Progress in Predicting the Immune Response to an RNAi Therapeutics

When in 2010 Tekmira decided to prematurely terminate its first clinical RNAi Therapeutics candidate, TKM-ApoB, due to unexpected immune stimulation in a phase I, it was disappointing in a number of ways.  Not only did it destroy TKM-ApoB right there, it also cast doubts on how predictive our preclinical immune stimulation tests really were and whether as a result, other candidates may suffer similar fates.  Apparently, using rodents or even non-human primates for that purpose had not been all that useful either. It is also said that the event served as a red flag to some regulators which wanted the technology being developed more slowly.

Tekmira, however, soon claimed to have discovered the reason why their PBMC-based assay did not pick up on the immunostimulatory potential of TKM-ApoB and reported that they had have found a more predictive test tube assay instead.  Apparently, using heparin during the preparation of PBMCs (sample enriched for certain immune cells from blood) was the culprit and a whole blood-based assay (including things like red cells and albumin) would reflect much more reliably the situation in people.

A nice paper by Coch et al. from the Hartmann group in Bonn, Germany, now provides comprehensive insight into possible causes for false positives and false negatives when using in vitro immune stimulation assays.

Role of serum proteins  

It is known that phosphorothioate oligonucleotides, a particularly widely used chemistry in the antisense field, rapidly bind to proteins in serum.  Given its abundance and sticky nature itself, albumin is thought to be an important target of such oligos.  The group thus found that their binding in whole blood sequestered them from the immune cells such that the immune response was significantly lessened.  By contrast, in the PBMC-based assay, these phosphorothioates stimulated a robust immune response.

This insight is obviously particularly important if the interest was in developing oligonucleotides that are immunostimulatory on purpose, especially for cancer and viral applications (false positive in PBMC).  Whether the finding that in whole blood assay phosphorothioate oligos may not exhibit immune stimulation can be used to conclude that they will be safe is another question as the blood-based assays may not be informative on what is going on in the tissues.   

Role of anticoagulants

Closer to the TKM-ApoB story, another important insight was that the nature of the anticoagulant can have a big impact on the outcome of the assay.  When drawing blood, and especially when employing a whole blood assay, it is important to use an anticoagulant as otherwise standing blood would start to clot. 

EDTA and heparin are standard reagents for this purpose.  EDTA has been recognized that it can distort immune stimulation assays and thus is not used.  The surprise (well not that surprising in hindsight) is that heparin, due to its negative charge, could displace oligos such as RNAi triggers from their delivery vehicles, including liposomal nanoparticles (LNPs).  It is often the delivery agents that bring the oligonucleotides to the immune receptors.  Moreover, nanoparticle formulations such as LNPs can facilitate multivalent interactions as they hold together and present a number of oligonucleotides simultaneously.  This in turn can potentiate the immune signaling.

Accordingly, what probably caused the immunostimulation of TKM-ApoB to be missed is that heparin at some point displaced the ApoB siRNA from the liposomes thus destroying the potentiator effect of SNALPs.  Importantly, replacing heparin with hirudin (think leeches) did not suffer from the same limitation.  Therefore, human whole blood assays with hirudin as the anticoagulants appear to be the simplest and most reliable preclinical innate immune stimulation assay for Oligonucleotide Therapeutics development.

Growing the database

Of course, these insights are only a first, albeit critical step.  The next step is to understand what degree of immune stimulation in the test tube relates to a likely adverse event in the clinic.  For this, it is necessary to go back and forth between the test tube and clinical observations and correlate the clinical phenotype (cytokine production, fever/chill symptoms) with the test tube response. Complicating matters, different persons have individual innate immunostimulatory sensitivities, and depending on the state of the immune system (e.g. existing infection) there will also be intra-patient variability.

   

On a more positive note, not all delivery/RNAi trigger formats are equally prone to stimulating the immune system with LNPs probably being one of the more challenged formats with regard to innate immune stimulation.  Even so, the clinical track record with SNALPs after TKM-ApoB (lowered doses of steroids with ALN-TTR02, no steroids apparently used in the TKM-EBOLA trial) suggests that the new assays are having a positive impact already.

Journal Club: Unraveling the Substrate Specificity of RIG-I and what it Means for RNAi Therapeutics

A paper by the Hartmann group in Bonn, Germany, takes the mystery out of the structural features of RNAs that activate the cytosolic viral innate immune sensor RIG-I and offers simple design rules for either avoiding, or in some cases intentionally inducing such activities with RNAi triggers. It is good news for essentially all types of synthetic siRNAi as practiced today, but requires re-evaluation of some (but not all!) DNA-directed RNAi approaches, and those approaches that depend on the preparation of RNAi triggers by in vitro transcription rather than by synthetic means.

Some of the first wave of RNAi Therapeutics candidates that were rushed into the clinic were most likely based on pre-clinical efficacy results due to the induction of non-specific innate immunity. As an aside, it always ‘surprised’ me for example how companies with no track record in nucleic acid therapeutic development would suddenly claim to be the first ones to enter an RNAi Therapeutics candidate into the clinic. Innate immunity is based on the recognition of pathogen-associated molecular patterns, PAMPs, by cellular receptors that then initiate a powerful signaling cascade leading to the successful defense against viral and bacterial infections, often resulting in the death of the infected cell itself. Therefore, to avoid mis-interpretation of RNAi data and to ensure safety, the RNAi Therapeutics field needs to take into account the structural and sequence-specific signatures of nucleic acid triggers of innate immunity.

There are two classes of nucleic acid receptors relevant to this discussion. The first one are the toll-like receptors (TLRs) 3, 7, and 8 which recognize certain types of single- and double-stranded RNAs mainly in the endosomes. This is important when RNAi triggers are delivered from the outside, but not for DNA-directed RNAi. The second one is comprised of cytosolic PAMP receptors that both synthetic and DNA-directed RNAi triggers may encounter. While PKR was initially thought to be the main cytosolic receptor relevant to RNAi, it more and more emerges that the RNA helicase RIG-I is what the field needs to be mindful of, and is the subject of the present paper by Schlee and colleagues.

Before these findings, it had been thought that any RNA with a triphosphate chemical group at the 5’ end would induce RIG-I. Blunt-end double-strand RNAs of the size of siRNAs were also thought by some to have this capacity independent of a 5-triphosphate modification. The present findings, however, show that the confusion about the exact RIG-I substrate structural features arose from the origin of the RNAs used in those studies. Since 5’-triphosphate modifications are not routinely offered by synthetic RNA vendors, it has been convenient to use RNAs generated through in vitro transcription by recombinant, purified phage polymerases which leave a triphosphate group at the 5’ end. Unfortunately, these phage polymerases have the property of generating additional species of RNAs aside from the desired one. It turns out that double-stranded RNAs, still with a 5-triphospate group, are one of those and that these are the ones actually recognized by RIG-I. The authors were thus able to show that well-defined synthetic single-strand RNAs with a 5’-triphosphate alone were not sufficient to induce RIG-I.

For RNAi Therapeutics the findings mean that RIG-I should not be a concern for essentially all synthetic siRNA therapeutics, as almost all of them are administered in a 5-hydroxylated form which are then 5’-monophosphorylated. Both modifications would abolish RIG-I activation. This is also good news for blunt-end ‘Atu RNAi’-type siRNAs as practiced by Silence Therapeutics which may have been previously suspected to trigger RIG-I. It is true, however, that in the context of 5’-triphosphates, the more traditional double-stranded Tuschl-type siRNAs which contain 3’ overhangs further diminish RIG-I activity even in a 5’-triphosphate context.

The picture is somewhat more complex for DNA-directed RNAi Therapeutics approaches. Historically, perfect complementary hairpin RNAs driven by RNA polymerase III promoters (Pol III) have been used. As these hairpins are destined to be exported into the cytoplasm, the cellular location of RIG-I, the minimally or not at all modified 5’ ends of such shRNAs, i.e. 5’-triphosphates, run the risk of triggering RIG-I responses. However, a simple mismatch of the 5’-triphosphate nucleotide with the opposite strand should abrogate most RIG-I activity and some of the widely used Pol III expression cassettes fortuitously carry such mismatches which do not appear to affect gene silencing. For RNA Polymerase II-driven DNA-directed RNAi Therapeutics, RIG-I should not be a concern for the reason alone that Pol II transcripts exhibit a 5’ modification that does not induce RIG-I. And finally, for trans-kingdom RNAi Therapeutics where the bacteria expresses the RNAi trigger, one may want to design shRNAs that are similar to the Pol III strategies. It is also good news that not any 5’-triphosphate RNA induces RIG-I, since bacterial transcripts are quite rich in 5’-triphosphates.

In summary, the paper by Schlee and colleagues indicates that innate immune activation by siRNAs in the cytosol is no show-stopper by any means. By contrast, since 5-triphosphates do not necessarily abolish the RNAi activity of double-stranded RNAs, bi-functional immunostimulatory siRNAs can be designed such as for antiviral and cancer applications- as demonstrated late last year by the same group in a collaboration with Alnylam. Of course, 5’-triphosphate dsRNAs may also be used independently of RNAi for the same reasons.

The Risk of Rushing RNAi Therapeutics into the Clinic

RNAi has always caught on very fast. It took only nine years for the Nobel Committee to recognize the importance of RNAi in medical biology and award The Prize for its discovery, and only 6 years following the discovery that siRNAs induce RNAi in mammalian cells by Tuschl to become an indispensable tool in the basic and applied studies of human gene function. Equally astounding is the fact that there are now close to 10 RNAi-based therapeutics that have entered the clinic since.

I have extensively described here before why I think RNAi has the potential to be the next great drug development engine, including the prospect of faster development timelines due to straight-forward mechanism of action and platform reproducibility. However, in the wake of Alnylam’s Q3 conference call announcing an insignificant delay in their RSV program, but a more open-ended delay in their liver programs, what I would like to do today is to point out the dangers of rushing RNAi Therapeutics into the clinic mainly borne out of the tension that exists between applying the best and safest science and satisfying investor demand for gushing clinical pipelines.

From the clinical perspective the ultimate danger is obvious: putting trial participants at risk, and disappointing patients’ expectations for a cure of their disease. From the perspective of running an early-stage biotech business that needs to raise money fairly regularly, the issues easily become more complicated. Although I admire the honesty and scientific intent that underlie statements like that by Nastech that one should not expect RNAi Therapeutics from your company until another 15 years, it certainly won’t capture the imagination of Wall Street. The easy way would be therefore to set your bar a little bit lower and signal to your potential investors that you deserve more money since you’ve been able to put so many drugs into the clinic in such a short period of time. A sophisticated biotech investor would know that these companies can be a good investment, although you do not necessarily want to stick it out until the Day of Reckoning comes.

The danger to the field of RNAi Therapeutics is therefore that as some of these rushed candidates come to a stage where they have to prove their safety and efficacy in large-scale clinical trials, a good number of them will fail, essentially because some of the Best Practices were not followed, including addressing cytokine induction issues, off-targeting profiles, RNAi delivery, and pre-clinical safety and efficacy studies that ideally include non-human primates.

Acuity Pharmaceuticals (now part of Opko Health) dazzled everybody when they came out of nowhere and can now claim to have been the first to put an RNAi candidate (for wet AMD) into the clinic. Unless they have changed the composition of their drug since study initiation, Cand5 appears to be an unmodified siRNA injected straight into the eye. This alone makes me wonder whether an optimized compound has been put into the clinic, and I have more confidence in a program run by Allergan and Sirna Therapeutics (Merck) targeting the same pathway for wet AMD, but with a modified siRNA formulation intended for slow release.

SiRNAs that induce cytokine responses may also have a number of additional biological properties, some of them even potentially beneficial for the disease at hand. Gunther Hartmann from Bonn, a scientist with a cytokine angle on oligonucleotide therapeutics, has even proposed at the recent OTS meeting to purposefully combine the immunostimulatory potential of RNAs (isRNA) with siRNA design. Cancer and infectious disease may be good areas to test this concept as isRNAs are thought to help the immune system in fighting related these diseases.

There has been similar discussion whether there would indeed be any harm if an RNAi therapeutic targeting the Hepatitis C Virus (HCV) had some concomitant interferon response. Isn’t interferon (and RNAi) nature’s first answer to viral infections and the mainstay of current HCV treatment regimens anyway? Similar arguments may also apply to RSV.

The fact that Alnylam is now focusing RSV-01 on adult populations makes me therefore wonder whether this was driven at least in part due to concern that the tender infant respiratory system may be more prone to overreact to a potentially immunogenic siRNA molecule than a lung hardened by years of air pollution. This siRNA is probably unmodified as it was this June that the first Alnylam compounds using ISIS modification patents moved into IND-enabling studies. Being unmodified from a pharmacokinetic perspective may not be that bad or even desirable in RSV, as RSV is an acute infection and long drug exposure may therefore have the potential to do more harm than good.

I should emphasise, however, that the early rodent RSV studies that form the basis of Alnylam’s RSV-01 and which have supposedly been replicated by the company, demonstrated sequence-specific antiviral activities. Furthermore, from Alnylam’s presentations one can assume that RSV-01 was carefully screened for cytokine induction in a number of human cell lines and animal models. I should add as well that the slight delay of the RSV experimental infection model studies is not the result of any of these considerations, but more simply reflects the fact that finding volunteers to be infected with a virus that gives you flu-like symptoms and requires you to be locked away from the outside world for a couple of weeks, is not that easy. However, 74 of the 88 subjects, I suppose mostly students, have already been recruited and we should hear top-line data early next year.

Alnylam’s conservative approach to drug development is further demonstrated by their delay of filing INDs for their liver programs, for hypercholesterolemia and liver cancer. While there is no doubt that with current systemic delivery capabilities it is possible to achieve potent gene knockdown in the liver, the safety and dose-response data so far would explain Alnylam’s caution into committing to a particular formulation by year-end as originally guided. Instead, I agree with their assessment that with new chemistries coming online, such as MIT’s lipidoids which formed the basis of the recent microRNA saturation data in Nature, it is wise to keep testing all of to find the formulations that offer the best therapeutic index. It would not be the first time that a drug for treating heart disease would fail in a large-scale trial because of unacceptable side-effects seen in a handful of participants. For what it’s worth and mindful of the business considerations about demonstrating human proof-of-concept of an RNAi Therapeutics with the hypercholesterolemia program, I wonder whether Alnylam should not go first with liver cancer anyway.

Needless to say, this cautious, data-driven approach not only benefits Alnylam the Science, but also Alnylam the Business. The importance of their scientific credibility through publications and conference presentations cannot be underestimated when it comes to their ability to execute on their business development goals, mainly in the form of lucrative license deals and access to enabling technologies. With a cash position of $468M, Alnylam is in a stronger position than ever to focus on the long-term success of the company and its shareholders.



Rosetta Genomics on Track to Bring the First Clinical RNAi-related Product to Market

Almost unnoticed in the microRNA diagnostics space, Rosetta Genomics reported this week that it had completed the pre-validation phase for its first microRNA diagnostic product scheduled to come into the clinic in the first half of next year. It would be exciting to see the first RNAi-related product have a direct clinical impact and, if successful, will fund Rosetta’s microRNA diagnostics and therapeutics programs with minimal shareholder dilution. The microRNA diagnostic is designed to differentiate between squamous and non-squamous lung cancer which is not always possible to tell under the microscope and an area of particular importance now that Genentech’s VEGF-targeting MAb Avastin showed life-threatening side-effects particularly in subjects with squamous cell cancer.

While RNAi Therapeutics has attracted most of the RNAi attention, microRNA-based diagnostics are set to become the first commercial success of RNAi-related products in the clinic. Their differential expression, scalability, and, equally important, potential relative stability advantages compared to protein and larger mRNA biomarkers means that microRNAs have the potential to become the biomarker platform of choice. The (near) future should tell.