Wednesday, September 3, 2008

RNAi Therapeutics and Innate Immunity- Keeping the Field Honest

As part of the RNAi Therapeutics review series in Human Gene Therapy earlier this year, former Protiva scientists Adam Judge and Ian MacLachlan (both now with Tekmira following the Protiva-Tekmira reunion) made some rather bold statements with regards to the interpretations of a number of pre-clinical RNAi Therapeutics validation papers (‘Overcoming the innate immune response to small interfering RNA’). As part of the same reviews series, in the risk section of “The Business of RNAi Therapeutics”, I also cautioned that some of the first RNAi Therapeutics candidates may show clinical efficacy, but not necessarily for all the right reasons.

The reason for this should not come as a surprise to anybody in the oligonucleotide therapeutics field: long known from the experience with antisense and other oligonucleotide therapeutics classes, oligonucleotides such as siRNAs have the potential to induce innate immune responses which can have antiviral and anti-angiogenic activity independent of their gene knockdown capacity. In fact, there are significant efforts to harness this biological property for therapy in its own right, particularly the TLR responses. Furthermore, the potential for inducing innate immune responses by synthetic and DNA-directed RNAi has been well documented since 2003 and many of the pathways involved elucidated. Nevertheless, one should not ignore the fact that while RNAi Therapeutics may actually be able to take advantage of such activity as part of synergistically acting immunostimulatory RNAi Therapeutics, the risk is that the oligo-dependent immune responses are quite complex and therefore often difficult to predict and in the worst case may cause serious adverse events.

Since many of the early RNAi Therapeutics validation papers involved antiviral and anticancer applications, it was therefore reasonable to suspect that some of the studies misinterpreted therapeutic effects as the result of RNAi gene knockdown when, in fact, innate immune responses accounted for the majority of the activity. In support, the Tekmira researchers now report that almost all of the unmodified siRNAs reported in a sample of such papers were immunostimulatory whereas a single siRNA that, somewhat disturbingly so, was used as the control siRNA in many of the studies proved to be the exception having no such detectable activity. I should add, however, that the assay conditions were rather stringent (types of cells used and high siRNA concentrations) and just because an siRNA may induce immune responses under these conditions does not prove that these were actually responsible for the treatment effect seen in each of the cited studies. Also, if TLR therapeutics history is any guide, oligonucleotides that elicit immune responses in small animal models, do not necessarily do so in primates.

Given its potential as a whole new class of therapeutics, the scientific and clinical bar for RNAi Therapeutics is set particularly high and reports like the effect of TLR3 stimulation by siRNAs on preclinical models for wet AMD and the present paper by Tekmira tend to get quite a bit of press. While they remind us of the complexities involved in establishing a functional new drug discovery platform, they should also be regarded as promoting that process. In fact, the handful of bona fide RNAi Therapeutics groups, pure-plays and Big Pharma subsidiaries alike, are already taking oligo-induced innate immune responses very seriously and have taken advantage of the rapid progress in the field by applying best practices for identifying and correcting these responses (modification, siRNA structure) in developing the latest crop of RNAi Therapeutics candidates.

The acquisition of former TLR therapeutics company Coley Pharmaceuticals by Pfizer for example may be interpreted as Pfizer investing in solving siRNA-induced innate immune responses as one of the main challenges for RNAi Therapeutics they had identified. Similarly, Sirna Therapeutics and Protiva in their prominent 2005 Nature Biotech paper on RNAi delivery in a mouse model of hepatitis B recognized the potential of unmodified siRNAs to elicit non-specific viral suppression and solved the issue by appropriately modifying the siRNAs. Around the same time, Alnylam somewhat quietly generated IP related to double-strand RNA immune stimulation that it then exclusively licensed to Tekmira. Clearly, the main players in the field have not chosen to ignore the issue, but have invested considerable efforts with tangible results.

But what about the current RNAi Therapeutics clinical candidates that have already entered the clinic? There are one phase III (Opko Health) and two phase II (Sirna/Merck-Allergan and Quark-Pfizer) siRNA candidates for the treatment of wet AMD that obviously have naturally come under increased scrutiny. As far as I am aware, all three of these are ‘unformulated’, intravitreally injected siRNAs with one of them, Opko’s, being an unmodified siRNA. While it is not clear how well the mouse TLR3 studies translate into humans, they certainly raise the concern that non-specific responses might be responsible for any thus far clinically observed therapeutic effects, particularly since in the recent Nature study gene knockdown by this route was very limited at best (cholesterol-conjugated siRNAs, however, administered by the same route were shown to mediate functional gene silencing in the same study).

As is the case with Alnylam’s lead candidate ALN-RSV01 for the treatment of RSV infection which has raised similar concerns, it will be important to be forthcoming in the interactions with the regulatory agencies such that safe trials can be designed based on our best understanding of the mechanisms of action of the different siRNAs. While I haven’t read the documents, it certainly wouldn’t be the first time if such non-specific effects were noted as potentially contributing to treatment. In the future, it would not surprise me at all to see openly declared immunostimulatory siRNA drug candidates enter the clinic. If, however, these issues are not addressed upfront, and should adverse events occur as a result, this could easily backfire and future trials rendered much more onerous- something that should be in nobody’s interest. As for the prospects of the individual drug candidates in question, even if non-specific effects contributed to the therapeutic efficacy of these candidates, as long as they are safe and well tolerated they may very well be viable drugs.

Finally, it is curious as to what exactly motivated Tekmira to re-test an entire battery of published siRNAs for their potential of inducing non-specific effects. It is possible that Tekmira has evaluated siRNA therapeutics for a number of the same applications like flu and wet AMD and were frustrated to see publications come out that according to their experience should have been artefacts (scientists tend to measure themselves by the number of publications and their impact factors and don’t like to see their own published work de-valued this way). Another part of the answer may also have been to keep the field honest at this early stage of RNAi Therapeutics drug development before long-term damage is caused: “However, surprisingly few of the reported studies have adequately tested, or controlled, for the potential effects of siRNA-mediated immune stimulation, making the many published claims of therapeutic efficacy a collective liability for the RNAi field that remains to be addressed.” By setting a rigorous new standard, Tekmira also signals their expertise not only in RNAi delivery, but also in siRNA chemistry and safety (like Coley, Tekmira has a long-standing interest in the use of immunostimulatory oligonucleotides for therapy). Supporting their claim, Tekmira/Protiva’s publications on abrogating TLR7/8 responses and SNALP RNAi delivery have proven to be extremely reproducible in many different laboratories.

The road to RNAi Therapeutics reality won’t be smooth. As much as it is important to tackle the scientific hurdles head-on, investors and the press should also make an effort to discriminate between ‘good’ and ‘bad’ science.

27 comments:

Anonymous said...

An excellent summary.

"…Alnylam’s lead candidate ALN-RSV01 for the treatment of RSV infection which has raised similar concerns…"

For those here who do not follow the relevant boards at IV, I would add that I posed a question concerning the mechanism of action of ALN-RSV01 to Alnylam's investor relations back in March following the publication of the Kleinman paper. In their response, they said

"…ALN-RSV01 shows specific anti-viral activity as compared with a LARGE NUMBER of control siRNAs and, further, ALN-RSV01 demonstrates anti-viral effects in vivo through an RNAi mechanism as proven by 5'RACE measurements." (emphasis added)

Assuming that Alnylam indeed controlled ALN-RSV01 efficacy by comparing to a LARGE NUMBER of control siRNAs, it would appear that any observed efficacy should be largely attributable to RNAi mechanism of action (the presence of which was demonstrated by the 5'RACE measurements).

"Finally, it is curious as to what exactly motivated Tekmira to re-test an entire battery of published siRNAs for their potential of inducing non-specific effects."

My view is that part of the motivation may have been the effort to pre-empt any immunostimulatory challenges that the current clinical candidates may face from adversely affecting the entire RNAi field. (We have seen the indiscriminate negative publicity generated by the Kleinman paper) But I think this is similar to what you describe as trying to keep the field hones.

Martin

Dirk Haussecker said...

Martin- I agree with you that good evidence has been presented by Alnylam that the antiviral effects they observe are the result of RNAi gene knockdown. However, in addition to mechanism of action, whether or not immune stimulation contributed to antiviral efficacy, the more important question might be related to safety. Although the phase I inhalation studies of ALN-RSV01 were largely successful, flu-like symptoms were observed at the highest dose level and it is not too far-fetched to suspect some kind of innate immune response to the siRNA. It's just something to be aware of and I would hate to see a Jesse Gelsinger-like event for RNAi Therapeutics.

Anonymous said...

@Martin:
"[...] RNAi mechanism as proven by 5'RACE measurements."

Although I do concur that RNAi "works"--obviously--, 5'RACE cannot prove the absence of unspecific TolR-mediated immonugenicity.

Also, 5'RACE is not a quantitative method; of course, there will be "some" RNAi activity and 5'-RACE will measure it regardless of the extent of RNAi activity.

In fact if you look at the recent Alnylam paper in PNAS 19-Aug-2008 you can see that the contrast is not great on the Fig.3 (5'RACE).

Semi-quantitative 5'RACE and immunogenicity studies are warranted.

Guy.

Anonymous said...

Great blog. You are bookmarked. I'm a postdoc in Dan Anderson's group and am still getting to know the field, so this will be a great way to stay on top of all the news. Thanks.

Anonymous said...

a non-scientific comment - could you change the background color? it is difficult to read with black background and white font.

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出張ホスト said...

女性会員様増加につき、出張ホストのアルバイトが不足中です。ホテルや女性の自宅に出向き、彼女たちの欲望を満たすお手伝いをしてくれる男性アルバイトをただいま募集しております。興味のある方はTOPページをご覧ください

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出張ホスト said...

女性向け風俗サイトで出張ホストをしてみませんか?時給2万円以上の高額アルバイトです。無料登録をしてあとは女性からの呼び出しを待つだけでOK、お試し登録も歓迎です。興味をもたれた方は今すぐどうぞ。

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By Dirk Haussecker. All rights reserved.

Disclaimer: This blog is not intended for distribution to or use by any person or entity who is a citizen or resident of, or located in any locality, state, country or other jurisdiction where such distribution, publication, availability or use would be contrary to law or regulation or which would subject the author or any of his collaborators and contributors to any registration or licensing requirement within such jurisdiction. This blog expresses only my opinions, they may be flawed and are for entertainment purposes only. Opinions expressed are a direct result of information which may or may not be accurate, and I do not assume any responsibility for material errors or to provide updates should circumstances change. Opinions expressed in this blog may have been disseminated before to others. This blog should not be taken as investment, legal or tax advice. The investments referred to herein may not be suitable for you. Investments particularly in the field of RNAi Therapeutics and biotechnology carry a high risk of total loss. You, the reader must make your own investment decisions in consultation with your professional advisors in light of your specific circumstances. I reserve the right to buy, sell, or short any security including those that may or may not be discussed on my blog.