Sunday, October 16, 2011

Liposomes Also Useful for Functional siRNA Delivery to Phagocytic Cells

The goal in developing a RNAi Therapeutics delivery technology is to achieve gene knockdown in a certain cell or tissue type. Once this is achieved, only our exploding genetic insights into disease limit the potential therapeutic applications. This is for example why we are seeing this SNALP-fuelled expansion in the clinical pipeline for indications in which gene knockdown is targeted in the liver.

In addition to the liver (SNALP), vascular endothelial cells (AtuPLEX, DACC), tumor cells (SNALP), skin (self-delivering rxRNAs), hematopoietic stem cells (lentivirus), and cells in the eye and CNS (lentivirus, AAV), RNAi Therapeutics delivery technologies have reached sufficient maturity to warrant their clinical development for knockdown in phagocytic cells of the immune system. In fact, there is already a clinical trial by Duke University that involves the ex vivo electroporation of siRNAs into dendritic cells for therapeutic cancer vaccine development (think Dendreon), and a first systemically administered example, Tekmira’s SNALP-EBOLA, is about to enter the clinic with an IND planned by year-end.

Probably fearing that the investing public has largely forgotten about such potential of Tekmira’s technology, Alnylam this week advertised two liposomal siRNA delivery studies, one published in Molecular Therapy (Basha et al.) and the other one in Nature Biotechnology (Leuschner et al.), which nicely characterized RNAi activity in phagocytic cells and provided a few tanatalizing examples of their potential therapeutic applications.

Leuschner et al: Interfering with Pathologic Phagocyte Recruitment by Knocking Down CCR2

The Leuschner at al. study, stemming from a collaboration between the Massachusetts General Hospital, MIT and Alnylam, used lipidoid lipid-formulated siRNAs to target chemokine receptor CCR2 in phagocytic cells. The lipid was the C12-200 cationic lipidoid that was shown in a paper last year to promote gene knockdown in the liver at much lower dosages compared to 1st-generation lipidoids (Love at al. 2010).

After demonstrating that the systemically administered liposomes were taken up by various phagocytic cell populations in the bone marrow, spleen, and the circulation, the researchers then focused in, for the functional part of their study, on the Ly-6high subset of these cells from the spleen. These were chosen because of the particularly efficient liposomal uptake in this cell population and their role in the pathogenic, CCR2-dependent recruitment to sites of local inflammation.

As a reminder, while such inflammatory recruitment is a vital part of our first-line defense and response to foreign invaders and tissue damage, respectively, they often end up doing their job too enthusiastically leaving behind pathologic scars, unwittingly promote cancers, or interfere with medical interventions such as transplantation.

Leuschner and colleagues studied four settings where interfering with Ly-6high through CCR2 knockdown might be beneficial (all mouse studies): ischemia-reperfusion injury as would occur following a heart attack; inflammation at atherosclerotic lesions; pancreatic islet graft survival; and reducing the number of tumor-associated macrophages which are thought to promote cancer. In all four settings, it was found that a moderate ~50% knockdown of CCR2 was sufficient to effectively inhibit inflammatory cell recruitment to the sites of disease and was accompanied by the hoped-for outcome.

While this is certainly encouraging news and seems to open up many therapeutic avenues just based on CCR2 targeting, the degree of knockdown suggests that while it should be possible to achieve therapeutic outcomes with well-chosen target genes, further improvements in efficacy are necessary for the particular formulation to be broadly applicable to other target genes that may not be as dosage-sensitive as CCR2 appears to be.

Basha et al.: Tekmira SNALPs Promote Gene Knockdown in Antigen-Presenting Cells

Unlike the Leuschner et al. study which focused on the potential therapeutic applications of gene knockdown in phagocytic cells, the Basha et al. studied the mechanism, efficacy, and safety of SNALP delivery to antigen-presenting cells (APCs). This paper was largely conducted in the laboratory of Alnylam's new friends from Vancouver.

Evaluating four SNALP formulations differing only in the nature of their ‘critical’ lipid- DlinDAP, DLinDMA, DLinK-DMA, or DLinKC2-DMA- it was found that three of them (DMA, K-DMA, KC2-DMA) were capable of promoting gene knockdown in antigen-presenting macrophages and dendritic cells- both in vitro and in vivo following systemic administration. Taking into account safety, the authors favored the formulation with the KC2-DMA lipid, the lipid of the Tekmira line-of-research that was shown last year in the Nature Biotechnology paper to be highly efficient also for gene knockdown in the liver (Semple et al. 2010).

Importantly, the present data showed that increased efficacy did not come at the cost of safety. Safety is all the more important here since, similar to the lipidoid formulations described above, the delivery efficacy is still much less compared to their use in functional SNALP delivery to the liver, meaning that higher dosages would have to be administered. Since hepatotoxicity could be a dose-limiting toxicity for these formulations, the demonstration that by increasing particle size, gene knockdown in the liver could be abolished without compromising gene silencing in APCs, suggests a strategy to increase the safety of APC-targeted SNALP applications.

The Basha et al. Study Employed Tekmira-Owned Lipids and Formulations and was Partly Funded by Tekmira

While the specific results were not really surprising in light of what has already been known about liposomal delivery, what really confused me was the fact that it was Alnylam that did all the advertising of the Basha et al. study with no word whatsoever from Tekmira. After all, the paper acknowledged Tekmira as a funder of the study and based on the Responses by Alnylam, including their Canadian subsidiary Alnylam-Canada (aka AlCana), to Tekmira’s allegations, neither Alnylam nor AlCana contest that all four lipids are owned by Tekmira. In fact, KC2 is the latest in the series and, as we know, the Nature Biotech paper in early 2010 was advertised by both companies (see here and here).

The acknowledgement:

The authors would like to acknowledge support from the Canadian Institutes for Health Research (CIHR) under U0P grant FRN 59836, as well as support from Tekmira Pharmaceuticals and Alnylam Pharmaceuticals. P.R.C. has a financial interest in Tekmira and receives grant support from Alnylam.

Sure, Alnylam has acted a bit irrational and confused when it came to Tekmira and its general understanding of what constitutes ownership. However, advertising a lipid for which there has been little controversy that it belongs to Tekmira and a study that was in part paid for by Tekmira, without so much as an acknowledgement of Tekmira sets a new standard. Perhaps as perplexing is the fact that Tekmira has not responded at all to these developments.

I entertain two explanations for this, explanations that I consider equally likely. The first one is that things have gotten so much out of control that Alnylam does not care any more about the liabilities of such press releases. If Tekmira needed to find an example of where Alnylam falsely advertised Tekmira’s technology as its own, one of Tekmira’s allegations, Alnylam just provided it with the most striking one.

The other explanation is that maybe things are pointing towards a settlement or merger of the two companies. This would explain why Tekmira seems to co-operate with Alnylam by not protesting about last week’s press release. It is also of note that Alnylam, as of October 8, is now represented by a new lead counsel in the ALN-VSP02 patent Interference case. Initially represented by Rothwell, Figg, Ernest & Manbeck, it is now the attorneys from Wilmer, Cutler, Pickering, Hale, and Dorr that have taken charge of the Interference for Alnylam…you guessed correctly, the same law firm representing Alnylam in the Big One with Tekmira. An alignment of these two cases would have always made sense. At this advanced stage of the Interference, however, the change seems somewhat surprising, but would make sense if it is now critical to have the two cases strongly aligned so as not to jeopardize a mutual understanding that may have been reached.


Anonymous said...

Dirk, what I am inferring from the change in counsel is that tkmr/alny will make up, hug, and work together. This will make both stocks go up, but I suspect tkmr more than alny. Thats what my tea leaves tell me.

Anonymous said...

It's obvious Tekmira had little involvement in the second research cited beyond any incidental funding (UBC and Tekmira used to be collaborators but no longer). This is still early stage research and whatever lipids are currently being used will be different if things ever progress to the clinic. Issuing their own PR or demanding that Alnylam "advertise" for them would be more than a little desperate.

Anonymous said...

Dirk, It's actually sad that your commentaries become more and more emotional. Used to read each and every one of them, and now am hugely disappointed... What happened?

Dirk Haussecker said...

Sorry that you feel this way. I believe I should just ignore the way Alnylam has tried and take away the technology from Tekmira. SNALP technology is critical for the near- to midterm future of the technology, and having closely followed the Alnylam-Tekmira relationship over the years, I allow myself to have an opinion on this important issue. Not doing so, or forgetting about it as time moves on, would be an omission.

Anonymous said...

Dirk - keep the candid approach coming. Whether we like the opinion or not is a separate matter. Your true thoughts on the RNAi scene are very much valued.

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