking about that concept on several occasions); and as we learn more about the biochemistry of endogenous RNA silencing pathways, modification is also increasingly used to increase the inherent biological specificity of RNAi. Unfortunately, it is surprising to me that compared to RNAi only very little, if at all, is reported about the specificity of antisense and I would be grateful if somebody here could point out pertinent studies that I should be aware of.

Targeted delivery may also avoid unnecessary drug exposure of non-target tissues. For unformulated antisense, no matter what the indication and target tissue, the biodistribution is essentially the same, and toxicities of the liver and kidney due to extended exposure to large amounts of the heavily modified antisense compounds is well known.

Certainly, improving the therapeutic index is an important issue for RNAi Therapeutics, too, but as the many transgenic mouse models which express ample and highly efficient RNAi throughout their life without causing overt toxicity attest, ultimately the improvement in the therapeutic index of RNAi is not limited by its very mechanism of action.

While it is a certainty that antisense companies will come out with 4th and 5th generation antisense technology, advances after decades of antisense research aiming to improve target mRNA recognition will only be marginal and based on trying out yet more nucleic acid modifications, although it appears to be a challenge to improve upon the efficacy of probably the most potent antisense modification that have now been known for a while, namely LNAs and their derivatives.

While RNAi efficacy in animals has already surpassed that of antisense for applications of the liver and lung as well as other tissues, I am confident that future advancements in RNAi will be more than marginal. For example, even as recent liposomal formulations achieve 90% gene knockdown in the liver at 1mg/kg, this still means that only about 1 in 10,000 siRNAs that have reached the liver makes it into the cytoplasm (assuming it takes about 1000 cytosolic siRNAs to achieve that level of knockdown according to a recent presentation by Phil Sharp). Alone a better understanding of the endosomal uptake of these nanoparticles, which is only in its infancy and starting to be explored, should allow for more than incremental improvements in the therapeutic index of RNAi Therapeutics.

And if you are still undecided on where the future is heading, numerous transfection studies in vitro where it can be assumed that equal amounts of antisense and siRNAs are present in cells, have shown that RNAi is quite a bit more potent on a mole-by-mole basis comnpared to antisense.

I am aware that some in the antisense community, including investors, may take offense with this blog, but since I am often asked about this issue, I think a more straightforward approach is better than to keep beating about the bush. And, of course, there is always the comment section.

Looking Ahead: Alnylam-Medtronic Alliance Stop-Go Decision Expected Soon

In early 2005, Alnylam and Medtronic announced a joint product development program to evaluate the use of Medtronic’s implantable drug delivery devices for RNAi Therapeutics applications of the central nervous system (CNS). If at the end of this development program a joint decision for the continuation of this program were made, Medtronic would take an equity position in Alnylam and additional investments following the achievement of certain milestones.

According to regulatory filings, the original decision making due date was April 2007, but was then postponed to July 2007. It is interesting to speculate that this delay may have been either caused by scientific uncertainties or is the consequence of drafting a comprehensive and therefore complicated co-operation agreement. Certainly, in development time-frames, 3 months do not seem to be sufficient to reasonably expect major new breakthroughs had there been fundamental scientific problems.

Medtronic realised early on the potential of RNAi as a new class of drugs that could be used with their drug delivery devices. Only shortly before the 2005 agreement, in August 2004, Medtronic published a patent on the treatment of neurodegenerative diseases such as Parkinson’s, Alzheimer’s, and Huntington’s, through intracranially delivered siRNAs using Medtronic’s implantable catheters (United States Patent 20040162255). Two years later, a similar patent was published for the treatment of neuropathic pain.

While pertinent in vivo data are lacking in these patents, it is clear that Alnylam already has amply tested Medtronic’s devices. Josephine Lai from the University of Arizona Health Sciences Center in Tucson, a Alnylam collaborator, for example has published strong in vivo data on intrathecally delivered siRNAs [Luo et al. Mol. Pain 1: 29 (2005)]. It is also of note that in this study, siRNAs were most efficient when formulated in the liposomal i-Fect transfection reagent from Neuromics. The same reagent was also successfully applied in rescuing mice from lethal flavivirus infection of the CNS [Kumar et al. Plos Med. 3: e96 (2006)].

One potential limitation with the local delivery of siRNAs and shRNA vectors such as AAV, however, is to achieve sufficient diffusion of the RNAi inducing agent to its target cells. That diffusion of macromolecules in the dense matrix of the CNS may also be an issue with a device from Medtronic similar to the one Alnylam is evaluating, is suggested by the failure of a phase II clinical experience of the neurotrophic factor GDNF for Parkinson’s [Salvatore et al. Exp. Neurol. 202:497 (2006)]. This particular experience, however, has to be taken with a grain of salt, as there appears to be some controversy about the interpretation of the trial results [Slevin et al. Annals Neurol. 59:989 (2006)]. A positive announcement on the Medtronic-Alnylam alliance may therefore indicate that it is possible to achieve sufficient siRNA diffusion.

Nevertheless, local delivery may not be sufficient for diseases where the whole brain has to be targeted, such as in viral infections and brain tumours. Here, systemic delivery solutions are needed. While the blood-brain barrier has historically been regarded as a major obstacle to achieving that goal, targeted approaches such as the rabies-peptide conjugation approach that I have highlighted in my June 19 Blog (“New Breakthrough in the Systemic Delivery of RNAi for the Brain”) and similar efforts make me hopeful that a range of both local and systemic delivery strategies will eventually prove successful in addressing a number of diseases of unmet medical need. These issues also highlight once more, that it will be critical to carefully tailor the delivery of an RNAi Therapeutic to its disease application.

Blog Watch: The ‘Old Vic’ blog talks about a possible takeover or significant RNAi Therapeutics alliance involving Silence Therapeutics: http://www.sharescity.com/2007/07/silence-therapeutics-takeover-rumours.html

New Breakthrough in the Systemic Delivery of RNAi for the Brain

One often cited challenge for the wide application of RNAi in the treatment of disease is the systemic delivery of the RNAi inducing agent to the appropriate target cells. Systemic delivery is the ability to deliver RNAi to target cells following intravenous or even oral administration, whereas local, also called “Direct RNAi” approaches for eye, lung, and mucosal diseases appear to be less of a challenge.

Neurodegenerative diseases and other brain-related diseases are an attractive area for the application of an RNAi therapeutic, due to significant unmet medical needs and the existence of well validated gene targets. The delivery of RNAi to the CNS, however, is challenging due to the presence of the blood-brain barried (BBB) which makes it difficult for macromolecules to exit blood vessels and enter the CNS. First generation RNAi therapeutics for the CNS may therefore rely on direct injection or device implantation as is illustrated by the current Alnylam-Medtronic collaboration. Systemic delivery, however, would have the added advantage that a therapeutic would be well distributed throughout the brain due to the extensive vascularisation of the brain. Local administration strategies often suffer from the fact that the therapeutic agent is restricted to the site of application.

The paper published this Monday online in the leading scientific journal Nature by Kumar et al. shows that the ability of the rabies virus to cross the BBB can be conferred onto an siRNA if it was non-covalently linked to a 29 amino acid peptide from the rabies virus. Although the mechanism by which this peptide achieves this is unclear, the authors show that once in the CNS it binds to acetylcholine-receptors on neuronal cells and concomitantly delivers the siRNA load to those cells. A number of endogenously expressed genes were thereby targeted achieving 30-70% knockdown efficiencies, including a 70% reduction in SOD-1 activity.

SOD-1 when mutated is linked to the debilitating neuronal disease ALS (Amyotrophic Lateral Sclerosis; aka Lou Gehrig’s) for which there is no real treatment. Interestingly, preclinical therapeutic effects have previously been achieved with lentivirally delivered shRNAs. It is therefore hoped that ultimately RNAi may offer the first treatment alternative for ALS that addresses the underlying cause of the disease.

In another impressive demonstration of the system, almost all mice infected with an otherwise fatal dose of Japanese Encephalitis Virus were rescued when they received the rabies-peptide siRNA combination. This follows a report last year by the same group that single siRNAs could also protect against the related West Nile Virus.

This report demonstrates that progress in the delivery of RNAi Therapeutics can come from unexpected sources and illustrates the force and commitment with which this technology is moving forward. It is reasonable to assume that one by one, similar solutions and improvements to the RNAi delivery challenge will come for other difficult-to-target tissues. Further improvements to the present system should also be possible, as the authors suggest, for example through covalent conjugation of the siRNA to the peptide to avoid premature dissociation of the siRNA from the rabies peptide. It is also tempting to imagine that high-throughput approaches such as one involving peptide phage display libraries could yield siRNA-peptide combinations for other tissues of the body.

The key will be that the results can be reproduced in another laboratory, is easy-to-use, and similarly non-immunogenic in man as it is in mice. In that case, I would not be surprised to see the technology being licensed by a major RNAi Therapeutics company. The proximity of Harvard to Alnylam’s Cambridge, Mass., headquarters certainly makes this company a prime candidate for that. It should also be noted that one of the co-authors, Beverly Davidson from the University of Iowa, an acknowledged RNAi expert with experience in brain-related RNAi applications, had been associated with Sirna Therapeutics prior to their acquisition by Merck.

A Framework for Progressing the Delivery of RNAi Therapeutics

The delivery of RNAi to the desired target organ is perceived by many to be the main hurdle before RNAi Therapeutics can have a universal impact on treating diseases. Arguably today already, it probably is possible to treat dozens of diseases for the eye and lung alone with current siRNA delivery methods. Furthermore, it is possible to efficiently knock down genes in the liver of primates, either with siRNAs formulations or viral gene therapy vectors. This is because the liver is the first pass organ for most drugs and highly vascularised thereby facilitating drug uptake.

The liver, however, demonstrates already some of the challenges we are facing. In the case of viral vectors, immunogenicity issues need to be addressed to make sure that immune responses against the vector will not abolish long-term gene knockdown. This is being addressed with efforts such as identifying novel serotypes or modifying pre-exising ones such as in the AAV field, but any of these solutions have yet to undergo testing in humans. For non-viral siRNA delivery, the main challenge at the moment is to get highly efficient gene knockdown without causing liver damage. It should be noted that limited liver damage is associated with a number of drugs on the market, however the degree of the damage needs to be tightly monitored. Needless to say, as with any drug, there is always a risk-benefit to consider. Alnylam published last year a study in Nature in which they show highly potent gene knockdown in primate liver. That in itself is a major achievement as only 2 years ago systemic delivery in humans was thought to be far on the horizon, and now it may happen much sooner. However, it was reported in the same study that there was a dose-dependent elevation of liver enzymes associated with the liposomally delivered siRNAs, in some cases quite high. Having established effective gene knockdown in primate liver, I would now expect the teams at Alnylam, Tekmira, Protiva, and Merck/Sirna to work on modifications of the liposomal delivery strategy that marries efficacy with little to no delivery-related side-effects

Beyond the liver, the reticuloendothelial system, kidney, intestines, and cancer, are probably next on the list of organs being targeted by systemic delivery. I expect more varied approaches to be taken to reach these organs, especially the use of targeting strategies such as adding cell-type specific targeting agents such as antibodies, peptides, or RNA-aptamers to a core delivery vehicle. This is an area where I see innovation particularly coming from the academic arena due to their freedom to explore. As a corporate strategy, however, I would be cautious in moving poorly characterised delivery strategies into the clinic, and put emphasis on further understanding and optimising promising technologies that exist today, such as the lipid-based technologies mentioned above. With scientists and funders working on all these fronts, it should be possible to gradually widen the scope for RNAi Therapeutics in a well-managed manner. From a commercial perspective, there are enough targets around now to focus on for the credible players in the field. I should add, however, that for orphan diseases or terminal diseases, more innovative strategies should definitely be considered for the clinic today.

Finally, I would like to appeal to all those involved in legal proceedings about who owns certain IPs in the delivery area. Please stop and realise that this is not helping anybody here, except of course for certain lawyers. It should be possible to set aside bruised egos and reach agreements with which everybody can live with that contributed to the development of a promising technology such as the liposomal delivery of siRNAs to mention just one example. Challenges such as liver toxicities associated with some of the liposomal vehicles are best solved when the scientists that co-developed and understand it best have access to.

The Challenge

Being hailed as the biggest breakthrough in biology of the last decade or two and being reproducible in the Petri dish is no guarantee that drugs based on RNAi will successfully make it as drugs. Three main hurdles need to be taken for this to come true: 1) efficient delivery of the RNAi agent to the target cells, 2) managing off-target effects, and 3) endowing RNAi molecules with drug-like properties.

The efficient delivery of the RNAi agent is frequently cited as the main hurdle to the wide application of RNAi. While local delivery such as needle injection into the eye for ocular diseases or inhalation for lung-related conditions has a relatively high likelihood of success, systemic delivery, e.g. needed to reach metastatic cancer cells hidden in the body, is a taller order. However, a number of delivery methods are being tested, some borrowed from older oligonucleotide-based technologies like liposomes, some more innovative. It is likely that no one-fits-all solution will emerge, but strategies that are tailored to the specific disease. Particularly interesting are approaches where a synthetic siRNA is coupled to agents such as monoclonal antibodies or RNA aptamers which can selectively target cells. Hence, the specificity of the siRNA is compounded by the specificity of its delivery. This should also help in reducing the likelihood of potentially harmful off-target effects.

Off-targeting, the suppression of non-targeted genes is mainly a consequence of the siRNA acting like a microRNA. MicroRNAs are related endogenous 20-24 nucleotide small RNAs that recognise their targets through less than perfect complementarity. Although this typically does not downregulate target genes as dramatically as an efficient siRNA might do, nobody can be sure that a 2-3 fold reduction in a “random” gene will not have adverse side-effects. I should stress that this specificity is probably still much better than many other drug-classes today, but we would like to do better, especially when human health and the substantial resources needed for the development of a drug are at stake. Helped by the knowledge of the human genome, bioninformatics already can winnow down the number of potential off-targets, thus reducing the likelihood of an adverse side-effect. More lately, however, and as demonstrated by the work of Dharmacon scientists, it has become possible to modify the siRNA such that it will lose much of its microRNA abilities while retaining potent RNAi-like cleavage potential. I view this as a particular exciting development.

Chemical modification of siRNAs can also help to enhance its drug-like properties such as half-life, metabolism etc. However, it was only quite recently shown quite by Alnylam Pharmaceuticals, viewed by many as the leading RNAi Therapeutics company, that siRNAs may knock down genes with a profile that may allow dosing every 2 to 4 weeks. This is a great relief and opens up RNAi for many more, particularly chronic applications such as hypercholesterolemia, than would have been the case if the knockdown was limited up to 4 days after administration. The latter is typically observed with cultured cancer cell lines and is due to dilution of the siRNAs following frequent cell divisions. For some genetic diseases, viral gene therapy vectors for the expression of hairpin RNAs that are processed to siRNAs by the endogenous RNAi machinery are a further important option. They should allow for the more long-term expression of RNAi effector molecules, although the risk-benefit equation is shifted for DNA-based therapies.

In summary, many strategies are being explored to develop RNAi as a safe and efficient therapy. While human trials for each siRNA will have to be performed to evaluate its specific therapeutic value, as the risk of RNAi Therapeutics gets more manageable each day, it promises to become one of the most specific and versatile drug class to date.