Stabilizing RNAi Triggers against Cytoplasmic Degradation Pays Dividends

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

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

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

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

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

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

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

RISC-optimized ultra-stable single-strand RNAi triggers

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

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

Antisense Therapeutics Dressing Up with RNAi Delivery

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

   

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

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

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

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

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

   

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

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

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

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

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

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

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

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

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

ISIS ssRNAi Challenge to Gold Standard RNAi Delivery Comes up Short

The esteemed editors and reviewers of CELL got it wrong this time.  Usually, when a high-impact journal like CELL decides to publish back-to-back papers on a given topic, it believes that they mark a turning point of some sort that will be cited all over.  In this case (Lima et al.; Yu et al.), the turning point would be nanoparticle, or more precisely SNALP-formulated RNAi delivery ‘out’, and unformulated, single-stranded RNAi ‘in’.

For those new to my blog: it is the double-strand feature that is a defining property of the RNAi mechanism.  While a single-stranded intermediate is generated in the process, numerous studies, including indeed the one by Lima et al., show that these are contrived, and consequently about 100-fold less potent ‘inducers’ of RNAi gene silencing.

It is therefore surprising that CELL would publish a confirmation of this.  What is new though is that the in vivo ssRNAi data involved unformulated ssRNAi application, whereas previous in vivo ssRNAi work by e.g. Merck involved LNP-mediated delivery (Haringsma et al., 2012; also covered on this blog here).  However, as detailed below, the efficacy was not impressive. To me, the main point of interest related to chemistry and how this sheds light on the basic RNAi mechanism, which actually made this paper enjoyable to read.  For example, the metabolically stable 5'-(E)-vinylphosphonate modification and the positive effect of 2’F on Ago binding.  Nevertheless, such biochemical detail is not the groundbreaking stuff that lands you a paper in CELL, but more something for the dedicated aficionado.

Does ISIS feel threatened by SNALP delivery?

The complexity of SNALP delivery, by which actually the difficulty of re-engineering SNALP technology without access to Tekmira’s trade secrets and know-how is meant, is held against the technology also in a commercially competitive sense.  If patient outcomes is the main goal, as long as you master complexity, isn’t that a good thing, especially in terms of the all-important length of market exclusivity (note that the main cost of SNALP delivery is still the siRNA ingredient)?

A common criticism of my writings is that I connect all things to Tekmira’s SNALP technology.  But read the Lima et al. paper and see for yourself how ISIS equates formulated RNAi delivery with liposomal delivery (start of the abstract e.g.): 

‘The therapeutic utility of siRNAs is limited by the requirement for complex formulations to deliver them to tissues. If potent single-stranded RNAs could be identified, they would provide a simpler path to pharmacological agents. Here, we describe single-stranded siRNAs (ss-siRNAs) that silence gene expression in animals absent lipid formulation.’  

Or

'However, in their current state, the therapeutic utility of siRNA is limited by the requirement for complex lipid formulations to deliver siRNA to peripheral tissues (Vaishnaw et al., 2010).'

It looks  like 1-billion market cap ISIS feels threatened by $40M market cap Tekmira’s SNALP technology after all and is a very interested participant in the frivolous patent infringement lawsuit against Tekmira (frivolous for the reason alone that Alnylam expressly congratulated Tekmira on the BMS deal).

SNALP requires 1000-10.000-fold less oligonucleotides

The assessment that the ssRNAi work by ISIS does not mark a turning point in systemic RNAi delivery is based on simple math.  1000 to 10.000-fold higher amounts of oligonucleotides were required to achieve equivalent knockdowns in mice: conservative 50microgram/kg/month for SNALP vs 50mg/kg/twice a week for ISIS ssRNAi.    

As with any drug, large doses increase the risk of causing toxicities.  In this case, it is particularly the accumulations of large amounts of phosphorothioated oligonucleotides in the liver and kidney that causes such concern.  Of course, SNALP LNP delivery is not entirely without its safety issues.  For example, in the clinic it still involves the use of transient immune suppression which may e.g. be prohibitive to their use in millions of patients with less severe forms of hypercholesterolemia.

Although assuming for a moment that the amount of required oligonucleotides should be irrelevant as long as it was safe, antisense technologies still suffer from poor cost of goods.  Last week for example, the CEO of another antisense company, Sarepta Therapeutics (formerly known as AVI Biopharma), wrote in an unsettling Open Letter to the Duchenne Muscular Dystrophy community that the company essentially cannot afford the large, almost nutritional amounts of oligonucleotides that are required for attempting a therapeutic splice correction.  Closer to home, instead of acknowledging their current cost of goods, ISIS Pharmaceuticals is only providing estimates for their future oligo manufacturing cost goals.

Extrahepatic tissues, which ones please?

Another claim by ISIS related to their ssRNAi tech was that it would be applicable beyond the liver ('broadly distributed and active in multiple organs'), also following systemic administration.  However, once again, this was directly contradicted by their own data which showed a maximally 35% knockdown (the type of from 100 to 65, not from 100 to 35 mind you) for such a tissue (the kidney) when administering…100mg/kg of oligonucleotides in mice. 

The accompanying Huntington’s Disease paper achieved ssRNAi-mediated knockdown in the brain.  This, however, was observed following non-systemic, intraventricular infusion of large amounts of oligonucleotides in small mice brains.

Alnylam was right in  terminating ssRNAi collaboration

Taken together, the publications explain why Alnylam decided two years ago to terminate their ssRNAi collaboration with ISIS.  In addition to using Tekmira’s intravenously infused SNALP LNPs, Alnylam has been working on GalNAc-siRNA conjugates as a subcutaneously deliverable alternative for gene knockdown in the liver.  Although the gene knockdown achievable with that technology still pales in comparison to SNALP LNP, it is about 10-fold more potent than ISIS’ ssRNAi, which goes to show that despite the disadvantages in cellular delivery of unformulated, rigid dsRNAs, their dramatically increased potency more than compensates for it.

At least Lima and colleagues and I agree on this point (opening statement of the introduction): 'RNA interference (RNAi) is a mechanism by which double-stranded RNA triggers the loss of homologous sequence (Fire et al., 1998).' [Emphasis mine]

Technology Trends: MicroRNA Inhibitors and Single-Strand RNAi

There have been developments in the areas of microRNA inhibition and single-strand RNA-mediated RNAi that might have strategic implications for delivery technologies and RNAi Therapeutics, respectively.

MicroRNA inhibition: naked antisense no more?

Currently, all development-stage anti-miR programs to my knowledge envisage the use of unformulated phosphorothioated antisense molecules with various high-affinity modifications such as LNA/LNA-type conformationally restricted nucleotides or 2’F and 2’MOE. To some degree, antisense and certain microRNA companies are making a living out of advertising that, unlike (most) RNAi Therapeutics, no intravenous administration was required.

At the same time, it is becoming clear that more complex structures such as Dharmacon’s miRIDIAN hairpin microRNA inhibitors or the tough decoys (also the synthetic versions that were newly developed in collaboration with Japanese RNAi behemoth Kyowa Hakko: Haraguchi et al. 2012) are considerably more potent on a per molecule basis. Because of their structural complexity, however, they would require delivery formulations for therapeutic use. It remains to be seen how often such formulations would need to be applied, but the early research by Haraguchi in tissue culture shows that the anti-miR effect with these structures can be relatively long-lived. Nevertheless, the in vivo pharmacology of these structured anti-miRs remains to be better explored, but I could imagine that especially for antiviral or oncology applications, the more rapid onset of action and the potentially improved targeting due to the delivery technology could yield positive surprises.

Single-strand RNAi Therapeutics: Stable 5’ phosphate and 2'F

A little more than a year after ISIS and Alnylam ended their ssRNAi Therapeutics collaboration (for which I believe Alnylam had greatly overpaid), ISIS and Merck have made progress in the area.

It had been well known based on particularly protein structural work that the 5’ phosphate modification in the guide strand is important for incorporation in the RNAi effector complex RISC. There has also been corresponding early evidence in ssRNAi research (Martinez et al 2002) that ssRNAs with a 5’ phosphate are more efficient inducers of RNAi, albeit at much lower efficicay compared to dsRNAi triggers. Notably, in the case of double-strand RNA-induced RNAi, prior 5’ phosphorylation is not necessary as this is efficiently accomplished inside the cells.

Based on work by ISIS presented at the Keystone conference in January it now seems that lability of the 5’ phosphate is partly responsible for the reduced efficacy of ssRNAi. Using a new 5’ phosphate mimic (and some other lipophilic modification strategies) that is much more stable than the natural counterpart, it was now possible, following subcutaneous administration, to achieve solid knockdown in rodents. However, the cumulative dose required to get there is still very high, in the range of 2^nd generation RNaseH antisense.

ssRNAi work just published by Merck (Haringsma et al. 2012) confirmed that the 5’ phosphate modification was important, but more importantly worked out the beneficial role of the sticky 2’-fluoro (2’F) modification in ssRNAi. The impressive efficacy-enhancing activity of the 2’-F modification (for which, I believe, Alnylam holds rights to an important patent via a license from ISIS, but a modification that has also raised genotoxicity concerns) was found to apply to both tissue culture and animal settings [Note: an earlier version mistakenly stated that the IP belonged to Alnylam]. Unlike the ISIS work, however, Merck studied ssRNAi in mice using SNALP-like liposomal delivery. It therefore seems as if Merck would need to extra work on adding chemistries such as phosphorothioates or conjugates so that its ssRNAi technology can be used in the ‘naked’ form. This is because the prospect of using ssRNAi in the naked versus formulated form would really be the only motivation for pursuing ssRNAi.

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)

Antisense or Guide Strand? That’s the Billion Dollar Question

As somebody who has spent the better part of my last decade in academic science, which in its ideal form is guided by the self-less pursuit of the ‘truth’, I must admit that I am somewhat irked when I read a paper on Argonaute 2 enzymology and find science being used also (though not exclusively) as a tool of corporate propaganda. You may think that this is nothing new at all. However, I’d like to use the latest publication by ISIS Pharmaceuticals on RNAi as an excuse to highlight a few of the issues that come up when ISIS Pharmaceuticals, by sheer repetition, claims that RNAi Therapeutics is an antisense technology.

In the paper by Lima and colleagues in the Journal of Biological Chemistry (‘The Binding and Cleavage Specificities of Human Argonaute 2’), the investigators study the effect of sequence length and modifications of what they call the ‘antisense’ strand on Argonaute 2 binding. What the authors call here ‘antisense’ is widely referred to by the rest of the field as the ‘guide strand’, that is the small RNA of the double-stranded RNA trigger that gets loaded onto Argonaute 2, the enzyme in RNAi that carries out the target mRNA cleavage. Admittedly, the ‘antisense’ is sometimes used in the RNAi literature to operationally describe the relative polarities of the RNAs involved (guide strand, passenger strand, target mRNA). However, when ‘antisense’ comes up 243 times within a manuscript, but ‘guide’ not at all, even as a verb, I believe it is a valid hypothesis that this cannot be a coincidence, but is calculated corporate speak.

I have explained before why RNAi by its very nature is a double-strand RNA technology, most tellingly illustrated by the fact that the discovery that what had been thought was mediated by antisense was actually mediated by the double-strand RNA contaminants of antisense preparations was awarded the Nobel Prize, so I will not repeat here myself. This is also not meant to criticize the scientific merits of the paper which furthers our understanding of the chemistries that could be used for the less efficient form of RNAi, namely single-strand RNAi and must be seen in the context of the recently announced ssRNAi initiative by ISIS and Alnylam, except maybe for the observation that the finding that single-strand RNA binds purified Argonaute 2 apparently better than double-strand RNA does not reflect Argonaute 2 loading in a living cell.

The point is that whether RNAi is labeled as an antisense technology or not can have significant consequences for the interpretation of the scope of patents, some of which date from before the discovery of RNAi in worms. It is all good for Alnylam for example to implicitly subscribe to ISIS notion as long as they have exclusive access to that IP as it helps constrain the freedom-to-operate of competitors such as Merck and Silence Therapeutics. However, when these early patents are ultimately not deemed as critical, can this leave Alnylam with patents that were pursued less rigorously so as not to conflict with the ISIS’ IP? One of the Tuschl patents comes to mind, I believe dubbed ‘Tuschl IV’, which for the first time explicitly described the use of single-strand RNAs for the induction of RNAi, but, to my initial surprise, had received very little attention among all the ssRNAi talk.

Aside from this political minefield, labeling RNAi Therapeutics as ‘antisense’ just does not do justice to the many ground-breaking discoveries this field has seen, and the people and institutions that made them and that you would think deserved their rightful place in RNAi history and a larger piece of the royalty pie. Specific modifications when used within an siRNA yes, but not the fundamental mechanism of action.

I leave it up to the reader whether this makes ISIS Pharmaceuticals an even better investment, and I must say they clearly deserve credit for their pioneering role in oligonucleotide chemistry and clinical application, and are now harvesting the financial fruits not only from their own drug development pipeline, but also from licenses for all types of therapeutic RNA approaches, including microRNAs (progress so far have exceeded my expectations, and according to a just published comments by Regulus’ CEO on HCV-miR122 this looks set to continue for the foreseeable future) and aptamers. Regardless, 243 times antisense does not make RNAi such.