Dicerna Exploits HBV Biology For Less Frequent RNAi Dosing

Despite recent findings by Arrowhead Pharmaceuticals suggesting that finite treatment with an RNAi medicine might be possible in the search for a (functional) cure of HBV, less frequent dosing is always a plus.  It now seems that fresh HBV entrant Dicerna may have stumbled across a way to reduce dosing frequency and at the same time uncovered an intriguing piece of HBV biology involving the mysterious HBV X protein (HBx).

Search for perfect RNAi target site uncovers important HBV biology

Gene knockdown trigger selection has become critical in a highly competitive field featuring not only Arrowhead and Dicerna, but also Arbutus, Ionis (along with partner GSK), and the Alnylam-Vir alliance in the Western World alone.  This is because ‘by rotten luck’, Arrowhead found that an RNAi trigger that was designed to hit all the HBV transcript did not take into account the absence of the target site on transcripts that derive from host chromosomally integrated HBV.    

The finding that in many patients, e-antigen negative patients in particular, most viral transcripts derive from integrated HBV, has to be considered the most important new discovery in HBV biology and  disease progression since the finding of NTCP as the viral entry receptor.

This has established the selection of a target site upstream of the integration breakpoint as the new industry standard.  Little did we know that the search of the perfect RNAi target site should yield another fundamental insight into HBV biology that could also be therapeutically valuable and provide for some important competitive differentiation for late entrant Dicerna.     

HBx-sparing RNAi trigger has dramatic impact on HBV core protein localization

When scanning the HBV genome for target sites, Dicerna found that an RNAi trigger that hits all HBV transcripts except for HBx was associated with considerably more sustained gene silencing following a single dose (>>8 weeks in a mouse model where GalNAc-RNAi is generally less long-lived compared to humans) compared to an RNAi trigger hitting all HBV transcripts without exception (~3 weeks until knockdown was considerably lessened). 

Further underlining the longevity of the effect, Arbutus’ LNP-based formulation was only able to produce 1 week of good gene silencing in the same (HDI; slide 6) mouse model.

Interestingly, this effect was correlated with striking differences in the cellular localization of the HBV core protein: when all gene products were equally targeted, core protein was predominantly in the nucleus.  By contrast, core protein was almost exclusively cytoplasmic when HBx was spared.  Since phosphorylation of the C-terminal domain is known to be important for HBx nuclear trafficking, it was speculated that HBx may bind in that region and thus mask the nuclear localization signal.  This would be useful during the late stages of a viral infection when core protein would be needed for pregenomic RNA packaging and ultimately viral release.

The reason why gene silencing would be shorter lived when core was in the nucleus is likely due to its stimulating activity on transcription from the HBV cccDNA which would increase transcript dynamics and thus more quickly dilute out the anti-HBV-loaded RNAi machinery in the cytoplasm.

Indeed, in a non-cccDNA-dependent mouse model (e.g. more reminiscent of e-antigen negative patients), this beneficial effect was not observed.  

In summary, while the mechanistic basis of the differential silencing phenomenon remains to be fully fleshed out by further experimentation, Dicerna has found a way to quite dramatically extend gene silencing and thus decrease drug administration frequency.  This is a useful competitive feature in antivirals in general where drug resistance due to missed doses is always a concern and also simply due to patient convenience.

Disclosure: long DRNA.

Alnylam Squares Off with Dicerna

Dicer-substrate RNAi triggers are often seen as a (probably cheaper) alternative to Tuschl siRNAs. Initially, based on a small sample size, it was even claimed that Dicer-substrates had superior potencies and prolonged durations of knockdown. Of course, Alnylam, considering Tuschl siRNAs to be its property, has recognized this and regards Dicer-substrates along with its corporate champion, Dicerna, a competitive threat. Although Alnylam has long claimed that Tuschl siRNAs are preferable over Dicer-substrates for various reasons, until now it has largely been Dicerna’s word against Alnylam’s word.

This has changed with a publication by Alnylam in the journal RNA which provides a comprehensive, and I believe fair comparison between the two structures (Foster et al 2012). Comparing large numbers of RNAi triggers both in vitro and in KC2-SNALP animal studies, the study shows that Tuschl siRNAs and Dicer-substrates are essentially equivalent in terms of potency and the duration of knockdown. However, when these structures are compared in terms of innate immune stimulation, Tuschl siRNAs had a very slight edge when unmodified sequences were tested. Of course, it is well recognized that chemical modifications are required and very effective at abrogating these immune responses. When these were applied, the potencies of Dicer-substrates were more likely to suffer than those of Tuschl siRNAs, and in a few cases innate immune stimulation was not entirely abrogated. The former can be explained by the additional requirement for the Dicer processing step which can be affected by chemical modification.

Overall, this means that it may take a little bit more effort to identify a suitable Dicer-substrate clinical development candidates, and there could be an increased risk in encountering unforeseen innate immune stimulations in humans. On the other hand, the study also suggests that for some genes it may be possible to find more potent RNAi triggers with Dicer-substrates, so that in an ideal world one would keep an open mind. I should also add that, not discussed in this paper, there are also other considerations which may favor one structure over the other.

Unfortunately, we are not living in an ideal RNAi Therapeutics world, but one in which patent trolls and IP freeloaders abound. The timing of the comparison study is particularly ironic since the freedom-to-operate of US-based Alnylam is very much in doubt, thus increasing the attractiveness of Dicerna's offering. This is because of the recent issuance of the Baulcombe patent in the US which has put essentially all the Tuschl siRNA structures, bar one, in a straitjacket. Until Baulcombe is sorted out, all buy-out and partnering efforts will be on hold, and if Merck gets exclusive rights to that patent...then Alnylam may well be toast.

Dicerna Ends Year on Positive Note

With the Notice of Allowance of a US patent application covering its basic Dicer-substrate RNAi trigger technology and a $5M milestone payment from Kyowa Hakko Kirin (KHK) due to the formal initiation of development work for a Dicer-substrate oncology candidate by its Japanese partner, Dicerna can look optimistically into the New Year. These events also symbolize some of the important shifts that have taken place over the last 3 years, namely the increasing choice between RNAi trigger technologies and the increasing importance of Asia in the industry.

Dicer-substrate patent

The patent that is about to formally issue covers methods related to knocking down genes in mammalian cells using certain 25-30 base-pair double-stranded RNAs that undergo Dicer processing before incorporation into the RISC gene silencing complex and target cleavage. While not gate-keeping for Dicer-substrates, the patent will give Dicerna considerable clout over this particular gene silencing approach and has an expiration of at least 2025 if not a year or two longer following patent term adjustments. It is interesting to speculate that the Allowance was also a trigger for the $5M milestone payment.

New oncology candidate formulated in KHK delivery tech

The press release on the $5M milestone noted that while the RNAi trigger of the candidate that is at the center of the $5M milestone payment is licensed from Dicerna (possibly targeting KLF5), the drug delivery tech would be KHK’s own. Based on comments made in early 2010 when Dicerna and KHK first announced their collaboration plus quite a few liposome-related patent apps by KHK, it is likely that the delivery tech relates to a cationic liposomal formulation. I would be a bit wary though about the novelty and strength of these liposomes since at least the patent apps merely claim cationic LNP formulations of chemical compositions that have been known for years in the art an manufacturing method that may be somewhat unique to KHK, but maybe too complex for industrial applicability. Moreover, the described approach lacked the scientific rigor that I would expect of a company that intends to enter clinical development soon.

Dicerna employs Tekmira SNALP delivery

Dicerna has also been saying to be developing delivery technologies, including LNPs and another (actively) targeted delivery system. Interestingly, in an analytical assay development paper that has just been published (Jiao et al., 2011), Dicerna employed Tekmira’s SNALP delivery technology. It is unclear whether the SNALPs were reproduced based on the literature or whether there has been some sort of collaboration between the two companies. However, since Tekmira has been investigating a variety of non-Alnylam RNAi trigger technologies, the latter hypothesis is not that farfetched. The Allowance should provide further impetus for the two companies to work together more closely.

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2010 in RNAi Therapeutics Starts Off with Big Pharma Validations

If the first week is any guide, 2010 could develop into one of the most interesting years in RNAi Therapeutics history and comes after a somewhat lackluster 2009. Both the Dicerna-Kyowa Hakko platform collaboration announced earlier this week and the pre-IND milestone payment Alnylam received today from Big Pharma partner Roche are important proof-points that Big Pharma/Biotech (BBP) continues to invest in RNAi Therapeutics.

The Roche payment due to the initiation of IND-enabling studies marks the first time that a BBP is close to entering the clinic with its in-house developed RNAi Therapeutics candidate. The strength and clinical value of Alnylam’s strategy to further monetize on its mainly RNAi trigger IP has been increasingly questioned by skeptical investors as around half a billion dollars in realized IP funding has apparently not been enough thus far for one of its BPP partner to enter the clinic. This event should also be a boost to Tekmira Pharmaceuticals both in terms of platform validation and financially, as Roche has stated before that its first 2 RNAi Therapeutics INDs will involve SNALP delivery and Tekmira stands to collect ~$9M for each of the 2 candidates it helps Roche to get to the IND stage. For speculations about the indication of Roche’s first RNAi Therapeutics candidate, read the recent blog entry here.

While the Roche news is a definite plus for Alnylam, the Dicerna news has somewhat negative implications as it could be interpreted that BPP is becoming more hesitant to pay the Alnylam premium for what had been considered a toll-gate into RNAi Therapeutics. This is particularly so because Kyowa Hakko once paid Alnylam $15M upfront for Asian rights for Alnylam’s lead RNAi Therapeutics program for the treatment of RSV infection, and very little has been heard about the Kyowa relationship after ALN-RSV01 has been dropped as a candidate for development in pediatric populations. [update] However, as a reader rightly points out in the comments section, Kyowa's 'slight' may also simply reflect the fact that Takeda is Alnylam's exclusive Asian platform partner until 2013 and that Kyowa Hakko was sufficiently eager to speed up the development of its internal RNAi Therapeutics efforts by partnering with a pure-play RNAi Therapeutics company such as Dicerna.

Listening to some of Alnylam’s recent comments, especially its more frequent comparisons of RiSC vs Dicer-substrates, it also appears that Alnylam is not claiming to control the IP around Dicer-substrates. dsRNAs of 25bp and longer may therefore be a tempting, cheaper alternative into RNAi Therapeutics. I still believe that while Dicer-substrates are of comparable potency to Tuschl-type siRNAs, they are more complicated and therefore more costly to develop (especially innate immunity and more restricted chemical space due to requirement for Dicer-cleavage), although they these should not be considered show-stoppers and Dicer-substrates may actually allow for unique delivery strategies and in that regard may be considered complementary. The relatively early stage of Dicer-substrates, however, may explain why the immediate financial benefit to Dicerna was limited ($4M upfront, the rest biotech bucks), but should help the VC-stage company to raise further funds.

Price competition in RNAi trigger IP in the face of financially struggling competitors such as Dicerna, recent IP uncertainties, and the relatively increasing value attached to delivery vs RNAi trigger IP may indeed be responsible for Alnylam’s missed guidance of two major deals in 2009. While SNALP/LNP delivery is making great progress, Alnylam may have to offer more high-quality delivery options to further command terms similar to the Roche and Takeda collaborations. The decision of Novartis, which is about to spend $50B on eye-care provider Alcon, to pay Alnylam ~$100M this year for the broad adoption of Alnylam’s full RNAi Therapeutics IP, will be a good indication of the perceived strength of Alnylam’s IP if not the maturity of the technology.

Both the Alnylam-Roche and Dicerna-Kyowa news further underline the strong momentum of liposomal delivery and RNAi Therapeutics for cancer as also indicated by the Silence-Intradigm merger. Liposomal and lipid-based deliveries were highlighted in the delivery capabilities of Kyowa Hakko and Dicerna, respectively, and the first target picked by Kyowa was for oncology. Antibody-directed targeted delivery, also seems to gather pace as Kyowa Hakko further high-lighted such capabilities in the press release ('POTELLIGENT').

The two news items thus should be a good prelude to two other events this quarter that could decisively shift sentiment on the RNA Therapeutics sector into positive territory, namely the clinical trial results from Tekmira and ISIS Pharmaceuticals on their respective ApoB-lowering drug candidates.

Some RNAi Therapeutics Trivia: Dicerna almost appropriately means 'digested' in the Malay language.

A new player in RNAi Therapeutics: Dicerna Pharmaceuticals

IN VIVO Blog (http://invivoblog.blogspot.com/2007/10/dicerna-crashes-rnai-party.html) reported today that a new RNAi Therapeutics company, Dicerna Pharmaceuticals, is about to debut. According to the same source, a $13M Series A financing round is expected to be announced in November. This company is aptly named after an enzyme in the RNAi pathway, as it is founded on the slightly unorthodox way to induce RNAi by providing synthetic Dicer-substrate siRNAs (D-siRNAs) of 26-30bp in length to effect gene silencing.

Dicer is this cool enzyme that digests ('dicerna' in Malay means: 'digested') long double-stranded RNAs into the shorter 21-23bp siRNAs with 3’ overhangs, the structure discovered by Tuschl and colleagues to efficiently induce gene silencing by RNAi in mammalian cells. Once delivered inside the cells, D-siRNAs are then processed by Dicer into 21-23bp effector siRNAs which then are incorporated into the RiSC complex to mediate gene silencing. Tuschl-like 21-23bp siRNAs are currently by far the most widely used method of inducing RNAi in human cells and fairly well understood.

In an elegant Nature Biotech paper in 2005 (Kim et al.: “Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy”), Drs. Kim and Rossi from the City of Hope, in collaboration with Mark Behlke from the nucleic acids synthesis company IDT, found that D-siRNAs can effect remarkably potent RNAi in human cell culture, often more potent than siRNAs of the same sequence. Importantly, in this and follow-up work they worked out some of the basic rules that would make D-siRNAs more practical inducers of RNAi such as better predicting strand incorporation and blocking one end of the dsRNA with non-RNA residues and modifications to force directional Dicer processing.

This Nature Biotech paper was accompanied by a similar paper from the Hannon group in Cold Spring Harbor which found that DNA-directed small hairpin RNAs (shRNAs) with double-stranded RNA stems longer than minimal 19-21base pairs similarly make them often more potent inducers of RNAi. Like Rossi and colleagues, it was speculated that this is due to biochemical coupling of Dicer processing to the RiSC effector complex. In addition to certain advantages in terms of potency, which I feel need further validation on a larger scale, D-siRNAs may in some instances facilitate RNAi delivery where covalent linkage of parts of the RNAi delivery system with D-siRNA is helpful as the active siRNA would be freed from the carrier by Dicer cleavage, although again it remains to be shown that the covalent attachment of e.g. peptides by itself is not inhibitory to Dicer processing.

[Erratum: The 2005 Hannon paper described the use of synthetic, not DNA-directed hairpins, with extended duplex length.]

In addition to these potential biological advantages, certainly a big part of the motivation that went into founding the company from an investors’ perspective is that Dicerna should be sufficiently distinct from the Tuschl siRNAs, a space clearly dominated both in terms of IP and know-how by Alnylam Pharmaceuticals. One can therefore expect that the new chairman and co-founder Douglas Fambrough from Oxford Bioscience Partners will do his best to make Dicerna his second Sirna Therapeutics, which he and his partners sold to Merck last year for a whopping return on their investment.

However, like with Sirna Therapeutics his claims of having freedom-to-operate will likely be clouded by uncertainty as there are a number of areas where Alnylam’s pre-dating IP will significantly overlap with Dicerna’s claims. This is not helped by comments, also cited in the IN VIVO Blog, of new CEO James Jenson stating that Tuschl’s landmark work had been conducted in flies, when Tuschl II –which by the way has issued and is exclusively licensed to Alnylam- is all about RNAi in mammalian cells, all this after laying the groundwork in work described in Tuschl I (also claimed by Sirna and CytRx, but has not issued) through amazing biochemical work in flies: Tuschl the prolific!

Importantly, Tuschl’s work as described in Tuschl II essentially discovered that RNAi operates in mammals and defined the basic rules of synthetic siRNAs. This, in my mind, should go a long way in the patent courts. In its worst case, D-siRNAs could therefore be regarded as simple pro-drugs of siRNAs. This also includes the 3’end overhangs which are thought to be beneficial for D-siRNAs since they are an important recognition element for Dicer.

Kreutzer-Limmer is another important cornerstone of Alnylam’s IP strategy, indeed important enough for them to buy the company (Ribopharma AG) that owned it very early on. Kreutzer-Limmer pertains to dsRNA-mediated gene silencing in mammalian cells, including predicted Dicer substrates, and although less well known in the scientific community due to lack of scientific publication, it is actually thought to have been the first demonstration of such gene silencing. Scientifically, my heart is with Tuschl’s detailed work, but Alnylam played it safe by just removing the uncertainty.

In addition, I would not be surprised if there wasn’t a note-book entry or publication that made use of long siRNAs either by design or accident. This would not be unlike early in the shRNA arena where scientists have made use of shRNAs with minimal and relatively long dsRNA stems alike.

Practically, the relatively small field of D-siRNAs will have to achieve what thousands of researchers around the world have done for siRNAs, namely coming up with siRNA design rules that consider all of potency, off-targeting potential, and the induction cytokines, as Rossi’s work has shown that these rules may differ from that of siRNAs. For these and other reason, I expect the complexity of developing D-siRNA therapeutics to be probably increased.

Nevertheless, I am curious to see more data come out that carefully characterize and compare the potencies of siRNAs and D-siRNAs. Comparative gene tiling studies would be an obvious experiment. This reminds me of the finding of hyperfunctional siRNAs, i.e. the odd siRNA that will be active in the low to mid picomolar range, and I could imagine a situation where efforts to find such siRNAs prove difficult for certain genes, while a D-siRNA is hyperfunctional, and vice versa.

I certainly look forward to Dicerna as a new member of the RNAi Therapeutics community. The science is certainly sound and innovative, and should be tested for use as a human therapy, which we all know would not happen without patent protection.

PS: This new development makes me wonder where that leaves Nastech Pharmaceuticals which has built so much of their RNAi program on Dicer substrates and is about to spin out mdRNA as their pure play RNAi Therapeutics subsidiary. It is clear that COH granted them 5 exclusive targets, but I am less sure about the other rights to Dicer substrates they had obtained.