An Exemplary RNAi Therapeutics Target

A paper by Alnylam and collaborators at Harvard and the MIT on a strategy to treat anemia recently appeared in Blood and involves an exemplary RNAi Therapeutics target gene selection which takes into account both the scientific and technical strengths and challenges of the technology (Querbes et al. 2012).  In addition, it is yet another demonstration of the powers of Tekmira’s SNALP technology and the various therapeutic opportunities that it can address with gene knockdown in the liver alone. 

Before going into the narrative of the paper and risk losing your attention, here are the main reasons why the target gene(s), PHD1-3, uniquely lend themselves to an RNAi Therapeutics approach (as opposed to competitive small molecules and recombinant proteins/MAbs) and why SNALP delivery is a particularly good match for them:

1. The target genes (PHD enzymes) are cell-autonomous negative regulators of the expression of a secreted factor (here: erythropoietin/Epo).  This means that even if efficient RNAi knockdown were only achieved in a subset of cells, the secreted factor that will be generated in this subset of cells can still act globally through the systemic circulation.  This is in contrast to other gene targets, such as certain intracellular targets involved in cancer cell proliferation, where efficient gene knockdown would be required in almost every cell to achieve a measurable outcome. 

2. Specific multi-targeting. In order to exploit a genetically intriguing mechanism for the treatment of disease (PHD regulation for the treatment of anemia), it was necessary to simultaneously knock down three gene family members.  Small molecule inhibitors with such multi-targeting activity would most likely cross-react with additional family members, increasing the risk of (A) obtaining confounding biological outcomes as other family members are more likely to play roles in related biological pathways than e.g. a spurious RNAi off-target event that is pathway-independent, and, of course, of (B) off-target toxicity.  The next time you hear about that exciting multi-targeting kinase inhibitor approach in oncology, remember that many of these were not multi-targeting by design and that there will be a number of additional kinases being hit that you will hardly hear about.  In summary, it is not just the capacity of RNAi Therapeutics for multi-targeting (e.g. as in the clinical candidates ALN-VSP02, TKM-EBOLA), but it is also the more ‘wholesome’ multi-targeting mechanism that is an advantage of the technology here. 

3. Tissue-specific delivery (here: SNALP and liver). Non-specific, but also specific targeting when it occurs in the wrong tissues, may cause toxicities.  In this example, because the gene family to which the target genes (PHD enzymes) belong play roles in various non-targeted biological processes throughout the body, there is great concern that a small molecule PHD inhibitor would cause a number of toxicities.  With regard to on-target toxicity in the wrong tissues, the HIF transcription factor that is regulated by the PHD1-3 enzymes plays an important role in cancer biology and inhibiting it all over the body may elevate the risk of developing cancer.  On the other hand, with the SNALP-formulated PHD siRNAs most of the knockdown can be directed at the desired liver so that the numerous on- and off-target toxicities in the rest of the body become irrelevant. 

4. Being an exclusively intracellular target means that it is not druggable by monoclonal antibodies = less competition. 

5. Recombinant protein therapy (e.g. rhEpo) often involves periods of supraphysiologic exposures which can be associated with adverse events (this is thought to be one problem with rhEpo therapy).  By contrast, (genetic) PHD knockdown allows for more physiologic regulation of the targeted biological pathway making it a potentially safer and also more efficacious therapy.  This advantage of affecting a more physiologic pathway compared to the competing recombinant protein approach is largely the result of having a larger target space to choose from.

Summary of paper

The motivation for the study by Querbes and colleagues is the fascinating insight that even in adulthood the liver continues to be a potential source of physiologically relevant levels of erythropoietin (Epo), a key red blood cell-stimulating factor that is almost exclusively produced by the kidneys in adults under normal conditions.  Nevertheless, it is the liver that is the site of Epo production until around birth when erythropoiesis switches from the liver to the bone marrow. At that point, hepatic Epo transcription is suppressed by the degradation of the important Epo transcription factor HIF following the activity of PHD enzymes 1, 2, and 3.  When these enzymes are removed, e.g. by genetic excision, the liver can produce similar levels of Epo as the kidneys even in adult mammals.

Clearly, constitutive genetic excision is not an option when it is important to achieve just the right level of a physiologic process (here: red blood cell volume).  The anemia drug development field in particular has been tarnished by companies and physicians having excessively pushed the use of high-dose recombinant Epo (rhEpo) and other erythropoiesis-stimulating agents (ESA).  In some patient groups, this has been shown to greatly increase the risk of thrombotic events and other side effects that may be unique to ESAs.

Oral small molecule inhibitors of HIF-related PHDs (HIF-PHIs) have been tested in clinicaltrials by Fibrogen for the treatment of anemia in end-stage renal disease along a more conventional treatment paradigm.  Although the clinical experience so far suggests a good safety profile, until larger patient numbers have been exposed for prolonged periods of time, there will always be concern about toxicities from the inhibition of on-target and off-target PHDs, especially in non-liver, non-kidney tissues.

By contrast, the SNALP-siRNA approach taken by Querbes and colleagues targets PHD1-3 almost exclusively in the liver by virtue of  short PEG-anchored SNALP* biodistribution.  Moreover, the nature of siRNA selection and off-targeting makes it much less likely that confounding or adverse off-targeting will be encountered.  

70-80% target gene knockdowns were achieved for PHD 1 and 2 and a somewhat less pronounced ~50% knockdown for PHD3 (probably due to a previously observed feedback mechanism) when measured across the liver in rodents.  Despite the incomplete overall knockdown, this resulted not only in several log-fold increases of liver Epo mRNA, but also pronounced, highly physiologically relevant levels of serum Epo.  These responses can be likely attributed to the RNAi Therapeutic candidate targeting negative regulators of gene expression.

Commercial potential

The anemia market has long been dominated by rhEpo and related protein-based ESAs.  Only recently, a peptide-based ESA by Affymax (Omontys/Peginesitide) was approved by the FDA as a new treatment option and should help bring down costs.

Although all these ESAs do a good job in elevating hemoglobin content, the opportunity for new agents is in doing just that, but with a better safety profile as all currently approved ESAs suffer from the above-described concerns arising from supraphysiologic exposures.  Thus, the hope with the new anemia drug candidates is that the targeting of alternative biologic pathways won’t suffer from the same limitations.  They may also increase the quality of the hemoglobin elevation, especially with regards to iron metabolism.

Despite the promising data, it is as yet unclear whether Alnylam will develop PHD inhibition to treat anemia.  Although refractory anemia is among the 5x15^TM programs, Alnylam hasindicated that it is more interested in modulating the hepcidin pathway for that indication (hepcidin affects erythropoiesis via iron metabolism).  I’m not sure though whether the choice is not partly 2due to the fact that Fibrogen seems to have a respectable IP position in PHD inhibition.  Incidentally, the corresponding author of the paper, Prof. William Kaelin from Harvard, has a financial interest in Fibrogen.  Also of note, some of the transgenic mice used in the study originated with Regeneron.

*Alnylam tight-lipped about specific SNALP formulation used in study

It is not surprising that, like all of Alnylam’s other commercially interesting applications, also the anemia program is based on Tekmira’s SNALP technology.  Because of the litigation between the companies, it is of interest that while the methods section described much of the SNALP formulation, it conspicuously failed to mention the identity of the ionizable lipid, although based on the observed potency it should be a ‘second’ or ‘third’ gen lipid:

‘siRNA Formulation in Lipid Nanoparticles

The LNPs were prepared with an ionizable lipid, disteroylphosphatidyl choline,

cholesterol, and PEG-DMG using a spontaneous vesicle formation procedure as

previously described at a component molar ratio of ~50/10/38.5/1.5 Ref 17,18.’ 

Ref 17: Akinc A, Querbes W, De S, et al. Targeted delivery of RNAi therapeutics with

endogenous and exogenous ligand-based mechanisms. Mol Ther. 2010;18(7):1357-1364. Study on the ApoE-mediated mechanism of SNALP delivery to hepatocytes (Tekmira claims this was their trade secret which Alnylam chose to publish and claim to be its own insight)

Ref 18: 18. Semple SC, Akinc A, Chen J, et al. Rational design of cationic lipids for siRNA delivery. Nat Biotechnol. 2010;28(2):172-176. Rational SNALP design paper by Tekmira (the contested ionizable MC3 lipid was derived from the rational design approach among others)

Alnylam said that it was planning on partnering the anemia program for phase I studies.  By disclosing their intention to partner at such an early stage, it almost seems like they have been in advanced talks with potential partners.  Otherwise, promising partners at such an early stage would only set the company up to disappoint.

I would therefore assume that Tekmira will be looking at this paper for evidence for whether, also in this case, Alnylam inappropriately shared SNALP reagents and insights with ‘3^rd parties’.  As a reminder, according to the Manufacturing Agreement between Tekmira and Alnylam, Tekmira is the sole SNALP supplier, also for pre-clinical research purposes, and Alnylam is prohibited from providing ‘third parties’ with such formulations (and insights).  

‘Third parties’ ought to include academic collaborators such as Prof. Kaelin and the other researchers from Harvard and MIT on this paper.  It is interesting to speculate that this was a reason for the secrecy around the ‘ionizable lipid’?