Arrowhead Research Breaks Up Dynamic PolyConjugates into Two

Arrowhead Research announced yesterday that it had received Notice of Allowance from the USPTO for a Dynamic PolyConjugate-related patent application (for systemic RNAi delivery).  Instead of it being your run-of-the-mill patent PR involving known technology that finally received patent protection, it was really about revealing Arrowhead’s fundamentally new approach towards DPC delivery (for my take on the original form of DPC, see here).

The patent, part of a series of patent filings that have come out recently, shows that under Roche’s ownership, the technology has morphed through a number of iterations from the original complex polyconjugate chemistry combining endosomolytic polymer, masking groups, RNAi trigger, PEG, and cell targeting ligand all in one molecule (schematic shows such molecule and presumed mechanism of delivery), into one where a RNAi trigger and the masked polymer, both targeted individually, are administered as separate agents. 

One problem with the original design has been that combining all the functional groups, including negatively charged RNAi triggers and positively charged polymers, into one molecule was not particularly easy.  The tendency to aggregate and poor yields made it a quite expensive and difficult-to-scale proposition.

Turns out that such complicated chemistry wasn’t needed after all.  As long as the RNAi trigger and the masked endosomolytic agent end up in the same place, it does not make much of a difference whether they are getting there as one molecule or separately.  In the example provided, namely for gene knockdown in hepatocytes, the RNAi trigger could be conjugated to either cholesterol or a cluster of galactose sugars, whereas the masked endosomolytic polymer was targeted to the hepatocytes by galactose.  Viewed differently, the polymer allowed the cholesterol-siRNA that apparently gets trapped in the endosomes when alone to be released into the cytoplasm.  Hence, the multi-fold increase in potency (Arrowhead Research says it’s 500-fold) over Alnylam’s original 50mg/kg cholesterol-siRNA report (Soutschek et al., 2004).

Another potential advantage of this separated approach is that it makes each component smaller, perhaps 'one day' enabling subQ dosing.  However, as mipomersen's FDA AdCom meeting briefing docs show, when it comes to subQ dosing, be careful what you wish for.

Manufacturing appears to have been one of the issues delaying the clinical translation of DPCs for quite some time (Arrowhead Research says they are ready to file an IND in Q2 2012 for Arc520 in HepB).  Toxicity, mainly due to premature unmasking in the blood instead of in the target cell endosome, seems to have been the other main reason.  Such premature unmasking also adversely affected circulation times, thereby rendering attempts to get beyond the liver, one of the original promises of DPCs, futile.  We will probably get word from the company soon what solutions it found for this challenge, but it seems that, based on the emerging patent literature (including non-human primate data; e.g. WO 2012/083185), masking the membrane penetrating peptide mellitin with endosomal protease-sensitive groups, is a promising approach.

Quark Biotech Dazzles RNAi Therapeutics World with Expanding Pipeline, Adds to Evidence that Delivery Walls to Kidney Crumbling

Yesterday, the US-Israeli company Quark Biotech announced the filing of their now third RNAi Therapeutics IND. The latest IND candidate, DGFi, is the siRNA knockdown of p53 in the kidney for the treatment of kidney transplantation-associated ischemia-reperfusion injury. This program follows a similar systemic kidney-p53 oxidative stress RNAi program, Akli-5, for acute kidney injury, and a local RNAi program (RTP-801i) for age-related macular degeneration targeting the apoptosis-related gene RTP801/REDD1 that came out of Quark’s own gene discovery program.

This likely makes Quark Biotech, which specializes in the discovery of disease-associated genes that it then targets by in-licensed RNAi technology, the company with the most RNAi candidates in the clinic, unless, of course, Merck has early clinical programs that we haven’t heard about. This is quite remarkable given that very little is known about Quark’s RNAi science as judged by the literature and presence at leading RNAi conferences. My own patent search for Quark-related RNAi delivery technologies failed, although their IPO documents stated that they had been building an IP estate around RNAi Therapeutics, including proprietary delivery technologies [Note: the planned ~$80M IPO failed last year due to a difficult market; instead the company earlier this year raised around $27M from private Japanese investors].

So I can only speculate as to the systemic delivery technology employed. Given that unformulated and unmodified oligonucleotides have the propensity to end up being rapidly excreted by the kidney, it is well possible that some of these siRNAs get functionally taken up for gene silencing. In fact, the ground-breaking systemic siRNA delivery paper by Soutschek and colleagues from Alnylam employing cholesterol-conjugated siRNAs showed that the lipophilic siRNA conjugate was taken up reasonably well not only in the liver, but also jejunum, heart, adipose tissue, the lung, and kidney, albeit at quite high 50mg/kg dosages. Similarly, a recent publication by the Natarajan group from the City of Hope, CA, showed that subcutaneous administration of ~20mg/kg cholesterol-siRNA reduced gene expression in the kidney by about 50-80% with promising therapeutic effects in a mouse model for diabetic nephropathy. It is, however, possible that the apparently intravenous formulation is composed of a nanoparticle as suggested by the second name of the Akli-5 progam, I5NP (NP=nanoparticle?). In any case, the evidence is growing that the fact that siRNAs like to go to the kidney could be exploited for treating kidney-related disease by RNAi, slowly clearing yet another organ for RNAi.

Although I feel more comfortable judging an RNAi Therapeutics company with some scientific data at hand, Quark Biotech’s speed of entering the clinic while others are humbly optimizing their own candidates, particularly with regards to delivery, warrants some attention. In addition to the AMD program, the company has licensed a second RNAi program to the emerging RNAi superpower Pfizer, a program for COPD likely to be administered by inhalation. Overall, the Pfizer relationship has brought in over $25M of realized funding as of the filing of the IPO documents in March 2007. Another important relationship exists with Silence Therapeutics, although a report earlier this year suggested that there might be some frictions in that relationship. This would be consistent with Quark having subsequently licensed IP from Alnylam as well as Quark’s ambitions of developing proprietary RNAi trigger IP, whatever that is supposed to mean. I guess by providing a little more transparency, Quark Biotech might be able to attract more investor interest for a second IPO attempt. With so many clinical candidates and more coming up, such a cash infusion could be necessary soon.

In other news: The Pharmalot Blog posted yesterday that the approval rate of innovative medicines continues to be anemic. Only 5 new molecular entities were approved by the almighty FDA in the year through May. It’s time for RNAi to contribute to the development of more innovative drugs addressing unmet medical needs, and the regulatory agencies and society as a whole to understand that overdone conservatism and by killing the profitability of drug development aren’t helping in that regard.

Journal Club: Alnylam and Collaborators Make Progress in Understanding and Optimising siRNA Uptake In Vivo

In yet another elegant paper, Alnylam and collaborators from the ETH in Zurich and the Rockefeller report this week in the journal Nature Biotech on the mechanism of siRNA uptake in vivo (Wolfrum et al.). This is significant, since a systematic approach to understanding siRNA delivery in vivo should most adequately address the delivery challenge that is considered by many to be the main barrier to the broad application of RNAi as a therapy. It is also a sign of the maturity of the RNAi field in general that no only are there now a variety of innovative delivery systems evaluated almost by the day, systems that show promise serve as leads for a detailed investigation of the underlying biology.

The study by Wolfrum and colleagues follows another high-profile publication 3 years ago (Soutschek et al.) where Alnylam scientists demonstrated gene silencing in mice following systemic administration of cholesterol-conjugated siRNAs. That study showed that although such siRNAs could silence genes particularly in the liver and gut, quite high amounts of siRNAs were needed (50mg/kg). By studying the uptake of the siRNA conjugates in these tissues, the authors not only hoped to understand why they functioned at all, but also to optimise their potency.

Efficient in vivo drug delivery requires favourable pharmacokinetics. Particularly, a drug has to be present in the blood for sufficient length of time so that it has a chance to accumulate in its target tissue. One reason for example why many experimental drugs fail is because they are rapidly excreted through the kidneys. This may often be prevented if the drug could interact with components of the blood such as the abundant lipoprotein particles.

Indeed, the authors find that siRNAs conjugated to cholesterol or other lipophilic molecules associated with the similarly greasy HDL and LDL lipoprotein particles. These would ferry them around in circulation and bring them into the proximity of cells that carry on their surface receptors for either HDL and/or LDL. Strikingly, pre-assembling the siRNA with purified HDL and LDL particles quite significantly increased the potency of the siRNAs. Furthermore, mice lacking either of the receptors for the lipoproteins were much less prone to gene silencing by the same pre-formulated siRNA particles.

In a further interesting twist, it was shown that siRNAs were not taken up by the cells as part of internalising lipoproteins, but that the siRNAs would take advantage of their proximity to the cell membrane during the docking, release, and re-docking process of their lipoprotein carriers with their receptors. Amazingly, through a combination of gene knockdown experimentation and blockage by antibodies, at least one of the actual entry routes for the siRNA was inferred to be the human homologue of the SID-1 gene that had earlier been shown to mediate systemic RNAi in the worm C. elegans.

Systemic RNAi describes the spread of an siRNA from one cell to another cell in the same or even different tissues. Systemic RNAi in worms and plants is associated with the amplification of RNAi, and both systemic RNAi as well as RNAi amplification were thought to have been lost during human evolution. It is therefore a surprise that SID-1 would still function in siRNA uptake, with demonstrated selectivity for siRNAs relative to other types of nucleic acids. This also raises the intriguing possibility that some sort of natural siRNA uptake should occur in humans.

Of more immediate importance, the present paper opens the door for the systematic screening of new lipophile-siRNA conjugates with improved association kinetics with lipoprotein particles, or even pre-formulation of such conjugates with lipoproteins or other natural or synthetic carriers of the blood. I look forward to what this line of investigation will yield next.