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:
- 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.
- 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.
- 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.
- Being an exclusively intracellular target means that it is
not druggable by monoclonal antibodies = less competition.
- 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 ‘3rd 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’?
2 comments:
That is good to hear. Thanks so much for keeping me informed. The published article had me wondering whether Tekmira's IP was involved or they were up to their usual, expected behavior pattern. Best wishes my friend. AKA Experiencedmentor
Thank you, XMan, for your support in this matter. I refuse to believe that Alnylam's management and BoD will get away with this. I'm confident that if the judge invests enough time, it will be hard not to see what game Alnylam has been playing. 3 1/2 more months to go.
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