RG-101 for HCV: Give Me 8mg/kg

Last October, Regulus Therapeutics announced stunning data from an ongoing trial of anti-miR122 RG-101 for the treatment of HCV infection.  Accordingly, 6 out of 14 patients administered a single injection of 2mg/kg of the GalNAc-conjugated antisense oligonucleotide dropped to what the company referred to as ‘below the level of quantitation’ (BLOQ) by day 30 and remained there as of the update (day 57 for those treated first in the study).

*BLOQ is not the same as 'undetectable'.  It is 'undetectable' for x number of weeks that technically defines a cure.

2mg/kg data looking ever better

The more I look at the data (here for the corresponding dataset), the more intrigued I get.  For starters, the 2mg/kg was only the starting dose in the patient cohort.  Moreover, a patient that had been more recently treated and had reached only 29 day in late October seemed to be on his way to BLOQ.  In fact, after his titers had dropped to below ~1000 IU/ml, viral decline seemed to accelerate as if a threshold had been reached. 

Overall, it seemed that as long as you got below ~1000 IU/ml, the virus would not rebound (at least until day 57).  Accordingly, the 5 patients that rebounded got close (1000-10000 IU/ml), but never below the apparent threshold.  The lack of overlap between the individual knockdown curves also suggest that viral rebound is not the result of stochastic escape mutations, as is often the case in HCV, but simply due to diminishing drug concentrations.

This leaves two patients which as of the update were still slowly declining as if the threshold rule was counterbalanced by declining oligonucleotide concentrations in the liver after day 30.

In summary:

Everybody responded irrespective of genotype and starting viral titers, of these…

…6/14 BLOQ;

…3/14 undecided;

…5/14 nadir of 1000-10000 IU/ml, then rebound.

A one-shot cure?

In the current marketplace, comprising of ~12M untreated HCV patients in the top 7 pharmaceutical markets plus hundreds millions more in the rest of the world, HCV treatment success has less become a function of viral cure rates achieved in clinical trials, but more one of compliance with the pill regimens.  Therefore, a therapy which would shorten current treatment regimens (say to 4 instead of 8-12 weeks), or even involve just one or two subcutaneous administrations in the physician’s office as part of routine checkup visits, would have great pharmacoeconomic impact.

The company is currently crunching the numbers for the next higher, 4mg/kg cohort.  In general, with RNAi and antisense therapeutics, what you see with increased doses is a slight acceleration of gene knockdown (thereby shifting the knockdown curves down), and, likely of more importance in this case, extended duration of drug activity.

The latter is supported by animal, including non-human primate data that showed that liver concentrations of RG-101 increase linearly with dose from 1-10mg/kg.  This means that for the 3 undecided patients in the 2mg/kg cohort, a doubling of the drug concentration could have easily pushed them BLOQ.  

Alternatively, a second dose of 2mg/kg on day 29 would have achieved that as also supported by animal data showing that viral nadir drops further when a second dose is given (on the left is an example from the challenging chimeric mouse model).

I am less sure whether the other 5 patients would have gotten there with 4mg/kg, but would be much more sanguine about it with a dose of 8mg/kg.

A mistake not to escalate to 8mg/kg

Unfortunately, Regulus Therapeutics stated last year that based on biomarker data which measures the activity of human genes naturally regulated by miR-122, 4mg/kg would be the highest dose to be tested in HCV.  This is because the biomarker response seemed to have saturated already by 2-4mg/kg.  This despite of the fact that they had established in the same phase I study that 8mg/kg is also safe and well tolerated in non-infected healthy volunteers.

I clearly think that stopping at 4mg/kg is a mistake.  First, I highly doubt that at least in terms of duration of biomarker activity, they did not see improvements with higher doses (show me the data).  Second, it is wrong in my opinion to assume that drug saturation with regard to microRNA activity against endogenously expressed genes is a good reflection of microRNA activity in the context of viral replication.  In this case, the rationale is further in question because of the special way miR-122 acts in HCV replication (increases, not decreases activity).

Tempering expectations

As we await the 4mg/kg data and an update on the 2mg/kg cohort in ‘early February’ according to recent company guidance, I should caution that the above scenario suggesting a one-shot monotherapy cure is within reach is obviously an unbelievably exciting best-case scenario.  There is a lot which could make the market react adversely to the upcoming data.  This includes emerging side effects, and responses that could even be worse than the 2mg/kg data, e.g. if some patients do not respond at all or if the company is right in their biomarker assumptions which due to chance could make the 4mg/kg data look worse numerically. 

Therefore, my base case for market-neutral data is that all patients will have to respond, and that the extent of the response will be similar to that seen with the 2mg/kg dose.  But then again, I’m looking at the data and keep thinking to myself…’8mg/kg might be the one-shot cure that could transform HCV treatment’.  In any case, the company has little to lose from dose escalating to 8mg/kg.

Anti-MiR122 Therapeutic Stuns HCV World with Single-Dose Efficacy Results

This morning, Regulus Therapeutics greeted us with amazing results from a phase I study of RG-101, an anti-microRNA 122 oligo for the treatment of HCV infection.  The results show that in the exploratory HCV-infected patient subgroup, a single dose of 2mg/kg of RG-101 resulted in a mean viral load reduction of 4.1log on day 29.  All responded with viral declines, with 6 and 3 of the 14 patients with viral levels below the level of quantitation on days 29 and 57, respectively.   

These results even exceed my own wildest imaginations (as discussed here yesterday) and I’m amazed how much this virus, in all patients, seems to have come to rely on this host-derived microRNA for replication and/or genome stabilization.

It is not clear whether increasing the dose to 4mg/kg, the pre-planned upper dose in the HCV-infected cohort for which dosing is ongoing will bring any additional benefit given that the biomarker data (host genes targeted by miR-122) from the healthy volunteers showed a plateau already at 2mg/kg, indicating the power of this GalNAc chemistry approach.  My guess is that the main benefit from a higher dose would be a decrease in response variability.

Interestingly, IL-28 status, frequently a predictor of treatment success, did not influence the results, nor did HCV genotype seem to have an impact (small numbers).  This further supports that RG-101 could fill some of the more attractive opportunities in the current HCV market.   

Regarding safety, mild and transient injection site reactions seemed most significant with no serious adverse events in the entire study, including the healthy volunteer cohorts (up to 8mg/kg).  This is also consistent with data for Alnylam’sALN-TTRsc which uses a similar GalNAc chemistry and where multiple doses up to 10mg/kg had been tolerated, with injection site reactions, especially at 10mg/kg, being the main safety finding.   

So what’s it all worth?  The results position RG-101 to facilitate a 4-week HCV dosing regimen (compared to typically 8-12 weeks currently), potentially in combination with a single direct-acting antiviral such as Olysio by Johnson&Johnson.  One or two injections maximum.  Great compliance, potentially pan-genotypic, ideal for the busy practicing physician who does not have the time nor inclination to know the ins and outs of each DAA.

In dollar terms, I’d like to think that with this drug profile, this overlooked compound and company are worth as much as what Merck recently paid for HCV drug developer Idenix: $3.85B. The market valuation of Regulus before the news: $300M.  Needlessly to say that I'm long the stock.

PS: GSK once had rights to a precursor compound of RG-101 which it did not exercise.  Importantly, at the time, Regulus’ anti-miR122 compound was not GalNAc-enabled.  This would have necessitated much more frequent dosing and higher dosages and resulted in less potent and more protracted viral declines, i.e. something that would not have been competitive in the current HCV marketplace.  But as often the case with Big Pharma and cutting-edge technology, today’s data clearly shows them wrong.  It has to be said though that GSK more or less got out of HCV which also would have explained GSK’s decision RE anti-miR122.

PPS: Congrats to Peter Sarnow and Catherine Jopling who in 2005 made the mind-boggling discovery that HCV relies on a microRNA for its replication.  I hope they will be handsomely rewarded for it.

ISIS/GSK and Tekmira Come Out with HBV Knockdown Plans

If you did not appreciate the value the pharmaceutical industry has come to place on the HBsAg knockdown concept for achieving a functional cure for chronic Hepatitis B (HBV) infection, the last two days will have woken you up. 

Yesterday, ISIS Pharmaceuticals reported that it had received a $7M milestone payment related to the development of an antiviral RNaseH development candidate (ISIS-GSK3Rx, aka ISIS-HBVRx) which, although undisclosed for competitive reasons, hasgot to be for HBV.  And today, Tekmira publicly announced that they will file an IND for an HBV-RNAi candidate in 2014 while hinting at the partnering potential of such a treatment candidate.

Arrowhead Research is thus not alone in their efforts any more.  Coincidentally, Arrowhead reported today the completion of their enrollment of the phase I single-dose, healthy volunteer study with ARC520, their DPC-delivered candidate for chronic HBV.  Accordingly, the dose escalation was able to run through all the pre-planned 6 dose cohorts up to the top dose of 2.0mg/kg. 

Apparently, there were no signs of significant dose-related toxicities.  The only finding of concern among the 36 volunteers, 24 of which received drug, was 2 cases of lightheadedness of uncertain clinical relevance.  As these occurred at the highest dose, it seems that the company suspects that it could have been drug-related although the study remains blinded for follow-up.

A dose of 2mg/kg without any serious adverse events or dose-limiting toxicities is a great start for DPC delivery technology.  This is especially the case when one considers that the single-molecule subQ version of DPC that I hope will form the basis for the upcoming pipeline candidates, except for the next one perhaps, will be much more potent than the two-molecule version of intravenously delivered ARC520 based on the non-human primate data presented at last year's OTS meeting.

With 2mg/kg of ARC520, I further believe that HBsAg knockdowns of over 90% are likely.  The biggest challenge going forward with this program will be setting a knockdown goal and getting the dose and dose frequency right.

For more about increasingly lively HBsAg knockdown for the treatment of chronic HBV, please follow my HBV Knockdown Blog.

Also today: Tekmira and Arrowhead Research Rapidly Filling Pipeline

In addition to chronic HBV, Tekmira further disclosed development plans for candidates addressing Marburg infection, alcoholism, hypertriglyceridemia and severe orphan glycogen disorders.  The presentation made clear that the company has not sat on its hands since the settlement with Alnylam and is close to having at least 4 drug candidates in active clinical development by early 2015 (TKM-PLK1, TKM-EBOLA, TKM-ALDH2, TKM-HBV).

Meanwhile, previously $200M market cap Arrowhead Research has succeeded with a $60M private placement without having had to offer a discount.  In my opinion, this reflects the broadened investor interest in the space and makes Arrowhead an even better investment as it can now, freed from financial constraints, immediately pursue some of the attractive liver targets with best-in-class solutions and beyond.

Wondering whether we are in an RNA Therapeutics bubble?  Then I suggest you get my OTS 2013 meeting report.  Dare I say that oligonucleotide therapeutics is a more capable and exciting technology than monoclonal antibodies already?  At least if you believe orphan diseases are the way to go in today's pharmaceutical drug development, there is no doubt about it coming out of the meeting. 

RXi Reports Dose-Related Knockdown Three Months Following Single Injection

RXi Pharmaceuticals today reported top-line results from the first of two phase I studies with RXi-109 in dermal scarring.  Intriguingly, the company claims to observe target gene knockdown three months following a single intradermal injection of their self-deliverable RNAi trigger (p=0.02) in a manner that was apparently dose-related. 

Such a drug-dependent and dose-related knockdown would exceed my expectations from this trial as stated in a recent preview here.  The reason why I merely expected to see a correlation between CTGF levels and phenotypic effect on dermal scarring being reported today is that I did not have high confidence that the tissue residence time of RXI109 would be prolonged enough to observe a bona fide RNAi knockdown. 

To wit, the tissue biopsy on which this data rests was obtained during a tummy tuck three months after the intradermal injection of the RNAi.  To assess whether there was an RNAi knockdown in such a single-dose study, I would have thought that an early time-point such as two weeks after injection would have been more appropriate, also because it is likely that some of the CTGF-producing cells might be proliferating in this setting (RNAi duration inversely correlated to proliferation status).

The notion that a correlation between CTGF levels and wound healing would be observed was based on CTGF reflecting tissue inflammation.  So regardless of whether there was an RNAi effect or not, you might expect to see such a correlation.  It is difficult, however, to explain a drug-dependent and dose-related target gene knockdown with this notion, except for by a rare coincidence.

These results then bode well for the multi-dose phase I studies from which results will be reported in time for the Investor and Analyst Symposium on July 12.  While safety was the primary focus of the first study (no adverse event on the early wound healing process confirmed), the effect of RXi-109 on wound healing will be the focus of the second study.  It should be added though that given the small size of the trials and the patient population which is not predisposed to scarring, spotting the difference will be tough.

Disclosure: long RXII.

New Clinical Results Put ALN-PCS Firmly in Business in PCSK9 Race

Alnylam reported on Friday updated results from the phase I single-dose, dose-escalation trial of its hypercholesterolemia candidate ALN-PCS02.  Importantly, the data demonstrate that ALN-PCS can produce clinically meaningful 30-40% reductions in LDLc (the ‘bad’ cholesterol) for much of the 28 study days of the study after only a single infusion of the drug.  This bodes well that ALN-PCS may be useful for the millions of patients that cannot achieve their LDL cholesterol goals despite the availability of statins.

After presenting solid PCSK9 knockdown results in January from subjects receiving up to 0.25mg/kg of the PCSK9 siRNAs which are critically enabled by Tekmira's SNALP delivery technology, the latest data include the results from subjects receiving 0.4mg/kg PCSK9 siRNAs after the decision had been made to increase the top dose based on the favorable safety-efficacy profile thus far.  This was important because despite the solid plasma PCSK9 reductions seen up to 0.25mg/kg (roughly 50% mean reductions throughout the 28 days), both the peak and average mean LDLc reductions until then had been less than 30% and 20%, respectively.  Probably not enough to effectively compete in the hot PCSK9 field.  

Based on the latest data, which included impressive >80% target plasma PCSK9 reductions, it is reasonable that ALN-PCS will be able to achieve average (=sustained) ~45-50% LDLc reductions in a once-every-4-week dosing regime.  This would put it just slightly behind Regeneron’s PCSK9-targeting monoclonal antibody REGN727 in terms of LDLc lowering.  It may be more difficult for ALN-PCS to achieve the LDLc lowering efficacy of Amgen’s PCSK9-targeting antibody AMG145 which has been hailed at the recent ACCconference to produce LDLc lowering of ‘up to’ ~60-80% at apparently pristine safety and tolerability.  I should add, however, that the warts-and-all data on AMG 145 have yet to be made publicly available.

In light of the intense competition in the PCSK9 space and a possible potency disadvantage for this surrogate marker, it will be interesting whether, firstly, the pharmaceutical industry (note, a larger partner will have to be found to financially shoulder the unavoidable outcomes trial), and eventually the market-place will adopt this new class primarily for their absolute LDLc reducing ability or in order for patients to achieve their lipid goals.  Some LDLc proponents, of course, stress that the lower the LDLc, the better.  On the other hand, whether a drug can lower LDLc by 50% or 70% won’t matter for many patients when it comes to simply achieving standard LDLc goals of 100mg/dL or even 70mg/dL.  In that environment, the focus in making the choice between competing drugs would shift to factors beyond mere LDLc lowering potential, particularly safety.

While ALN-PCS is mainly suffering from the fact that it is given following pre-treatment with corticosteroids, something that also had important impacts on the PCSK9 and LDLc efficacy data, the once much-touted safety profile of the PCSK9 antibodies have taken a significant hit with the publication of phase II data from REGN727 (McKenney et al. 2012).  In addition to one serious adverse event, a case of potentially life-threatening inflammation of blood vessels (leukocytoclastic vasculitis) that, however, readily resolved after giving corticosteroids, there was an apparently dose-dependent, adverse event-related increase in the rate of discontinuations, with ~15-20% discontinuing treatment in the biweekly dose cohorts in study 11565.  Next to the case of vessel inflammation, the AE-related discontinuations involved cases of neutropenia, fatigue, injection site rashes, chest pain, and headache/nausea. 

Amgen has yet to publicize more detailed data for AMG145.

Next to multi-dosing and the safety and efficacy impacts of omitting corticosteroids in future studies, another important question for ALN-PCS that remains to be answered in the upcoming trials will be testing the candidate in patients on statins.  REGN727 encountered problems here as patients concomitantly taking statins experienced an LDLc rebound effect after around 2 weeks.  Whether this is a class effect of agents merely blocking extracellular PCSK9 (such as antibodies), instead of inhibiting the synthesis of both intra- and extracellular PCSK9 such as ALN-PCS is an interesting question.  

This mechanistic difference could also be the big wildcard when it comes to the ultimate arbiter of clinical utility, the outcome trials: despite the clear evidence that LDLc levels are inversely related with cardiovascular events, with all the complicated feedback loops of lipid biology and related diseases, mechanistic differences could easily erase 20% differences in LDLc lowering.  An indication that these theoretical considerations are translating into the clinic may be deduced from the observation that (in Alnylam's words) ‘importantly, ALN-PCS demonstrated consistent clinical activity toward both PCSK9 and LDL-C independent of baseline levels of PCSK9, highlighting the unique mechanism of action for a PCSK9 synthesis inhibitor.’

For Alnylam, the near-term challenge will be thus to find a large partner, probably without a stake in PCSK9 yet, willing to take the risk with RNAi Therapeutics mainly for the differentiation it offers over the other PCSK9 approaches.  A stumble of the monoclonal antibody class could easily mean that ALN-PCS becomes the lead candidate in the PCSK9 market, estimated by many to be a multi-billion dollar one. 

Tekmira Reports First Knockdown in Man Following Systemic RNAi Delivery

Tekmira today reported very encouraging results from their first clinical study with SNALP siRNA delivery targeting apolipoprotein B for the treatment of hypercholesterolemia (PRO-040201, aka SNALP-ApoB). While the ~20% ApoB/LDL-c knockdown following single dose administration and evidence for flu-like toxicities at the highest tested dose level indicate that the present formulation is not suitable as a therapeutic, the company is rightly optimistic that based on these results, follow-up formulations that it intends to enter into the clinic later this year will be able to provide significant improvements in the absolute dose and therapeutic index. This is because the new knowledge can now be applied to the rapid progress that has been made in finding much more potent SNALP/LNP formulations (see recent PNAS paper and ILS2009 presentation) since the present formulation was locked down 2 years ago. In addition, much has been learned since on how to predict and mitigate SNALP siRNA-triggered immunostimulation in humans (extensive review). I therefore agree that it was probably the scientifically and financially best decision by the company to stop this trial at this stage and focus their resources on the next formulation.

When I first adopted SNALP/liposomal delivery as my pet systemic siRNA delivery approach 2-3 years ago, my main outstanding nightmare stemmed from an unexpected early-stage clinical disaster during a phase I trial by the same company (Protiva branch) almost a decade ago employing related stabilized cationic liposomes for the delivery of plasmids (SPLPs). In that trial, severe flu-like symptoms were observed at the very early and low dosages and the was trial terminated and the drug never heard of again. Instead of giving up, this has stimulated them to undertake almost heroic and industry-leading work aimed at mitigating immunostimulatory effects of liposomal nucleic acid delivery. It is thus quite remarkable that after just seven years after stumbling on siRNAs as more promising payloads for the technology, Tekmira has now demonstrated unambiguously functional liposomal siRNA delivery following systemic (intravenous) administration, with first, transient flu-like symptoms observed at what should be much higher dosages compared to the SPLP trial.

It is worth bearing in mind that the decision to stop the trial was not because the one case of flu-like adverse event was deemed a serious one, but because they felt that at this point they had already learnt what they wanted to learn and given the superior performance of the next-generation formulations it would therefore not have been ethical to further expose patients to a drug that will not be developed for commercialization (all my interpretation). It also might have risked tainting the asset with little to gain. To put the toxicity into context: in the most recent published clinical results on ISIS’ mipomersen targeting the same gene, 70% of patients receiving drug exhibited flu-like symptoms at similar 20% ApoB/LDLc knockdowns.

Equally encouraging is the fact that the knockdown, which based on the natural intra-person lipid variability and the pharmacokinetics of the reported knockdown (fast onset as expected for an RNAi drug) was almost certainly due to drug, occurred at dosages where the company expected to start seeing efficacy based on pre-clinical animal studies. One cannot underestimate the importance of being able to predict efficacious dose (and toxicity) based on these models, particularly at the early stages of evaluating such a novel technology where very little human pharmacological experience has been obtained.

The trial design involved cohorts of 4 patients for each dose level: one treated with placebo and three with study drug. While 8 cohorts had originally been planned, the trial was stopped in approximately the 6th or 7th cohort. According to the company, at this point the dose had been escalated to somewhere in the 0.6mg/kg ballpark and is thus consistent with IC50s for SNALP-siRNAs in non-human primates of ~1mg/kg at that time. Considering that according to a presentation at the International Liposome Society a month ago by Pieter Cullis, scientific founder of Tekmira, SNALP-like formulations have now reached IC50 potencies in non-human primates of ~0.03mg/kg, i.e. around 1.5 magnitudes better, there is every reason to believe that significant improvements in absolute dose and therapeutic index can be achieved with the follow-on SNALP-ApoB candidate. Strengthening this claim, similarly potent formulations have just recently surfaced from a study supposedly (Alnylam IR: I still cannot find the paper) published in PNAS by Alnylam and collaborators at the MIT on lipidoid-containing SNALP-like particles (allow a delay of at least a year from experimental finding to publication).

While all this sounds good, a critical question to ask is what gives them also the confidence that the new formulations (= more potent liposomes + less immunologically active siRNAs through modification) will not perform worse immunologically than the first one, thereby eliminating some of the benefit from the improvements in SNALP potency. The ability to answer this question would hinge on the ability to sensitively detect immunostimulation and translate such findings pre-clinical models into humans. The JCI paper last year by Tekmira scientists on the application of SNALP for distant tumors in mice showed that Tekmira indeed understands how to detect such responses even in the absence of cytokines in the serum (tissue-based PCR assay for IFIT1 mRNA). And from all the presentations I have seen, another important piece of the puzzle in being able to translate from animals into humans is the good correlation between immune responses seen in in vitro human PBMC immuno-assays and responses in animals. In combination, these elements allow for efficiently modifying siRNAs until predicted immune-related tox in humans have been minimized. Through such knowledge, Tekmira was able to provide a good explanation for why some non-human primates exhibited liver toxicity in the 2006 Nature study (the first demonstration of systemic RNAi in non-human primates) even in the absence of outward immune activation and remedy it through siRNA modification. It is interesting, however, that in the present clinical trial the presumed immune stimulation appeared to have (thankfully) been uncoupled from liver enzyme elevations as an absence of liver toxicities was noted. Although the reasons behind these are unclear, this uncoupling lends further credence to the notion that also efficacy and liver toxicity can be uncoupled: the mechanism that allows for functional entry of the liposome itself does not cause hepato-cytotoxicity.

According to a conversation with the company that I had following yesterday’s release of top-line results, it also appears that they have developed what appears to be a separate assay that they have not disclosed yet, and that should be even better in predicting immune responses in humans. Although, in the absence of seeing the data, I will have to take the company by its word here, as a proven leader in liposomal siRNA delivery, I’d like to give them the benefit of the doubt.

Looking ahead

The company indicated that the new formulation will go into the clinic later this year, although discussions with the FDA have yet to determine the exact regulatory requirements for this (e.g. an abbreviated IND package would make sense IMO). Like the first trial, the second one is likely to be a single-dose one, and a multi-dose trial in humans would similarly require additional pre-clinical data. All this makes it clear that the main value of this program to the company is that ApoB is an exceptionally good target to most rapidly develop insights into the entire SNALP-siRNA delivery platform. The market reaction tomorrow, however, will show whether this is shared by investors who may be disappointed by the delay on ApoB and may have also hoped that SNALP-ApoB could find a development partner soon. However, I would think that most invested in Tekmira as a leader in systemic RNAi delivery and are equally encouraged that the company is now able to build real data which have for the first time demonstrated unambiguous systemic RNAi gene silencing in Man. In addition to investor perceptions, the financial repercussions of these results also depend on what value such pioneering work will fetch from potential partners that are interested in the broad development of the RNAi Therapeutics platform.

Whether Tekmira’s enthusiasm is shared by current partners will be seen by their clinical actions. As such, Alnylam until recently reiterated that it was still on track to file an IND for the liver-directed SNALP-TTR. Since Alnylam apparently was aware of the status of SNALP-ApoB in November already (see RNAi Clinical Experience chart in this slide presentation), Alnylam also seems to be comfortable that the more potent SNALP formulations will have clinical applicability given Tekmira's results. I look forward to learning about the TTR trial design, including whether multi-dose will be attempted, and there should be results by the end of this year. In addition, the fact that we have just learned that Roche entered IND-enabling studies, most likely with a SNALP product, further validates Tekmira’s decision. The fact, however, that the IND submission of SNALP-PLK1 has been moved into the second half of this year represents a slight delay, and it remains unclear whether this might be related to today’s news.

In any case, today makes me feel like we have witnessed possibly the beginning of the most important line of RNAi Therapeutics clinical research thus far. Many more data-points on clinical SNALP-siRNA delivery should accumulate over the next two years.

Disclosure: I have financial interests in the company, largely by holding TKM shares.

Note added in proof: Scientific papers by Tekmira and Alnylam on the development of significantly improved SNALP formulations:

1) By combinatorial chemistry screen (December 2009 PNAS paper)

2) By rational design of lipids (January 2010 Nature Biotech paper)

Santaris Initiates First Clinical Trial for a MicroRNA Therapeutic

Another notable milestone has been reached in the history of small RNA-based therapeutics. Yesterday, Santaris announced that it had commenced a clinical trial for SPC3649, an LNA-based antisense molecule against miR-122 for the treatment of Hepatitis C. The phase I trial will be a double-blind, randomized dose-escalation study involving 48 healthy male volunteers.

MiR-122 has been shown to facilitate HCV replication as well as to have a role in cholesterol synthesis. Because of these potential applications and its expression in the liver, the tissue that can be best addressed with today’s systemic delivery technologies for nucleic acid-based therapeutics, miR-122 has become the favorite target for first generation microRNA-based development programs.

Santaris has made rapid progress and appears to be leading anti-miR-122 efforts, at least based on the published literature, with most prominently a recent Nature study demonstrating miR-122 antagonism in non-human primates, but also a nice transcriptomic study looking at changes in gene expression following miR-122 inhibition in mice. Santaris is using so-called LNA-“mixmers” which consist of a combination of interspersed DNA and LNA nucleotides and a phosphorothioate backbone and appear to act by sequestering the microRNA in the cytoplasm.

In mice, this LNA-antimiR-122 was quite a bit more potent than a competing technology developed originally by Alnylam and now owned by Regulus Therapeutics (‘antagomirs’) which were thought to induce the degradation of miR-122, possibly by RNase H. While these differences may also relate to the tissue concentrations and tightness of hybridization achieved by the various chemistries, my impression is that sequestration rather than RNaseH is the more promising approach to antagonizing microRNAs while for mRNA inhibition by antisense the opposite may be the case (e.g. morpholino versus gapmers). Moreover, the exact mechanism of action of the antagomir is still somewhat controversial, and I am curious to learn about the potency of antagomirs in vivo when formulated into SNALP-like particles.

A remarkable aspect of the LNA-antimiR-122 studies in non-human primates was the long duration of activity, up to 100 days after 3 loading doses of the antisense as judged by the lowering of plasma cholesterol. However, at this time point, the shift in the Northern blots which was taken to directly reflect microRNA sequestration, was not observed at this point. This could either mean that the Northern blot shift may represent an experimental artifact due to hybridization of antisense to miR-122 following sample preparation, and/or that the cholesterol lowering had persisted after LNA-antimiR-122 has ceased to sequester miR-122.

Of course, while the speedy technological progress is encouraging, there are a number of risks associated with this program. One is that it is unclear whether successfully inhibiting miR-122 in a chronic hepatitis C infection in man will have a meaningful effecgt on HCV burden. Compared to targets in RNAi Therapeutics trials, little is known about the actual role of miR-122 in HCV replication. Unlike in tissue culture liver cell lines which generally express less miR-122, miR-122 is a very abundant microRNA in the liver and may not be as rate limiting for HCV repliation in vivo. The argument, however, that this drug candidate lacks in vivo validation could be applied to a range of other HCV therapeutic programs, some of which have proven to be successful. The development of more convenient animal models for HCV infection over the next 5 years should change this and benefit not only the development of miR-122-based HCV therapeutics, but particularly RNAi-based HCV antivirals.

The Nature studies have also shown that anti-miR-122 may have limited utility for the treatment of hypercholesterolemia as its inhibition downregulated the “good cholesterol” even more than it did the “bad cholesterol”. On the other hand, given its abundance in the liver, it is likely that other indications for miR-122 antagonism will emerge and for which the Santaris phase I trial could be leveraged.

As far as the business strategy is concerned, maybe Santaris ought to solve the IP issues surrounding miR-122 for HCV once they initiate HCV-specific studies, as related IP fundamental to such an application has been exclusively licensed to Regulus and may thus limit the partnership value of this promising program.