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)

RNAi Clinical Trial Started for Genetic Skin Disorder Pachyonychia Congenita

This week saw the initiation of a clinical trial investigating the use of siRNAs for the treatment of Pachyonychia Congenita (PC). The study is sponsored by the PC Project, a public charity devoted to serving the needs of the ~500 people worldwide affected by this rare genetic disorder, and led by Sancy Leachman from the University of Utah. The siRNA itself, TD101, is the fruit of a collaboration between the International PC Project and the Californian RNAi-focused start-up TransDerm with ties to the shRNA-company SomaGenics.

PC is one of the dominant-negative epithelial fragility disorders caused by the mutation of a keratin gene. There are over 20 keratin genes in the human genome, with two different types forming heterodimers in the assembly of the keratin intermediate filaments which are important for the structural integrity of epithelia such as of the skin. A mutation in one of the dimerization partners may disrupt the organization of the filaments, which in the case of PC results in thickening of nails and skin of the palms and soles. Apart from the obvious cosmetic consequences of the disease, pain due to stress on the palms and soles is a major symptom of the disease for which no specific treatments exist.

Previous studies suggest that a 50% reduction in the mutant protein should get rid of the molecular aggregates caused by the filament assembly defect, and even the total loss of the mutant keratin should be well tolerated due to the expression of compensatory keratins. RNA- and DNA-based treatments offer the best opportunity for a specific treatment as they can address keratins directly and should be able to distinguish between mutant and wild-type genes. After considering a number of technologies, the PC Project has chosen RNAi due to its potential specificity, relative straight-forward mechanism of action which should accelerate drug development.

This is supported by two related publications from the consortia (Hickerson et al. (2007): Single-Nucleotide-Specific siRNA Targeting in a Dominant-Negative Skin Model; Smith et al. (2008): Development of Therapeutic siRNAs for Pachyonychia Congenita) which demonstrate the ability of siRNAs to specifically down-regulate the mutant keratin with an almost complete reversal of the aggregation phenotype. This is shown in both in vitro tissue culture and in vivo mouse footpad models involving the use of keratin-reporter genes.

While these studies provide proof-of-concept of RNAi for the treatment of PC, the in vivo studies were limited to the knockdown of reporter genes co-transfected with the siRNAs, rather than targeting endogenous genes in the skin epithelium. It is difficult to judge from such studies the overall delivery efficiency since cells that take up the reporter gene are likely to take up the siRNA as well, even when most of the remaining cells have not taken up any of the siRNA. (note: similar reservations apply to the recently initiated phase I RNAi studies for HBV by Nucleonics which are also heavily based on co-transfection experiments.)

The mouse models involved intradermally injected, unmodified siRNAs in a simple PBS buffer. While TransDerm is working on a topical lipid-based “gene crème”, it appears that at least for the phase Ib studies the siRNAs, targeting one of the more common mutations in PC, will be intradermally delivered by needle injection. One concern here is whether the area that such a delivery method can reach is sufficient to alleviate the symptoms of PC. Another is that using unmodified siRNAs in PBS over stabilized formulations will unnecessarily sacrifice some of the efficacy as well as necessitate more frequent treatment. Clearly, there is room for improvement, but the phase Ib studies should provide precious clinical data and inform future treatment strategies.

In addition to more sophisticated delivery methods and formulations, such strategies could also involve DNA-directed RNAi where autologous skin transplants and/or stem cells of the skin are stably corrected ex vivo, e.g. by lentiviral transduction of shRNAs, and then reapplied to the patient. This may be preferable to repeat needle injections.

TransDerm hopes that providing proof-of-concept for an RNAi Therapeutic of such a rare skin disorder with little commercial potential will be a stepping-stone for addressing much larger patient populations. This illustrates once more the value of proof-of-concept studies in general for RNAi since the ability of deliver one RNAi treatment could be rapidly expanded to delivering many more RNAi Therapeutics for the same tissue.

It will be interesting to see whether TransDerm, which by the way has opted for a classical Tuschl siRNA rather than a synthetic shRNA like SomaGenics is using, will be successful not only in the clinic, but also in their corporate strategy. In the absence of any RNAi core IP, the development of RNAi delivery technologies for the skin is probably their best shot in creating shareholder value.

PS: I wonder what happened to Sirna Therapeutics’ dermatology program which in their first development program aimed for siRNA-mediated permanent hair removal. The prospect of RNAi dermatology products is certainly exciting, especially if it were possible to develop convenient delivery methods such as topical cremes.

SomaGenics Reports on the Use of shRNAs for the Treatment of HCV

At the end of a day packed full with fun RNA science from the San Francisco Bay Area, Brian Johnston, President and CEO of SomaGenics presented some of his company’s data on the use of shRNAs for the treatment of HCV, historically one of the early therapeutic targets for RNAi.

Most shRNA approaches are based on DNA-directed expression of RNA hairpins in the nucleus that are then fed into the RNAi pathway. Somagenics’ approach is slightly different in that they are introducing the RNA directly into cells. Concentrating more on the science than IP issues here I only want to briefly remark that this probably overlaps quite a bit with the Hannon patents (licensed to both CytRx and Alnylam) on the use of shRNAs as RNAi triggers, both transcribed and synthetic.

The scope of the experiments were limited either to in vitro culture studies or its glorified in vivo counterpart, the hydrodynamic co-transfection experiment. Here, a large volume of RNA-containing liquid is injected into the tail vein of a mouse in a brief period of time, physically forcing the RNA into hepatocytes. It is of interest, that unlike the findings of the back-to-back papers of the Hannon and Rossi labs more than two years ago, hairpins (directed to the conserved HCV IRES) with minimal stems, 19 base-pairs, were generally more effective than those with extended 25 base-pair hairpins. While it has to be said that compared to the Hannon and Rossi papers which argued that the long stems that were processed by Dicer would increase RNAi efficacy, the sample size here was quite limited, it nevertheless shows that the processing of hairpins are often not as predictable as one would wish based on microRNA biology.

Johnston further reported that the shRNAs were at least as efficient as the corresponding siRNAs with some of the IC50s in the low picomolar range. I am curious how this will translate into in vivo uses for example because shRNAs, unlike classical siRNAs and Rossi-type two-stranded Dicer-substrates, contain the double-stranded RNA within one molecule which should make this structure thermodynamically quite stable. A potential disadvantage is that it is so much more expensive to synthetically make the ~42-55 nucleotide hairpin RNAs since cost and purity of manufacturing RNAs increases more than linearly after you reach a certain size, say 25 nucleotides. While these studies mostly used RNAs generated through biochemical synthesis in the test tube using recombinant phage RNA polymerases, for the clinic they are working together with Agilent to generate the “same” RNAs to scale. I say the “same” here since the phage polymerase leaves a triphosphate 5’ end while chemical synthesis does not. This is not a trivial issue here since 5’ modifications are known to influence RNAi processing. We will therefore have to wait how the transition from in vitro transcribed to synthetic shRNAs will affect the reproducibility of their data and potential concerns from the FDA. Similarly, some mention was made that shRNAs may tolerate RNA modifications less well than siRNAs, probably because they are subject to additional processing steps.

The use of more challenging animal models is also warranted before entering the clinic. It is therefore important for them to find suitable delivery solutions which they apparently have only started to. It was good to hear though hearing him mention Protiva/Tekmira’s SNALPs and Mirus Bio’s Dynamic PolyConjugates as probably the most advanced RNAi delivery platforms to the liver, reflecting my views on what’s out there in the literature. Overall, I like the fact that multiple RNAi approaches are in the pipeline to tackle HCV, namely siRNAs: Sirna/Merck; DNA-directed shRNAs: Tacere/Benitec and Nucleonics; and now synthetic shRNAs: SomaGenics. And maybe Tekmira/Protiva should harness the power of SNALP and officially nominate HCV as a clinical development program. I hope that the next 2 years should finally see programs moving into the clinic after HCV RNAi had been delayed in the wake of company-specific issues (Benitec; Merck/Sirna vs Protiva).