Silence Therapeutics DACC Lipoplexes for Lung Endothelial RNAi Delivery

After having shown for some time now deep and long-lasting gene knockdown in lung endothelial cells, Silence Therapeutics has finally published (Fehring et al. 2014) more detailed chemical and pharmacokinetic information on the DACC formulation.  This formulation could be useful for indications such as cancer involving the lung and acute lung injury.

Chemistry: cholesterol replaces helper phospholipid DPhyPE

Atuplex has long been the workhorse delivery technology of Silence Therapeutics.  Atuplex has been shown to target pretty much all vascular endothelial cells independent of tissue/organ system and enables the company's lead candidate, Atu027 for the prevention of cancer metastasis currently in phase Ib/IIa in combination with gemcitabine in pancreatic cancer.  Unlike the four-lipid formulation pioneered by Tekmira, Atuplex consists of just three lipids:

-          the cationic lipid AtuFECT01 for cell attachment and penetration (proprietary);

-          a pegylated lipid to prevent aggregation;

-          and the helper phospholipid DPhyPE for structural stability.

To my surprise, the DACC formulation contains the same cationic and pegylated lipids as Atuplex.  The main difference is in replacing the helper phospholipid DPhyPE with another helper lipid, in this case cholesterol.  With this change (and some adjustments in the lipid ratios), 40% of the DACC ends up in the lung with the concomitant silencing of genes in resident endothelial cells.

Silence Therapeutics’ strategy of utilizing a given cationic lipid in new lipid combinations therefore stands in contrast to previous efforts by the likes of Tekmira and Dicerna which have focused on the discovery of new cationic lipids to increase potency and the therapeutic index.

Rationale of DACC over Atuplex for lung endothelial RNAi not fleshed out

Although the new research clearly shows that the lung is the most important physical sink for DACC, the study did not investigate whether this translates into a tissue-specific knockdown effect as well [correction 28Jan14: Figure 4 of the paper does show preferential knockdown in endothelial cells of the lung versus other tissues].  This would be an important additional safety-related rationale for using DACC over Atuplex for lung endothelial gene knockdown, especially for target genes that might have critical functions in normal physiology as well. 

Atuplex, in contrast, has previously been shown to mediate lung endothelial gene knockdown, not just in mice (as DACC in this study), but also non-human primates.  What is more, the previous Atuplex studies showed comparable (~70-80%) knockdowns at ~10-fold reduced dosages (0.3mg/kg vs 2.8mg/kg).  It will therefore be important to test whether lower dosages are feasible for DACC knockdown in non-human primates, potentially with the use of slow infusions instead of bolus injections as was the preferred method in the present study.  And as is my pet peeve when it comes to Silence’s delivery technologies, why not attempt them with other, non-AtuRNAi trigger technologies when a factor of 2 could make all the difference in whether you have an acceptable therapeutic window or not.   

It should be noted, however, that unlike Atuplex, DACC appears to have a more reliable dose-response and seems to be tolerated at up to 6mg/kg so I trust that Silence has good reasons for choosing DACC for programs such as acute lung injuries (ALI). Based on the data revealed by the company thus far, it appears that the ALI candidate Atu111 is the prime candidate to be Silence's next clinical development candidate.

Silence Therapeutics Signs Lung Delivery Deal with Mystery Partner

As Tekmira is struggling to regain possession over its delivery technology from mighty Alnylam, it is foremost Silence Therapeutics' business development that is benefitting from having one of the most advanced, clinically tested, and commercially uncontested systemic delivery technologies in RNAi Therapeutics. Less than a month after closing a deal with Dutch company InteRNA under which Silence’s AtuPLEX lipoplex delivery technology will be evaluated for the delivery of microRNA cancer therapeutics, the company has just announced another delivery collaboration, this time for the related DACC delivery system. Like AtuPLEX, DACC targets vascular endothelial cells, but unlike AtuPLEX it does so with a high preference for the vascular endothelia of the lung.

Curiously, the identity of the partner was not disclosed, although it was mentioned that it was a ‘Top 10 Pharma Company’. 'Top 10 Pharma Company'....didn't Takeda just become Number 10 with the acquisition of Nycomed? Moreover, the Nycomed acquisition included a fresh COPD drug, a pulmonary disease with an inflammatory component for which the DACC delivery system could very well be useful.

You can imagine that Takeda is a bit hesitant to talk about RNAi Therapeutics, maybe having overpaid a bit for what Alnylam really sold them, but with their significant investment in RNAi already it has every incentive to get things moving while Tekmira and Alnylam are fighting it out. Maybe following this rationale, Takeda and South Korean company Samyang announced earlier this year a somewhat surprising delivery collaboration.

The press release also noted that it is the partner that will provide the RNAi triggers. This pretty much excludes Silence’s existing Big Pharma partners AstraZeneca and Dainippon Sumitomo, the latter of which is not a Top 10 Pharma anyway. On the other hand, Takeda licensed RNAi triggers from Alnylam only for the metabolic and oncology fields, something that would probably add to the apparent sensitivities. I highly doubt that Takeda will hand over another $50M to Alnylam for pulmonary.

Of course, it is always possible that RNAi Therapeutics has gone so much out of fashion that Big Pharma companies are embarrassed to be publicly associated with it. Not only that, their investors shun the word ‘Research’ like the plague.

More seriously, the data that Silence reported for the DACC system has been quite impressive. Working on a model for acute lung injury, it has demonstrated potent and persistent gene knockdown in the pulmonary vascular endothelium following a single intravenous administration. While less is known about the clinical safety profile of DACC, it can be considered somewhat de-risked by the fact that its chemistry seems to be very similar to the clinically proven AtuPLEX system (see the highly encouraging safety profile of their phase I Atu027 clinical candidate so far).

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)