The Time for the U(nusual)siRNA Strategy Has Come

As Tekmira and Arrowhead Research will unveil their next RNAi Therapeutics development candidates later this year, an interesting question will be whether these will involve one of their RNAi trigger options that some consider to be unencumbered by fundamental IP related to traditional designs (esp. the Baulcombe and Tuschl II IP).  These decisions could have important strategic consequences for the competitive landscape, from targets and indications to Big Pharma involvement.   

Support for freedom-to-operate claim

One of these designs is the usiRNA from Marina Biotech.  These comprise at least one ‘unlocked’ nucleic acid monomer (UNA) in the double-stranded RNA molecule.  While I have reservations about the scientifically tenuous claim (see here why) that UNAs are not to be grouped with most of the other nucleotide modifications for RNAi use because they lack an intact ribose group, usiRNAs were held to be sufficiently non-obvious and of specific utility that the USPTO issued fairly broad claims in 2012.  Moreover, Marina Biotech once commissioned an external IP lawfirm perform a freedom-to-operate analysis on usiRNA, and (surprise, surprise) came to the conclusion that, indeed, usiRNAs have FTO.

Overcoming target picking limitations

This view seems to be shared also by others in the industry. Notably, Roche RNAi (now part of Arrowhead Research) in 2009 gained access to Marina’s usiRNAs, meroduplex siRNAs, and Dicer-substrate RNAi triggers.  This came as a surprise given that Roche had spent over $300M just two years earlier to gain access to RNAi trigger IP held by Alnylam.  Given that none of the three licensed RNAi trigger forms and related IP poses any FTO threat to traditional Baulcombe-Tuschl designs, the most likely explanation for the move is that it was about allowing the company to escape the target picking limitations under the license from Alnylam.  This included the 31 targets exclusively held by Novartis, some Tekmira exclusive target picks, and some targets pursued by Alnylam that Alnylam exempted from competition.  Whether the last of Alnylam’s Big Pharma licensees, Takeda, might pursue a similar strategy is an interesting question.

Facilitating platform partnerships

When Alnylam and ISIS sued Tekmira for infringing on their RNAi trigger IP by collaborating with Bristol-Myers Squibbs on RNAi delivery, it became a priority for them to have access to or control over non-Alnylam RNAi triggers.  As a consequence, they obtained an exclusive license to Halo-Bio’s multivalent RNAi triggers (more than two strands).  Subsequently, they gained access to Marina’s usiRNAs, including the ability to sublicense.   This now puts them in the position to engage in platform partnerships with Big Pharma companies that do not have access to Alnylam IP.

The same strategy would likely also apply to Arrowhead Research with its various RNAi trigger options that it inherited from Roche, especially if Alnylam provided Roche with only product-specific sublicensing rights, if at all.  As RNAi Therapeutics enjoys a return of pharmaceutical interest, this one-stop-shop option by the two leading delivery companies could be critical to bringing new companies into the space.

And for Alnylam, these developments would not only diminish the royalty it might earn from licensing its IP, they could undermine their own product candidates, including ALN-PCSK9 (hypercholesterolemia) and ALN-AT3 (hemophilia).  Accordingly, the preclinical data strongy suggest that subcutaneous DPCs can do everything that Alnylam’s GalNAcs can do, only much more potently and with less frequent dosing. 


 

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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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.