RNAi Therapeutics Financial Viability Looking Up Following String of Clinical Results

By: Dirk Haussecker

Note: A PDF version of this article is available at myfirstnameDOTmylastnameATgmailDOTcom

Abstract

Shortly after the 2006-8 period of exuberance during which access to capital was easy, the RNAi Therapeutics industry found itself in a financially difficult position. At the roots of this change were the eventual recognition of some poor science, clinical setbacks, and the tension arising from the more gradual progress of science and impatient markets. Clinical validation of RNAi-mediated gene knockdown following systemic delivery was seen as the only way out of this situation. This review summarizes how such critical validation was provided by a series of recent clinical results from the ALN-VSP02, Atu027, ALN-TTR01, and ALN-PCS02 development programs. These results are expected to reinvigorate investments in the technology.

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Background

In the spring of 2011, the RNAi Therapeutics industry had just gone through two financially very stressful years. A few large pharmaceutical companies (‘Big Pharma’), among them Roche and Pfizer, made high-profile exits from the space [1], pure-play RNAi Therapeutics companies were crashing, and optimism gave way to a sense that RNAi in humans may take much longer to achieve than once thought due to the delivery challenge and the risk of causing immune stimulations [2]. This negative view of the technology and its financial prospects came sudden. After all, it was only in 2006-8 that the medical importance of RNAi had been recognized with the Nobel Prize in Physiology or Medicine [3], and Merck and Roche alone accounted for almost $2B of investments in the technology.

The argument can be made that the RNAi Depression was catalyzed by the US Housing Crisis and the ensuing global financial turmoil towards the end of 2008. This triggered a retrenchment of capital from high-risk innovation, capital that failed to return to the RNAi space. The seeds, however, had already been planted during the RNAi Therapeutics hype phase. As often is the case in such periods, it attracted the attention of the notorious promotional biotech schemes which in turn were readily followed by the fast money crowd, all of which, of course, did not mix well with the more gradual pace of technological progress. The scientifically leading pure-play RNAi Therapeutics companies can be accused as well for having catered to the cravings of fast money by over-promising on timelines and pushing programs into the clinic which were not adequately mechanistically validated (e.g. clinical candidates based on the local administration of naked, sometimes entirely unmodified siRNAs to the eye and respiratory epithelium). Similarly, the scientific community and journals can be blamed for failing to better police the quality of the science that got published [4,5]. Consequently, considerable investments were made (and wasted) with little discernment to what was deserving science and what was not. When it was realized that only a very few of the claimed technical solutions had clinical potential, the equally erroneously conclusion was drawn that the technology as a whole had poor prospects instead of realizing that only a very few successful platform technologies can already create considerable value.

With investors tiring of ‘promising’ pre-clinical results and refusing to put more money into RNAi Therapeutics development unless clinical validation was seen, it was up to the science to prove itself in Man. If not, probably all of the significant pure-play RNAi Therapeutics companies, possibly with the exception of Alnylam, would find it impossible to raise capital on acceptable terms. Fortunately, despite the attendant contraction in the number of new development programs, some of the early capital meant that four candidates in particular had entered clinical development in time to be the industry’s chance at unambiguously validating RNAi in Man: ALN-VSP02, Atu027 ALN-TTR01, and ALN-PCS02. If this could be achieved, it was likely that capital would return to the space. If not, the added wait before such clinical validation could come would have been a great setback to the industry, a setback from which it might have been difficult to recover from financially.

ALN-VSP02 and Atu027: clinical safety of two leading delivery technologies

Together with CALAA-01 (sponsor: Arrowhead Research), Alnylam’s ALN-VSP02 and Atu027 by Silence Therapeutics were the three leading RNAi Therapeutics candidates in cancer, with enrolment starting in 2008-2009. Not only were these candidates important in their own right for their medical and commercial potential, they were supposed to clinically validate the three distinct systemic delivery platforms on which they were based: the cyclodextrin-containing polycation RONDEL technology (CALAA-01), the AtuPLEX lipoplex technology (Atu027), and SNALP liposomes (ALN-VSP02). In addition to cancer, together with hepatic applications the commercially most critical application of RNAi Therapeutics in the near to medium term, AtuPLEX has potential for endothelial cell-directed gene knockdown in general, and systemically administered SNALP also for liver and phagocytic cell-directed gene knockdown.

CALAA-01 enjoyed a year head-start and reported a first data update in a high-profile paper in April of 2010 [6]. Attracting the widespread interest was the fact that by taking tumor biopsies, the investigators were able to demonstrate, with the help of the 5’ RACE assay on tumor biopsies, that RNAi had occurred in target tissues. Moreover, evidence was provided that consistent with this functional finding, RONDEL nanoparticles could be detected in the biopsies. On the other hand, the data on the target knockdown was more ambiguous. Although the RNA analysis suggested measurable gene suppression, the protein analysis did not fully support that. The early promise was only short-lived, however, as a subsequent 2010 ASCO presentation (Abstract No: 3022) showed ample innate immune stimulations which forced the company to concede in late 2011 that at least another phase I trial was necessary in an effort to better manage them. In retrospect, it seems obvious that a major omission of the program was in leaving the RNAi trigger (targeting the M2 subunit of ribonucleotide reductase, RRM2) chemically unmodified, meaning that the risk of inducing such responses was quite high. In the absence of evidence for clinical efficacy and unexplained trial delays (it has taken ~3 ½ years to conclude enrolment), the CALAA-01 phase I results were thus unable to positively impact perceptions of RNAi Therapeutics.

An important step forward in that direction was made with the 2011 ASCO presentations of the fully enrolled phase I study of ALN-VSP02 and a quite encouraging interim update for Atu027. The data suggested that Atu027, an endothelial cell-directed multilamellar cationic lipoplex containing an RNAi trigger against PKN3 [7], was surprisingly well tolerated as dose escalation had reached dosages at which gene knockdown efficacy could be expected based on the preclinical animal data. Dose escalation has been ongoing since and has exceeded predicted RNAi-functional doses- and still no dose-limiting toxicities or serious adverse events were claimed as of December 2011. The reason why this is somewhat unexpected is that the positively charged lipoplexes may have been considered prone to induce various innate immune responses [8], also because no immune suppressive regime was used in the trial. It is possible that the extensive 2’-O-methylation of the AtuRNAi-type trigger partly accounted for that. Activations of the alternative complement pathway, however, were noted, although these were claimed to be clinically not significant. Tumor responses by stringent RECIST criteria meanwhile remain to be demonstrated. Nevertheless, the overall safety profile and well-behaved pharmacokinetics have encouraged further investments in the AtuPLEX and related cationic lipoplex delivery platforms from Silence Therapeutics and can be considered a meaningful step forward for the field.

Of the three candidates, the most ambitious phase I study was that for ALN-VSP02, a SNALP formulation that includes two siRNAs, one against VEGF and KSP for anti-angiogenic and anti-proliferative mechanisms of action, respectively. Enrolling 41 patients with advanced solid cancer with liver involvement, this study included a battery of tests, including biopsies to test for target mRNA cleavage and siRNA tissue concentrations, the measurement of tumor blood perfusion as an indicator of anti-VEGF activity, and various other pharmacological parameters. The study succeeded in demonstrating that this SNALP formulation was fairly well tolerated in these advanced cancer patients at dosages of up to 1.0-1.25mg/kg (ASCO 2011 poster #3025). Among the dose-limiting toxicities were a liver failure with subsequent death at 0.7mg/kg, two cases of transient grade 3 thrombocytopenia at 1.25mg/kg, a grade 3 hypokalemia at 1.5mg/kg, and four grade 1-2 rigor/chills at 1.0mg/kg (one case) and 1.25mg/kg (three cases). The death at the 0.7mg/kg dose was deemed to be possibly related to study drug and occurred in a patient where the tumor burden in the liver was quite extensive. The enrolment criteria were subsequently adjusted to exclude similar patients with a greater than 50% tumor burden in the liver.

Based on the pre-clinical studies (AACR 2009 poster #B204), knockdown activity could have been expected at dosages of 1.0-1.25mg/kg. Consistent with this notion, the 5’ RACE for VEGF (but not KSP) RNAi cleavage performed on the biopsies revealed RNAi activity. In terms of functional activity, the Ktrans, a measure of tumor blood perfusion, declined by 40% or more in over half the patients that had received one or more doses and was consistent with anti-angiogenic activity of the drug. It has to be said though that the Ktrans response was not nearly dose related. Evidence for dose-responsiveness, however, was provided when categorizing patients according to RECIST criteria of tumor responses. Accordingly, only one in 13 patients treated with up to 0.4mg/kg had stable disease for two months or more. This compared to 12 of the 24 given higher doses. Moreover, there was a 70% partial tumor response in an endometrial cancer patient at 0.7mg/kg who stayed on study drug for months; at the 1.0mg/kg dose, the recommended dose for further studies, 7 of 11 achieved stable disease. Despite these preliminary signs of activity, there remain questions about the choice of the target genes, particularly the suitability of VEGF as an RNAi target and the absence of detectable KSP cleavage. Moreover, systemic SNALP delivery to even tumors in the liver is thought to require extended blood circulation times in order to harness the EPR effect, yet the half-life of siRNA in the blood was only in the 15-30 minute range. This is consistent with the relatively short C14-PEG lipid anchor in this formulation. Notwithstanding, the ALN-VSP02 study was a stringent test for the safety of SNALP delivery and added considerable clinical pharmacokinetic experience to this technology. Among the latter was the detection of amounts of siRNAs in normal liver which strongly indicated [9] that gene knockdown with SNALP was possible for liver-expressed genes.

ALN-TTR01 and ALN-PCS02: most impressive demonstrations of RNAi in Man

Despite the largely acceptable safety and promising pharmacokinetic data from the Atu027 and ALN-VSP02 studies, the field still lacked black-and-white evidence for target gene knockdown following systemic delivery. The SNALP-enabled TKM-ApoB by Tekmira in early 2010 was close to providing such evidence. Unfortunately, concomitant with the achievement of slight ~20% ApoB reductions, moderate immune stimulations were observed at 0.6mg/kg in the dose escalation trial, causing the company to terminate the trial (http://clinicaltrials.gov/ct2/show/NCT00927459?). The burden thus fell onto ALN-TTR01, another SNALP-enabled RNAi Therapeutics targeting the liver-expressed transthyretin gene. Mutations of this genes frequently cause familial amyloidotic polyneuropathies and cardiomyopathies which shorten the lives of ca. 50,000 patients worldwide.

This time implementing transient immune suppression (corticosteroids and H1/H2 histamine receptor blockade) as a precautionary measure, the trial began enrolling patients in June 2010 in Europe. A little more than a year thereafter, Alnylam presented almost full trial data at the November 2011 FAP conference in Kumamoto, Japan. It came as a great relief that at the highest, 1.0mg/kg dose the 5 patients exhibited a mean reduction of serum TTR of 41% following a single intravenous infusion. One patient exhibited a text-book 81% RNAi-type TTR reduction at nadir (week 1), with pronounced knockdown persisting out to 4 weeks (50%). Without ifs or buts, this was clear demonstration of effective RNAi in Man. Equally important, except for mild-to-moderate infusion reactions which were readily managed by simply slowing the rate of infusion, no meaningful adverse events were reported. As that trial is currently being wrapped up with more patient data expected for the 1.0mg/kg dose cohort, the sponsor Alnylam is aiming to further enhance the competitive profile of its TTR candidate and has filed a CTA for clinical trials with a new, ALN-PCS02-type SNALP formulation for which equivalent gene knockdown can be expected at 10-fold or more reduced dosages (patent application WO 2010/144740 A1). The goal here is to achieve a more potent knockdown with a higher margin of safety and a once every month or two dosing frequency.

The RNAi clinical dataflow culminated in early January 2012 with Alnylam announcing dose escalation data for the phase I study of ALN-PCS02 for the treatment of hypercholesterolemia. As predicted, equivalent knockdown to ALN-TTR01 were obtained at much reduced dosages with this improved ‘MC3-type’ SNALP formulation from Tekmira: ~60% mean peak reductions in serum PCSK9 for the 0.15mg/kg and 0.25mg/kg dose cohorts. As PCSK9 antagonizes ‘bad’ LDL cholesterol removal from circulation [10], its inhibition was accompanied by ~35% reductions in LDL cholesterol. Due to the favorable safety profile (rashes were noted, but these were likely related to route of administration as they also occurred in the placebo cohort), dose escalation is expected to proceed. At the higher dosages, more robust, less variable knockdown can be expected for both PCSK9 and LDL-cholesterol. This would put it in a favorable competitive position vis-à-vis the PCSK9 monoclonal antibody competition (e.g. REGN727/SAR236553).

The most significant limitation of the ALN-PCS02 study turned out to be the use of transient immune suppression. These caused short-lived (+65%) spikes and (-25%) depressions of PCSK9 and LDL-cholesterol, respectively. While transient immune suppression should be acceptable for many of the initial indications of high unmet medical need for which SNALP-based therapeutics are largely being developed at the moment, especially if once every month or two dosing can be achieved, they can not only complicate the analysis of studies such as ALN-PCS02, but would also restrict the eligible patient population for a condition such as hypercholesterolemia. Introduced as a precautionary measure following the TKM-ApoB experience, the question is whether it is possible to do entirely without it as SNALP potency has improved [11], lipid-specific toxicities been minimized, and more predictable innate immune stimulation assays been introduced as presented by Tekmira at a Drug Information Association (DIA) meeting on March 23, 2010, in Bethesda, MD.

Conclusion

The clinical results not only provided the long-awaited clinical validation and are a boost of confidence for the entire RNAi Therapeutics industry, but they directly de-risk two of the most important systemic delivery technologies: SNALP and AtuPLEX. The results with SNALP in particular set the stage for a forceful expansion of this delivery platform with already 5-6 candidates in active clinical development: ALN-VSP02, ALN-TTR01+02, TKM-PLK1, TKM-EBOLA, and ALN-PCS02. More still are expected to enter the clinic over the next two years. The following months should also add to the SNALP clinical experience in the form of results from the fully enrolled and dose-escalated ALN-TTR01 and ALN-PCS02 trials, an update on Tekmira’s cancer therapeutic candidate TKM-PLK1, and safety data from the TKM-EBOLA volunteer study. Since the ASCO 2011 presentation, Atu027 has attracted commercial interest in the form of various technology evaluations of AtuPLEX and related delivery technologies from Silence Therapeutics (partners: InteRNA Technologies, Mirna Therapeutics, an undisclosed Japanese ‘Top Ten’ global pharmaceutical company, and miRagen).

The space, however, awaits confidence expressed in the form of a more major financial commitment by a larger pharmaceutical company. This might break the gridlock caused by Roche’s decision to stop in-house RNAi Therapeutics development, a decision so powerful that it essentially caused all capital to retrench to the sidelines or leave RNAi Therapeutics entirely. What should not be lost is that the current situation also represents an attractive technical risk-financial reward opportunity for those companies that dare rely on their own scientific instincts rather than follow the herd. This includes mid-sized pharmaceutical companies and those in the newly emerged and emerging economies which are playing an increasingly important role in RNAi Therapeutics. As access to capital normalizes, platforms in addition to SNALP and AtuPLEX will likely emerge and help further expand the therapeutic reach of RNAi Therapeutics.

Abbreviations

5’ RACE: 5’ rapid amplification of cDNA ends; Ktrans: volume transfer coefficient; RECIST: Response Evaluation Criteria of Solid Tumors; RONDEL: RNAi/Oligonucleotide Nanoparticle Delivery; siRNA: small interfering RNA; SNALP: stable nucleic acid lipid particle.

References

1. Ledford H: Drug giants turn their backs on RNA interference. Nature 2010, 468: 487.
2. Robbins M, Judge A, Ambegia E, Choi C, Yaworski E, Palmer L, McClintock K, MacLachlan I: Misinterpreting the therapeutic effects of small interfering RNA caused by immune stimulation. Hum Gene Ther 2008, 19: 991-999.
3. Zamore PD: RNA interference: big applause for silencing in Stockholm. Cell 2006, 127: 1083-1086.
4. Tolentino MJ, Brucker AJ, Fosnot J, Ying GS, Wu IH, Malik, Wan S, Reich SJ: Intravitreal injection of vascular endothelial growth factor small interfering RNA inhibits growth and leakage in a nonhuman primate, laser-induced model of choroidal neovascularization. Retina 2004, 24: 132-138.
5. Aouadi M, Tesz GJ, Nicoloro SM, Wang M, Chouinard M, Soto E, Ostroff GR, Czech MP: Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation. Nature 2009, 458: 1180-1184.
6. Davis ME, Zuckerman JE, Choi CH, Seligson D, Tolcher A, Alabi CA, Yen Y, Heidel JD, Ribas A: Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 2010, 464: 1067-1070.
7. Aleku M, Schulz P, Keil O, Santel A, Schaeper U, Dieckhoff B, Janke O, Erdruschat J, Durieux B, Roeder N, Löffler K, Lange C, Fechtner M, Möpert K, Fisch G, Dames S, Arnold W, Jochims K, Giese K, Wiedenmann B, Scholz A, Kaufmann J: Atu027, a liposomal small interfering RNA formulation targeting protein kinase N3, inhibits cancer progression. Cancer Res 2008, 68: 9788-9798.
8. Yew NS, Scheule RK: Toxicity of Cationic Lipid-DNA Complexes. Adv Genet 2005, 53PA: 189-214.
9. Landesman Y, Syrzikapa N, Cognetta A 3^rd, Zhang X, Bettencourt BR, Kuchimanchi S, Dufault K, Shaikkh S, Gioia M, Akinc A, Hutabarat R, Meyers R: In vivo quantification of formulated and chemically modified small interfering RNA by heating-in-Triton quantitative reverse transcription polymerase chain reaction (HIT qRT-PCR). Silence 2010, 1: 16.
10. Horton JD, Cohen JC, Hobbs HH: Molecular biology of PCSK9: its role in LDL metabolism. Trends Biochem Sci 2007, 32: 71-77.
11. Semple SC, Akinc A, Chen J, Sandhu AP, Mui BL, Cho CK, Sah DW, Stebbing D, Crosley EJ, Yaworski E, Hafez IM, Dorkin JR, Qin J, Lam K, Rajeev KG, Wong KF, Jeffs LB, Nechev L, Eisenhardt ML, Jayaraman M, Kazem M, Maier MA, Srinivasulu M, Weinstein MJ, Chen Q, Alvarez R, Barros SA, De S, Klimuk SK, Borland T, Kosovrasti V, Cantley WL, Tam YK, Manoharan M, Ciufolini MA, Tracy MA, de Fougerolles A, MacLachlan I, Cullis PR, Madden TD, Hope MJ: Rational design of cationic lipids for siRNA delivery. Nat Biotechnol 2010, 28: 172-176.

ISIS TTR Results Show 1st Gen SNALP Already on Par with RNase H Antisense

ISIS today reported knockdown results from a phase I trial with its RNase H antisense compound ISIS-TTRRx against the transthyretin gene for the treatment of TTR amyloidosis. The results show that while high 81% knockdowns can be achieved, very large amounts of oligonucleotides (400mg weekly for 4 weeks) had to be given. When clinically more relevant doses were given (200mg as in mipomersen and the next lower dose from 400mg), a mean knockdown of 44% was achieved.

As you will remember (SNALP Works!), the RNAi Therapeutics candidate ALN-TTR01 last November showed a mean reduction of 41% of serum TTR from baseline following a single 1.0mg/kg infusion. In light of yesterday’s results with ALN-PCS02 where a supposedly less potent RNAi trigger was employed and a ~60% knockdown was achieved with 0.25mg/kg, it seems likely that the Tekmira SNALP-enabled ALN-TTR02 which is about to enter clinical development and which employs a formulation similar to PCS02 will show the type of 70-80% knockdown at 0.25mg/kg.

We have yet to learn more about the safety profile of ISIS-TTRRx which is being developed under an option agreement with GSK. ISIS stated in their press release that they were planning to enter pivotal studies with this compound soon. If it is with the 400mg dosage, they may be tempting fate. It might be interesting to try 300mg.

As you know, these days I’m strongly advocating RNAi Therapeutics as the technology is currently being sold at much below the worth of the science which has progressed steadily over the years despite the up and downs in public perceptions. Today’s results by ISIS show that the new darling in nucleic acid therapeutics can certainly knock down genes in the liver (and some other tissues). They also show, however, that even the initial SNALP formulations are already on par with it in terms of efficacy at the higher end of tolerability (not talking about other aspects such as absolute differences in dosage or route of administration).

SNALP Works!

The long wait is finally over: Systemic RNAi delivery has been proven in Man. No 'ifs' or 'buts'. Alnylam just announced that Tekmira’s SNALP-enabled ALN-TTR01 reduced target transthyretin (TTR) protein levels in ATTR patients in a dose-dependent manner- without causing an elevation of liver enzymes or serious adverse event (SAE).

Following preliminary evidence in Tekmira’s TKM-ApoB study, this is the long-awaited moment where there is no doubt that successful systemic RNAi delivery has been achieved. With already about half a dozen SNALP-based programs in or very close to the clinic, this result de-risks a major segment of the RNAi Therapeutics pipeline and should stimulate further investments in SNALP-enabled RNAi Therapeutics development as well as provide a boost of confidence to the entire sector.

Today's presentation at the FAP meeting in Kumamoto can be found here. For a more detailed background on ALN-TTR01 for ATTR, please read yesterday’s blog entry.

In the phase I study (single-dose, dose-escalating), 5 patients received 1mg/kg, the highest planned dose. At this dose, there was a mean reduction of 41% in serum TTR levels from baseline. Despite the small patient numbers and natural TTR variability, this was stat significant at p=0.02 relative to placebo. While this level of knockdown may not seem dramatic and may or may not be useful for eventually achieving a therapeutic effect in ATTR patients, it is important to keep in mind that the other liver-targeted SNALP programs employ formulations that should be at least 10x more potent than the one used in TTR01 and probably also slightly better tolerated. As such, this was a stringent test for the safety and tolerability of SNALP technology and bodes well for the commercial potential of particularly the liver-targeted SNALP pipeline following TTR01, including TTR02 for which an IND is expected by year end.

The time course of TTR protein suppression in one patient provided a picture-book example of bona fide RNAi knockdown in Man. Consistent with the now extensive experience with SNALP in pre-clinical animal models, including non-human primates, this patient exhibited a rapid onset of knockdown (63% reduction at 48 hours) which became a peak knockdown of 81% a week after drug administration with 50% suppression still being observable 4 weeks after this single dose. There is no doubt that this was an RNAi-mediated response.

Preclinical repeat dosing studies have shown that in order to maintain the same level of gene suppression, one can reduce the amount of drug given at subsequent doses. This also means that perhaps giving patients two or three loading doses of 1mg/kg within a week or so may allow one to achieve the 70-80% knockdown with ALN-TTR01 not just in select patients.

Importantly, the safety profile seems to exceed even my own expectations. Unless Alnylam will reveal major immune stimulations in the upcoming conference call (at 8.30am Eastern Time; note added in proof 11/23: none were revealed), the only mild-to-moderate adverse reactions seemed to be infusion reactions that were experienced by 3 out of the 23 TTR01-treated subjects and which was well controlled by simply slowing the infusion rate. As the same was seen in the SNALP-enabled ALN-VSP02 study, it seems that this risk factor is indeed well manageable. What is more, even at the high dose of 1mg/kg, there were no signs of liver toxicity as evidenced by increases in liver function tests. As SNALP-RNAi for ATTR will be a chronic treatment, this should be the major safety focus in the future.

Whatever Alnylam decides to do with ALN-TTR01 (I expect it to remain on the shelf as a viable alternative pending TTR02 results), today represents a milestone in the history of RNAi Therapeutics and I do believe that we have seen the bottom in RNAi Therapeutics. Credit belongs to Alnylam for pushing ahead with the SNALP clinical studies. Alnylam has a truly gifted, and in many ways inspirational drug development team, but it is probably also this talent that has made them blind to what they are actually entitled to. My sympathies are therefore with Tekmira today as it has developed (and owns) the delivery technology that is turning out to be quite literally the savior of RNAi Therapeutics: SNALP. Clinical results eventually follow strong science.

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Phase I Study of ALN-TTR01 in Transthyretin Amyloidosis- A Preview

(For a discussion of the phase I results, see here).

Alnylam is about to reveal results from its phase I study with ALN-TTR01 for the treatment of transthyretin amyloidosis (ATTR) at the Nov20-22 FAP meeting in Kunamoto, Japan. This blog provides a brief overview of the rational for RNAi Therapeutics in this disease and the importance of this particular study for the field of RNAi Therapeutics.

ATTR is an autosomal dominant amyloidotic disease due to point mutations (>100 possible) in the TTR gene. These mutations cause protein misfolding and aggregation into fibrils that, depending on which tissues they accumulate in, can cause various organ dysfunctions, most notably polyneuropathy (FAP), cardiomyopathy (FAC), and gastrointestinal/nutritional defects. For those that develop the disease, death is common 5-15 years following the emergence of disease symptoms (usually between 30 and 50 years of age). Although there is genotype-disease phenotype overlap, the most common mutation, Val30Met, strongly predisposes to FAP, while the Val122 is associated with FAC. About 10,000 patients suffer from FAP and 40,000 from FAC. As a rare genetic disease, it occurs in clusters, with FAP cases for example concentrated in Portugal, Sweden, and Japan.

Until recently, the only accepted treatment has been liver transplantation for FAP where removing the source of the mutant TTR in blood serum can reverse the polyneuropathy. Pfizer just got European approval for FAP with its small molecule TTR conformational stabilizer Vyndaqel based on slowing the rate of peripheral neuropathic impairment.

There exists, however, great need for additional therapies as Vyndaqel actually missed the primary endpoint in its pivotal study (the FDA did not accept the NDA for review earlier this year), and because liver transplantation is ineffective for FAC. The latter seems to be due to wildtype TTR still being able to deposit into pre-existing plaques, for example in heart tissue, at a rate that is higher than the turnover of the amyloidotic plaque. In fact, the amyloidotic potential of wildtype TTR is illustrated by the fact that it frequently causes spontaneous amyloidosis in elderly people (senile ATTR).

RNAi Therapeutics Approach to ATTR

The contribution of both wildtype and mutant to disease pathology, the dynamic turnover of plaques, and the fact that TTR knockout mice have the same life expectancy and fertility as their wildtype littermates and are otherwise essentially asymptomatic, makes RNAi Therapeutics a highly attractive treatment approach for this disease. TTR is involved in the transport of vitamin A and thyroxine in the blood, but it appears that in the absence of TTR these carrier functions are compensated for by other carrier proteins in the serum. What is more, essentially all the life-limiting pathologies are caused by TTR that is expressed in the liver, and with Tekmira’s SNALP delivery technology, RNAi can address the relevant gene expression.

TTR01 vs TTR02

One source of confusion that I expect to affect the financial markets tomorrow stems from Alnylam developing two candidates for ATTR, ALN-TTR01 and ALN-TTR02, the difference between the two candidates being in the SNALP lipid composition. TTR01 is the subject of the present trial and is based on an early DLinDMA lipid-containing formulation. It was shown to be effective in knocking down TTR in non-human primates with an ED50 of around 0.3-0.4mg/kg. As the highest dose in the phase I study was 1mg/kg it is reasonable to expect there to be evidence for TTR knockdown in the ALN-TTR01 trial. I should warn, however, that because of the small patient number in each dose cohort and the natural intra- and inter-patient variability of TTR levels in the serum, the pharmacodynamic outcome measure in this trial, the pooled numbers may not give us a straightforward 'stat-significant' answer.

Because of the rapid developments in improving the efficacy and tolerability of SNALP technology, it is therefore almost assured that Alnylam will drop TTR01 and prioritize TTR02 which takes advantage of these developments and for which the filing of an IND is imminent.

Consequently, the importance of tomorrow’s results for the RNAi Therapeutics field lies in providing proof-of-concept for RNAi knockdown following systemic delivery at OKish tolerability. That’s it. It also sets up the results from the phase I studies with ALN-PCS02, the PCSK9-targeting hypercholesterolemia candidate, which are expected to be reported by the end of this year. As PCS02 uses one of the more recent SNALP formulations, this will be the SNALP candidate that has to shine both in terms of knockdown efficacy and safety/tolerability.

ATTR Economics

If you had any doubts as to the commercial potential of ATTR, it is worth noting that Vyndaqel, Pfizer's just-approved TTR drug, is expected to be priced at more than 100,000 Euros per patient year and that most (known) FAP patients are in healthcare systems that will still bear such costs. Pfizer last year paid $200M in upfront considerations for FoldRx, the original developer of Vyndaqel, with another $200M in contingent milestones. Although FoldRx has a mission of developing other protein folding-based drugs, this price tag was essentially for a drug with results from a pivotal trial that failed to meet the primary endpoint and for which approval was far from certain. Considering Alnylam’s cash position, this one registrational drug candidate valued FoldRx higher than all of Alnylam- although you might justify that with the mounting existential risks stemming from Alnylam's alleged theft and misuse of Tekmira's SNALP technology.

Given these economics, it is not surprising that other companies have similarly recognized the commercial potential of ATTR. Importantly, ISIS Pharmaceuticals will also present progress with its antisense candidate for ATTR (ISIS-TTRRx) at the meeting. This candidate entered clinical development in May and is financed by GSK which retains an option for its exclusive license.

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RNAi Therapeutics Investors Hoping for a Merry Christmas

I’ve just come back from working at the Starbucks across my street which strongly reminded me that Christmas was just around the corner. Christmas this year in RNAi Therapeutics is synonymous with data releases by Alnylam from its transthyretin amyloidosis (ALN-TTR01; data presentation November 20-22 in Japan) and hypercholesterolemia (ALN-PCS02; release of top-line results by year-end) phase I clinical trials. These have the potential to demonstrate, for the first time, direct and physiologically meaningful target gene knockdown following systemic RNAi delivery, and thereby have the potential to turn around still negative RNAi Therapeutics sentiments and depressed valuations.

Some of the anticipation can already be felt in the form of appreciating share prices of Alnylam and Silence Therapeutics, together with Tekmira the companies most directly exposed to the current RNAi Therapeutics dataflow, and the financial analyst-investment community which have turned noticeably bullish on Alnylam. Only Tekmira, the inventor of SNALP technology that powers ALN-TTR01, ALN-PCS02 and 5 other candidates in or close to clinical development, has not participated in the rally by failing to find investors willing to defend its stock after taking on well-connected Alnylam.

In assessing the data, a primary focus will be on whether dose escalation was able to proceedeup to the highest planned doses (1.0mg/kg for ALN-TTR01 and 0.25mg/kg for ALN-PCS02) and whether, despite the small number of patients at the high dose levels, there are clear signs for target gene knockdown. 50% target gene knockdown in both cases would be reasonable goals, and probably also necessary ones to have the desired impact. In the case of ALN-PCS02 there should also be at least a 30% reduction in ‘bad’ LDL-cholesterol, the intended pharmacologic outcome of a PCSK9-targeting agent. In terms of safety, the absence of grade 3 adverse events or worse would be highly welcome, of course, as we would be the absence of consistent and clinically meaningful innate immune activations.

Santaris’ anti-miR122 HCV Drug Continues to Impress

MicroRNA Therapeutics seems to have found its poster child already with Santaris’ miR122 LNA antagonist for the treatment of HCV. In an oral presentation at The Liver Meeting which is just wrapping up in San Francisco, the company reported robust dose-dependent anti-HCV activity in a phase IIa study, with close to a 3-log mean reduction of HCV RNA from baseline and viral load below detection in 4 of 9 patients at the highest dose of miravirsen (7mg/kg). The corresponding abstract marking a milestone in microRNA Therapeutics by reporting first clinical activity of an microRNA Therapeutic was released in early October (click here for commentary). There is no doubt that this drug candidate works as expected/hoped for, and unless the future of HCV treatment is in all-oral combos, anti-miR122 with its uniquely differentiated mechanism of action looks like a valuable addition to the fast-moving field of HCV care.

Confidence in SNALP Safety Building

Alnylam Pharmaceuticals disclosed in their Q1 2011 results that it will expand the dose escalation of the phase I study of ALN-TTR01 in TTR amyloidosis, increasing the target dose in that study from 0.4mg/kg to now 1mg/kg. This puts the company in a good position to demonstrate, unambiguously, that systemically administered SNALPs can knock down genes in the human liver. This dose expansion is an important indication that, as we learn more about the clinical performance of SNALP delivery, confidence in its safety is growing.

Given yesterday's news, the phase I study being conducted in Europe should have almost fully enrolled its first 24 patients up to 0.4mg/kg. Regulatory applications for this study were filed in late 2009/early 2010, yet it took over half a year for the first patient to be dosed in July 2010. At that time, Alnylam revealed the 0.4mg/kg target dose. This was somewhat confusing and made me even speculate that TTR01 may involve a ‘generation 1.5’ formulation as it had been Alnylam’s stated goal to demonstrate proof-of-concept of gene knockdown with this study. 0.4mg/kg, depending on the gene, is about where you’d expect to start seeing efficacy with the initial SNALP formulations, so stopping at 0.4mg/kg would have been slicing it quite thinly. Indeed, the ED50 (dose of 50% drug efficacy) for ALN-TTR01 in non-human primates is around 0.4mg/kg.

In retrospect, the original conservative dose escalation schedule might have been due to safety concerns surrounding SNALP delivery. Around the time of the regulatory submissions, Alnylam knew of one death in their liver cancer trial with ALN-VSP02 (also using SNALP delivery) which occurred some time after a patient with extensive liver mets from pancreatic neuroendocrine cancer received a second infusion of 0.7mg/kg SNALP. Dose-limiting toxicities were not observed in that trial until then up to 0.4mg/kg. Although patients in that trial are quite fragile and autopsy revealed that the patient may have in fact died as a result of too much drug efficacy (extensive necrosis of the liver mets), it was a potentially drug-related severe adverse event nevertheless. ALN-VSP02 was subsequently escalated to the robust dose of 1.5mg/kg, with 1.25mg/kg being my predicted maximally-tolerated dose.

Shortly thereafter, in January 2010, Tekmira stopped early their phase I hypercholesterolemia trial for TKM-ApoB after one trial participant at the 0.6mg/kg dose level became hypotensive and suffered from general flu-like symptoms after receiving a SNALP formulation. Because the necessary safety/tolerability profile for a hypercholesterolemia drug differs from that of a cancer drug, Tekmira did the right thing and stopped the trial and take advantage of the technology progress in the SNALP delivery field with the aim of re-entering clinical development with a formulation with presumably improved potency and robust long-term safety. Just as for ALN-VSP02, 0.4mg/kg was the dose level up to which no significant adverse event was observed with TKM-ApoB.

Since no good deed goes unpunished in the competitive area of siRNA delivery, these events were quickly made out to prove that ‘SNALP delivery is toxic’ adding to the SNALP confusion caused by Alnylam's lipidoid line of work. Regulators may not be as emotional, but it is understandable that they and Alnylam took a cautious approach in selecting their initial doses. They were, however, clever enough to design an adaptive trial that would allow for further dose escalation depending on the safety findings up to the 0.4mg/kg level.

The enrollment of the anticipated additional 8 patients should therefore be a very good sign that the safety profile so far has been highly satisfactory, setting the scene for convincingly demonstrating TTR knockdown in the liver as measured by plasma protein levels. Alnylam plans to present top-line data from the completed study in the third quarter, hopefully building on good ASCO data on SNALP-based ALN-VSP02.

There has been some debate around whether the clinical development of SNALP technology was started pre-maturely. Yes, you can try and aim for perfection (that may be what Merck and Roche are waiting for), but by taking the necessary clinical precautions, Alnylam and Tekmira were able, in a timely manner, to gather much clinical data on the SNALP delivery platform that is really starting to pay off now as their synergistic value becomes obvious. Important months for RNAi Therapeutics are ahead of us.