ISIS Pharmaceutical Reveals Dynamic PolyConjugate Efforts

When Arrowhead Research made a splash 1 ½ years ago at the Boston OTS meeting in late October 2012 with impressive subQ DPC knockdown efficacy in monkeys, there was a large number of meeting participants crowding the speaker after his presentation.  Although I sat a few rows away, the most eager questioner was an employee from ISIS Pharmaceuticals who managed to beat the Merck representative to the podium!

There were times when I believed that the interest of large companies in the technology of small companies boded well for partnership potential.  It has become clear to me, however, that while this may be true to some extent, the first instinct by the large guys is to get close to the innovators so that they can appropriate as much of the technology as possible without paying a dime.

While I have long shelved Merck into that category and DPCs will now be a research priority at Alnylam following their acquisition of Sirna/Merck, a now published patent application by ISIS Pharmaceuticals on melittin-based, GalNAc-targeted single-stranded antisense delivery (ssRNAi and conventional ASO) demonstrates that ISIS ticks no different.

With a priority date of WO 2014/089146 A1 of December 4, 2012, this poor employee and collaborating patent agent had to stitch together a patent application in just a month after returning home from the conference.  Unsurprisingly, the patent application lacks any actual experimental data to support the 'ínvention'.

I don’t want to be too cynical about this as this is how the industry and the patent system function.  It is an important lesson for small companies though that they should not get overly excited about interest they generate for their technology and they should only engage in deeper relationships once they have established that the larger party is not just trying to steal their technology by pretending to collaborate with them.

It is this unwillingness to share and attendant mistrust that in my experience impedes much business development that makes great sense on paper.  This ultimately delays timelines, leads to litigation, and makes the pie smaller for everybody.

And for the future of gene knockdown in the liver…if ~5mg per week may be possible for GalNAc-targeted gen 2.5 RNaseH ASOs, one can only wonder what a melittin-type escape mechanism would add to its potency, and safety. 

Comment on Seeking Alpha bear article on ISIS Pharmaceuticals

Last night, a detailed bear article on ISIS came out that shook the market in after-hours trading.  In summary, the thesis rests on the poor safety profile, especially immunogenicity of gen 2.0-based KYNAMRO (2’MOE gapmer) and that this is likely to translate to all other gen 2.0 drugs based on shared chemical composition and the (in my opinion idiotic) claim by the CEO of ISIS that KYNAMRO is a success and is representative for gen 2.0.  Talk about shooting yourself in the foot.

While I agree that the KYNAMRO data have raised legitimate questions around the safety of gen 2.0 phosphorothioate oligonucleotides, the bear article conveniently ignores the abundant clinical data that have emerged since for a number of other antisense drugs based on gen 2.0 chemistry.  These support the claim by ISIS that through improved screening, they are now able to better weed out the sequences that will likely prove immunogenic in the clinic. 

Importantly, in oligonucleotide therapeutics in general, while nucleic acid chemistry has a great influence on whether a molecule is immunogenic, it is the exact sequence composition that ultimately decides whether this is actually the case.

This is illustrated by the recent phase II clinical results for ApoCIII (no discontinuations noted), Factor XI, and GCGR.  Not only were robust gene knockdowns achieved, in sharp contrast to KYNAMRO, the company claimed that they were no flu-like symptoms, chills and other symptoms indicative of the immunostimulatory potential of oligonucleotides.  By contrast, about 1/3 in the KYNAMRO studies exhibited such events, with even higher numbers in the open-label extension phase.

Granted, given that these were 13-week studies, it is impossible to disprove the thesis that things are bound to get worse over the long-run.  However, no flu-like symptoms versus 1/3 of patients exhibiting flu-like symptoms in studies of comparable duration is a dramatic difference and allows one to extrapolate that the safety of the follow-on drugs will similarly be greatly superior to KYNAMRO over time.

While I had been tempted to let the Seeking Alpha article by Dr. Anonymous pass as raising legitimate concerns, the blatant failure to mention the more recent experience with gen 2.0 and the after-hours action last night makes this article suspect and outright useless given the lack of new information or insight.

OTS 2013 Meeting Report- Now Available

Want to learn how Oligonucleotide Therapeutics are emerging as the 3rd major drug discovery engine after small molecules and recombinant proteins?  If so, then read my report on the 9th annual meeting of the Oligonucleotide Therapeutics Society held this year from October 6-8 in Naples, Italy.  My hope is that by reading the report, corporate decision makers, investors, and scientists will learn about the key dynamics in the field to help them strategically allocate their time and capital.

Please refer to the Meeting Agenda for the presentations at the meeting.

Table of Contents                                                                                          Page

I.                    Introduction                                                                                  2

II.                  Themes and Trends                                                                     3

A.      Multiple high-impact therapeutic opportunities               3

B.      Thinking small: focus on oligonucleotide chemistry        4

C.      New antisense applications mushrooming                       5

D.     Few new oligo inducer structures and delivery                7

E.      The Duchenne muscular dystrophy phenomenon               9

III.                Highlights from the various oligonucleotide modalities              10

A.      Aptamers                                                                          10

B.      Immunomodulatory oligonucleotides                                11

C.      mRNA Therapeutics                                                         12

D.     RNAi Therapeutics                                                           13

E.      LncRNA Therapeutics                                                      17

F.       Therapeutics splice modulation                                       19

G.     MicroRNA Therapeutics                                                  22

H.     RNaseH Antisense                                                            23

Pricing 

(15% discount for those that have ordered the 2013 RNAi Therapeutics Investment Guide)

Drug development companies up to 10 employees: 250 Euros/300 US dollars

Drug development companies over 10 employees: 500 Euros/600 US dollars

Other companies (e.g. financial institutions and investment funds): 500 Euros/600 US dollars

University libraries: 125 Euros/160 dollars

Individuals (private use only): 100 Euros/130 US dollars

Prices exclude 19% VAT for German customers (corporate and non-corporate) and non-corporate customers in the European Union.

Payments either by bank transfer or paypal.

When ordering, the following information is required:

Full name and address

Billing address if different  

Use (corporate, individual etc)

Order by contacting me at myfirstname.mylastnameATgmail.com
My first name: dirk; my last name: haussecker

Bullish on RNAi Therapeutics

Attending the Oligonucleotide Therapeutics Society meeting last week in Boston, I could sense a new bullishness around RNAi Therapeutics like I haven’t in a long time.   

Big Pharma: Tipping the Scales in Favor of RNAi Therapeutics

Over the last year, we have been witness to the clinical successes of Tekmira’s SNALP delivery that literally saved the sector.  After nightmarish years, these results had investors enjoy the doubling and tripling of the share prices of companies like Alnylam, Tekmira, and even non-SNALP players such as Silence Therapeutics.

For the next leg of expanding investments in RNAi Therapeutics, a renewed, publicly visible commitment by Big Pharma would be important.  With Genzyme taking a license to Alnylam’s transthyretin amyloidosis program, a first step in that direction has been just made.  On the platform side, it is worth noting that the two most significant Big Pharma players in RNAi Therapeutics, Merck and Novartis, were also represented at the conference.  One can only hope that the current RNAi Therapeutics clinical and scientific tailwind will give their internal champions the ammunition to push the technology into the clinic in the next 2-3 years and therefore escape the Sword of Damocles that surely must have been felt dangling above them.

Unfortunately, if these two companies, like so many others it seems, big and small, insist on using ‘their’ own delivery technologies, chances for that will be much reduced.  With all due respect, but the presentation on RNAi delivery by Merck was just a review of the most advanced systemic delivery technologies, SNALP (Tekmira), DPCs (Arrowhead Research), and GalNAcs (Alnylam) exemplified by Merck’s homebrew versions.  Somebody needs to show me the math behind it- I just don’t get it.  The only explanation for me is pride and the resistance against collaborating after having invested internally so much.

A re-commitment towards RNAi Therapeutics should also be at the expense of ‘naked’ RNaseH antisense.  Although in his keynote speech, Alnylam’s CEO made a point of congratulating ISIS CEO Stan Crooke (sitting in the first row) on the recent mipomersen Advisory Panel, there was considerable talk during the conference about antisense-related toxicity, also as regards the high-affinity versions.   And even Stan Crooke could not help but admit that the RNAi Therapeutics results have ‘exceeded [his] expectations’.  However, since ISIS claims ownership over RNAi Therapeutics, as it does indeed over most of oligonucleotide therapeutics anyway, his pain should be limited if indeed he believes what he is saying [note: Dr. Crooke in discussing the safety of mipomersen went as far as saying that there has been no imbalance in its safety profile compared to placebo, and when there was an imbalance, it was in favor of mipomersen...for a starkly different view, see here].

A Breath of Fresh Air in Delivery

A conference highlight were the new results from Arrowhead Research on their new DPC delivery, a conjugate approach.  Potent gene knockdowns in the liver with an apparently reassuring safety profile in non-human primates using subcutaneous delivery is certainly deserving of some serious attention.  Having had systemic delivery of synthetic RNAi triggers almost for themselves for the last few years, Tekmira seems to be finally getting some company- although in terms of validation, SNALP is still years ahead and more de-risked.  Such increased diversity of approaches should also be good for attracting general interest to the sector as it would be viewed as more vibrant and with more disease opportunities.

5 Reasons to be Bullish on RNAi Therapeutics

1)      Clinical results show that RNAi Therapeutics in Man can be made to work (Tekmira’s SNALP delivery);

2)      RNAi Therapeutics ideally suited to address orphan disease, the hottest category in drug development;

3)      RNAi Therapeutics has taken the lead over RNaseH antisense for gene knockdown;

4)      Big Pharma coming back to RNAi Therapeutics;

      5)      Vibrancy of sector increasing (e.g. recent results on Arrowhead’s DPC technology). 

A Side Note on the Conference

To some degree reflecting the increasing maturity of Oligonucleotide Therapeutics, the 4-day program did not include important programs in the field.  Not only was Tekmira notably missing, but also efforts such as Dynavax’ important HepB vaccine candidate that could soon allow oligonucleotide ‘therapeutics’ to touch many lives.  Moreover, the industry-academia balance was tilted in favor of industry like I have not seen before.   

Impressions from the Abstracts of the 7th Annual Meeting of the Oligonucleotide Therapeutics Society (Part 2)

The OTS Meeting has just finished and it looks like it was a success in that meaningful progress, especially on delivery, but also specific development candidates was presented. As the press releases by Tekmira and Alnylam today showed, the abstracts do not necessarily reflect the full progress of the respective studies as these have been submitted months ahead of the conference and also because some groups may want to hold back with the most exciting data points.

For this reason, I will first summarize what I consider to be the highlights of today’s press releases, and then complete my review of the abstracts following a blog post from last week.

Tekmira and SomaGenics report ~300-fold knockdown of HCV in chimeric mouse model

In last week’s post I had mentioned that Tekmira and SomaGenics reported in their abstract #30 the successful use of 'short shRNAs' together with SNALP LNP delivery technology in a model of HCV. This model comprised of an HCV-luciferase reporter gene driven by a liver-specific promoter which is delivered on a plasmid before the application of the LNP-formulated shRNAs. A 90% knockdown at 2.5mg/kg was noted with these somewhat unorthodox RNAi triggers. This type of system is somewhat similar to knocking down a 'normal' gene in the liver, and as such the abstract did not suggest that studies were also performed in the context of real viral replication.

Today’s press release, however, revealed that the companies went an important step further, namely that they applied LNP-shRNAs in a chimeric mouse model for HCV infection. As a reminder, animal models of HCV infection are hard to come by. The best system may be the chimpanzee model, but it is obvious that this system does not lend itself to large numbers, is very costly, and it has been challenging also for ethical reasons to conduct these types of studies. More recently, chimeric mice have been developed in which the mouse liver is repopulated by transplanted human cells under selection pressure. Unlike the hepatocytes of mice and almost all other animals, these chimeric livers can sustain a form of HCV replication.

Having demonstrated 2.0-log and 2.5-log HCV viral knockdowns following one and two administrations of LNP-shRNAs, respectively, is therefore intriguing news. Similar to Santaris’ anti-miR122 antagonist, such a treatment should have significant potential to increase cure rates in treatment failure patients and those patients with genotypes not served well by current therapies.

Alnylam reports more potent LNPs and liver cell-targeted GalNac-siRNA conjugates

Alnylam issued an OTS-related press release today on three different lines of RNAi delivery research, two concerning LNPs, one to siRNA conjugates. As ionizable SNALP LNPs already incorporate a hepatocyte-targeting mechanism (ApoE), the interesting aspect of the GalNac-siRNA conjugates (Abstract #32) is that they may better lend themselves to subcutaneous administration compared to the mostly intravenously delivered LNPs- although this may be less so if the LNP delivery efficiencies are in the single to low double-digit micrograms per kg. The 5 mg per kg ED50 of GalNac-siRNAs puts them right around where some of today’s RNaseH antisense technologies are just in terms of amount of oligonucleotides administered.

Alnylam claims that these numbers make GalNac-siRNA conjugates serious candidates for clinical development. Still, because of the superior, 1000-fold increased efficacies of LNPs and the less frequent dosing that can be achieved with them, this comment may be Alnylam trying to portray themselves as having multiple realistic delivery options, when in fact they are relying for essentially all of their relevant pipeline candidates on Tekmira’s LNPs- my prediction is also that the 5th and final 5x15TM candidate will be SNALP delivered. This cautionary note may also apply to the reported 0.002mg/kg ED50 MD1 lipidoid formulation, developed in collaboration with Professor Dan Anderson from the MIT, although I have yet to see the lipid structures and formulations to really conclude this.

The news on the ‘3^rd generation reLNPs’, probably belonging to the SNALP line of research, similarly relates to further increases in LNP potencies. Here, a ~10-fold increase was reported in therapeutic window over ‘2^nd generation LNPs’, including those containing the contentious MC3 lipid, with an ED50 of below 0.005mg/kg and being well tolerated at the much higher 10mg/kg dosage. Whether Tekmira has a claim also on reLNPs remains to be seen.

Abstract #35: Preclinical development of sd-rxRNAs for fibrosis and retinal disorders (RXi Pharmaceuticals)

RXi Pharmaceuticals reports interesting progress with their self-delivering siRNA for dermal scarring with more than 2 weeks of CTGF gene silencing following single intradermal administration. Also based on Excaliard’s antisense work, CTGF seems to be a quite promising gene target for an orphan indication of solid market potential. RXi Pharmaceuticals would probably aim to improve upon Excaliard’s candidate by increasing the extent and duration of CTGF knockdown, making it potentially a once or twice drug administration approach instead of one involving multiple administrations every couple of days.

RXi expects to file the corresponding IND later this year.

Abstract #43: Characteristic aspects of skeletal muscle as a target organ for siRNA (Dainippon Sumitomo and Koken, both Japan)

I found this abstract particularly interesting because it indicates that Japanese pharma company Dainippon Sumitomo has a quite broad interest in RNAi Therapeutics. As a reminder, Dainippon Sumitomo is one of Silence Therapeutics’ larger RNAi target and delivery collaborators and we should hear about that particular relationship soon.

Koken meanwhile is a company which has an interest in providing delivery solutions for RNAi Therapeutics using its collagen-derived atelocollagen formulation.

Abstract #59: Dicer-substrate siRNA exhibit improved guide strand selection and stronger RISC Loading Complex formation compared to canonical siRNA (Rossi lab, City of Hope)

This study concerns the molecular comparison between Dicer-substrate RNAi triggers, as also practiced by Dicerna, and more conventional (Tuschl-type) RNAi triggers. The abstract suggests that Dicer substrates were more potent and more specific than the competition. A big sweeping comparison between these structures remains to be performed and published to put this issue to rest.

Abstract #69: Glucan particles for selective delivery of siRNA to phagocytic cells in mice (University of Massachusetts)

Glucan-encapsulated siRNA particles (GeRPs) made headlines and raised a few eyebrows a few years ago when a study published in Nature claimed these to be a viable oral delivery technology for targeting gene knockdown in phagocytic cells throughout the body. RXi was the licensee to that technology.

The present update concerns combining GeRP-like particles with the Endoporter peptide-based delivery system. The micrometer-sized GeRP particles are meant to be taken up by phagocytes with the amphiphilic Endporter chemistry supposed to overcome the ‘last mile’, that is RNAi trigger escape into the cytoplasm which has been a challenge especially with phagocytic cells. In principle, these ‘first-localize, then escape’ two-component formulations are reasonable to develop. Whether this particular one can solve the problem, again, remains to be seen. It is probably noteworthy that the present abstract does not mention the oral delivery of these particles.

Abstract #72: RNA-based pancreatic cancer therapy by local delivery of ant K-RASMT siRNA (Silenseed, Israel)

The Israeli company Silenseed has been quietly pursuing the development of its siG12D LODER slow-release siRNA matrix for the local treatment of pancreatic cancer (gene target: mutated KRAS). This candidate is unique in the clinical RNAi Therapeutics staple in that it uses a biodegradable polymer matrix to locally deliver siRNA not at once, but over a sustained period of about 8 weeks. The matrix is placed into the pancreatic tumor mass by ultrasound-guided procedure.

While the concept is sound in principle, it is still not clear whether Silenseed has considered all of its technical requirements such as the intracellular delivery of the RNAi trigger following its local release and distribution. It would not appear an insurmountable challenge, but I have seen groups pursuing promising concepts, but really addressing half the technical requirements only.

Abstract #97: miRNA turnover in Dicer knockout cells (Monash University, Australia and IDT)

This is an interesting abstract as it addresses the inherent stability of small silencing RNAs in mammalian cells. This stability is responsible for allowing us to often observe 4 weeks and more of RNAi gene silencing in non-dividing tissues after single RNAi trigger administration in vivo. With the more potent LNPs, every two months (intravenous) dosing is already within reach.

In this particular system, the authors removed the critical microRNA enzyme Dicer by genetic ablation in mouse embryonic fibroblast. With this, the observed decline in miRNA abundance reflects the decay of the largely RISC-incorporated single-stranded miRNAs following Dicer knockout. An average miRNA half-life of ~5 days was observed this way. It is likely that knocking out the microRNA pathway upstream of Dicer at the Drosha step would have yielded even slightly prolonged half-lives and this would have better reflected the fate of double-stranded RNAi triggers. It will be critical to find out by how much these half-lives can be further increased using siRNA chemistry, as Merck has done, and how much of it is determined by the protein turnover of the RISC complex which ultimately may be a rate-limiting step difficult to meddle with.

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)

Impressions from the 3rd Annual Meeting of the Oligonucleotide Therapeutics Society: Day 1

The 3rd Annual Meeting of the Oligonucleotide Society (OTS) got under way in the stylish Langenbeck-Virchow Auditorium situated in the centre of a culturally vibrant Berlin. The OTS brings together the many disciplines in the rapidly expanding field of oligonucleotide-based drug development, ranging from aptamers, ribozymes, antisense, immunostimulatory nucleic acids to RNAi. Reports from clinical studies of these treatment modalities and oligonucleotide chemistries were at the centre of today’s lecture sessions.

Brett Monia from ISIS Pharmaceuticals chaired the first session and set the stage by reporting that while there are only four approved oligo-based therapies, more than 50 are currently in clinical development. Many of these are designed for cancer applications, but infectious, metabolic, and inflammatory disease applications are emerging as major new focus areas. This may not only be due to considerable unmet medical needs, but also because these may offer better success rates for clinical development. A few talks on cancer therapies with ASOs (antisense oligos) documented the challenges of designing smooth clinical trials for cancer therapies. One problem is the heterogeneity of cancers and matching the gene target with the right cancer. Early trials on a given compound typically involve small patient numbers with a wide spectrum of cancer manifestations. Although safety trials, almost anecdotal evidence of biological activity in a few patients then often guide the design of later phases of clinical development. Clearly, for cancer, there is a need for the development of more reliable biomarkers as early indicators of therapeutic efficacy and for patient stratification, which may be something that Rosetta Genomics’ microRNA diagnostics may be able to facilitate.

Focusing on diseases like viral infection and metabolic disease where efficacy can be routinely evaluated in early stages of clinical development (viral titers, LDL-cholesterol, glucose) should therefore be lower hanging fruits for the next wave of oligonucleotide-based therapeutics. This transformation in the industry is illustrated by ISIS’ new focus on metabolic disease and its out-licensing of the more challenging ASO applications. Rosanne Crooke recounted the clinical experience with 301012, ISIS’ lead compound for the treatment of hypercholesterolemia. As I wrote earlier, one major concern with targeting ApoB100 is the risk of steatosis of the liver due to inhibition of a protein required for the export of triglycerides and which has indeed been observed by a number of groups targeting ApoB100 by RNAi. Possibly motivated by the existence of individuals with null mutations for PCSK9, the new target of choice for hypercholesterolemia, with no evidence for adverse effects due to lack of the LDL-receptor processing protein, a conference participants asked about the existence of ApoB100 knockout mice. Interestingly, no such mice exist, because ApoB100 would have additional roles during development.

Despite reports of steatosis due to knockdown of ApoB100 with RNAi, Sirna Therapeutics is yet another biotech company interested in targeting it for lowering cholesterol. In a talk about siRNA delivery to the liver, Barry Polisky, CSO of Sirna Therapeutics/Merck also noted that in Sirna’s experience, there are genes that can be knocked down extremely well (e.g. ApoB100) and some that are relatively refractory to knockdown by RNAi. Although anecdotal, given the extensive experience of Sirna Therapeutics with screening for effective siRNAs by tiling them across entire genes, and similar findings by Qiagen and others, it will be interesting to determine what makes a gene a good target for RNAi, particularly whether it involves its normal regulation by microRNAs.

Polisky further focused on the need for siRNA modifications to make RNAi therapeutics a reality. Currently, Sirna’s standard siRNA design seems to involve si(R)NAs that lack essentially all 2’OH groups, except for the three 5’ nucleotides of the guide strand, while the passenger strand has 5’ and 3’ modifications that avoid it to be loaded into RiSC to reduce off-targeting. Other speakers and posters also noted that since most nucleotide modifications had been developed with antisense therapies in mind, new modifications may exist for optimal RNAi efficacy, particularly in vivo. Indeed, new synthesis strategies are being developed that will allow this to be tested, such as the 6-membered carbohydrate substitutions of ribose reported by Piet Herdewijn from the University of Leuven, Belgium.

Polisky further characterized siRNA delivery to the liver as efficient, albeit not optimal. Progress is clearly illustrated by the fact that >30% of siRNA formulated into liposomal nanoparticles now end up in the liver with first-pass kinetics with no evidence for toxicity in the 1-9mg/kg range (mostly rodent studies), whereas >99% of unformulated siRNAs are rapidly filtered out by the kidney.

The liver is also the organ where the first microRNA-based therapies are likely to be targeted. Like Regulus and Rosetta, I was amazed, or maybe not, at how many groups are currently working at abrogating miR-122 function for treating either hypercholesterolemia or HCV infection. LNAs, owned by the privately held Danish company Santaris, appear to be a particularly promising oligonucleotide class for achieving potent microRNA down-regulation in vivo. Similarly, antisense strategies based on LNA appear to rival siRNA potency in a number of settings, and it will be interesting to learn more about the toxicologies of these compounds, which would certainly be facilitated if Santaris decided to enable to field by allowing better access to their technology. It also highlights the need for specialized chemistries for therapeutic targeting of microRNAs which in my mind is something that Regulus ought to consider.

With regard to delivery outside the liver following systemic administration, PEI-formulated siRNAs (see recent PolyPlus-Alnylam deal) are now being used successfully by a number of groups for lung delivery. Other interesting delivery data was presented by a Portuguese group in a poster on RNAi in the brain. By non-covalently adding transferrin protein to cationic liposome-siRNA particles, they were able to efficiently target neuronal cells both in vitro and in vivo which, similar to cancer cells (see Calando’s cancer cell targeting with transferrin), are studded with transferrin receptors.

PS: A nuisance to most scientists in the field, immunostimulation by oligonucleotides is exploited by Dynavax as an adjuvant for vaccines. Poor adjuvants activity lead to insufficient immune responses and the need for multiple booster injections affecting compliance. HBV vaccination is a case in point, and as a consequence around 50% of patients in the US fail to successfully complete vaccination. Very high HBsAg antibody titers after only two injections of recombinant HBsAg with an immunostimulatory oligo from Dynavax as adjuvant, instead of alum, suggests that they may be able to greatly improve on those numbers.