Recognizing and Addressing Critical RNAi Off-targeting Issue

In late 2016, Alnylam discontinued its alpha-1-antitrypsin program due to observed increases in liver transaminases. A year later, its HBV program was also terminated with the goal of advancing a new compound with increased targeting specificity.  

Apparently, close to half of RNAi triggers largely picked based on potency cause hepatotoxicity in rodent studies.  It's been clearer than ever that Alnylam, if not the industry as a whole needed to pay more attention to minimizing off-targeting and not just focus on potency in candidate selection.

Formal presentation of their findings of the causes underlying the toxicity  have now been published in a paper titled ‘Selection of GalNAc-conjugated siRNAs withlimited off-target-driven rat hepatotoxicity’.  

In an elegant series of experiments, the authors first sought to disprove that other steps in the delivery pathway that were possible candidates for the observed toxicity were responsible.  They then went on to show that it was indeed microRNA-type off-targeting that were at the root.  

Endolysosomal Constipation

GalNAc-conjugated RNAi triggers need to be highly stabilized to be active.  As a result, they reach high, antisense-type tissue accumulation, largely through sequestration in the endolysosomal compartment.  This has led to the concern that toxicities may arise from interfering with endolysosomal functions in the liver.

Strongly arguing against this possibility, when a toxic GalNAc-conjugated RNAi trigger with standard 2’-O-methyl and 2’-F-chemistry and partial phosphorothioation was rendered incapable of being taken up by the RISC RNAi effector complex, liver toxicity was abrogated without much change in the tissue accumulations of the respective oligos in rodents. 

Still, it will be important to further follow this risk in humans, especially when there is long-term, chronic exposure given that the present studies lasted for ‘only’ 5-9 weeks.  Also, patients with pre-existing morbidity in the liver may be particularly susceptible to adversely reacting to the accumulation of RNAi triggers in the endolysosomal compartment. 

Modification Chemistry

One concern with the high degree of chemical modification of the RNAi triggers which are employed to stabilize them in the absence of nanoparticle delivery formulations is that the modification chemistry could be toxic.  Rival Ionis Pharmaceuticals e.g. had previously claimed that 2’-F-modified antisense and RNAi constructs are genotoxic- at least in cell culture system.  Alnylam, however, had already disputed this claim and now argues that this is only the case with single-stranded, phosphorothioated formats Ionis is using.

The fact that preventing RISC uptake abrogated toxicity already exonerated chemistry and degradation intermediates as culprits since such abrogation in itself does not change overall RNAi trigger metabolism (note that RISC-incorporated RNAi trigger represents only a very minor fraction of overall tissue RNAi triggers).

To further rule out this possibility, the Alnylam scientists varied the degree of relative 2’-O-methylation and 2-F-modification to see whether this impacted toxicity in the rodent models.  There was no correlation between the type of modification and toxicity.

So at least for acute treatments, 2'-F modification does not appear to be a relevant tox issue for RNAi Therapeutics.  Based on the recent demonstration that 2'-F metabolites can be incorporated into endogenous RNA and DNA, there still remains a need to be vigilant about this modification regarding its potential to be genotoxic, i.e. promote cancer.

Competition with endogenous microRNAs

Years ago when I joined as a post-doc in the Kay lab in Stanford, colleagues of mine had found that high levels of RNAi trigger small hairpin precursors expressed from DNA templates could trigger severe toxicity in mice and that this could be traced back to these precursors interfering with normal microRNA function. Subsequently, studies to investigate the same were performed using synthetic RNAi triggers and modest competition with endogenous microRNA function was sometimes seen.

In contrast to the elegant investigations above, Janas and colleagues from Alnylam offered only circumstantial evidence to rule out competition.  In particular, they found no correlation between the degree of RISC occupation of the exogenous RNAi trigger and toxicity. 

It would have been interesting to study e.g. whether delivery of the RNAi triggers resulted in the de-repression of known targets of endogenous microRNAs to get insights into whether further investigation of this issue had merit.

MicroRNA-type Off-targeting

By contrast, the authors found strong evidence that it is microRNA-type off-targeting that is the culprit for the toxicity of many RNAi triggers in vivo.

To wit, in addition to directing the cleavage of mRNAs with near complete sequence complementarity (~20 base pairs), RNAi triggers may also participate in microRNA-type mRNA interactions based on limited sequence complementarity (mainly ~7 nucleotide seed) with more subdued silencing impact and without the canonical RNAi cleavage.   

Importantly, a reversir molecule with the function to soak up RNAi trigger-loaded RISC and consequently interfere with its silencing activity, reversed the toxicity.  Parenthetically, if competition with microRNAs were the problem, this ‘freezing’ of the RISC complexes would not have been expected to relieve the toxicity since it would not have freed RISC for loading the endogenous microRNAs.  

Supporting evidence came from seed swapping experiments where it was the seed from the toxic RNAi trigger that dictated toxicity. 

Practical solution

Since microRNA-type off-targeting does not translate well- if at all- between species (it doesn’t take much to change a microRNA target into a microRNA-refractory sequence), simply screening RNAi triggers in rats to weed out the toxic ones does not help much.

Fortunately, following a decade of studying microRNA off-targeting in RNAi, one chemical strategy in particular has risen to the top to minimize microRNA-type off-targeting.  Based on the hypothesis that the limited sequence interaction in microRNA target recognition renders it more susceptible to weakening the binding energy compared to a fully, or almost fully complementary RNAi target, incorporating energetically less favorable nucleotide residues were found to greatly minimize microRNA-type interactions while leaving RNAi on-target recognition intact.  

I  consider studies by Jackson et al. (2006) and Vaish et al (2011) to have been seminal, and long underappreciated, if not downplayed by RNAi industry leaders in this regard.

Similar to such prior efforts, Alnylam incorporated an ‘empty’ placeholder residue (glycol nucleic acid, GNA) which cannot base pair.  This indeed greatly reduced off-targeting as assessed by transcriptomics and, importantly, abrogated liver toxicity.  

In the end, the risk of clinical failure due to microRNA-type off-targeting can never be fully excluded before studying the RNAi trigger in humans.  Still, reducing clinical attrition due to this from 30-40% to more like on the order of 5% is well worth the effort.

Clinical studies with the new candidate for AAT by Alnylam will be the first direct test of this hypothesis.

Wave Rides Coattails of Ionis, Strikes CNS Deal with Takeda

Today, Wave announced the licensing of their (stereopure) antisense technology to Japanese Big Pharma Takeda for their application in the central nervous system (CNS).  In return for $110M in upfront cash, a $60M equity investment and other biobuck goodies, Takeda receives an option to co-develop/commercialize ASO candidates for the treatment of Huntington’s Disease (HD), Amyotrophic Lateral Sclerosis (ALS), and type III Spinocerebellar Ataxia (SCA3).  Takeda also receives opt-in right to additional preclinical programs.

This deal has clearly been driven by the outstanding data gathered by rival Ionis and its corporate partner Biogen in applying phosphorothioate antisense chemistry for diseases of the CNS.  This not only includes the remarkable efficacy and commercial success seen with splice-skipping oligo Spinraza for spinal muscular atrophy (SMA), but also preclinical knockdown and biodistribution data from many other CNS programs. 

Also of note, Ionis and Roche disclosed last December statistically significant knockdown of huntingtin in people with HD, the same indication as Wave’s lead neurology program which has entered clinical development.  Important additional color for the Ionis HD candidate, especially relating to the actual depth of target knockdown is to be presented  at the upcoming CHDI conference at the end of this month.

To wit, the efficacy of exon skipper Spinraza relied on gain-of-function where minor (say 20%) target engagement may already translate into significant clinical benefit.  Most other programs, however, aim at inhibiting a disease-causing molecule from being made.  Here, you may want more like something of the order of 50%+ target engagement. 

Another wrinkle is that the target cells in the CNS will differ from indication to indication whilst antisense efficacy is not homogeneous throughout the CNS, so the SMA and HD program data will not have de-risked all the indications.   

Nevertheless, it is crystal clear that phoshorothioate oligos hold tremendous value for the many diseases of the CNS of high unmet medical need for which the industry is more and more relying on gene-targeted approaches after years of failures with small molecules.  And kudos to Wave for retaining substantial commercial value to the licensed programs.  

So thank you, Ionis, for making it exceptionally easy for them!

Happy Gene Therapy Talk Presaged Market Correction

In January, many biotech stocks went parabolic shattering all-time-highs.  Now they have started crashing back down to earth.

The run-up was partly driven by real fundamentals.  These include corporate tax reform in the US, a new FDA commissioner (Scott Gottlieb) who makes all the right decisions (speeding access to innovative medicines, fostering generic competition, amenable to rational discussion etc), and a voracious appetite for innovative pipelines in the pharmaceutical industry which has manifested itself in a number of chunky, multibillion dollar acquisitions. 

At the same time, ever-increasing valuations in a deceptively low-volatility stock market environment has emboldened analysts, investors, and managements alike to proclaim that the golden era of curative medicines is upon us.  Gene therapy and genome editing in particular got hyped beyond reason as if all of a sudden critical bottlenecks in tech development had been solved.  CRISPR companies Editas Medicines and CRISPR Therapeutics, without a single drug candidate in the clinic, were bid up to $2B valuations although access to genome editing tools have become a commodity and cannot be considered gate-keeping tech any more.

Not much changed technology-wise

I understand that gene therapies have made tremendous inroads into the pharmaceutical marketplace with the first 3 gene therapy approvals in the US over the last year, one for an eye disease by Spark Therapeutics and two CAR-T cell cancer therapies going after the same target.

While I acknowledge these successes, what is really surprising here is that it took that long to get there given that the related delivery and genetic technologies (AAV, gene insertion, immune cell transfer) have been in existence for a decade or two already.  In that light, the recent Happy Talk around gene therapy seemed overdone.

The CEO of one genome editing company, Sangamo Therapeutics, is a great example of this divergence between scientific progress and clinical reality.  

Having come on board only a year ago, the good Dr. Macrae looks as if he is having a steep learning curve ahead of himself.  The reason I got the impression is that he likes to grandiosely talk about how the company's AAV capsid shuffling will enable them to cure all these CNS disorders while making great progress with LNPs to address the rest of humanity's afflictions.

Well, LNPs have gotten 10x more potent every year for the last 15 years so we must have reached homeopathic doses by now; and looking at the literature, AAV shuffling hasn't changed all that much since I completed my post-doc in an AAV lab in Stanford.  Or maybe Jim Wilson has grown exceedingly gun-shy when he resigns from his well-paying function at AAV company Solid Biosciences over concerns of toxicity from high systemic AAV doses and possibly also his finding that delivery to a certain tissue in the mouse does not predict same delivery in humans.

Certainly, there are also tailwinds supporting gene therapy that had not been around a decade ago.  As alluded to above, society and regulators have finally warmed up to a very sensible approach to medicines in the genomic era.  Moreover, there are established paths now to effectively develop such therapies and the challenge of producing large quantities of viral vectors is gradually being addressed.

Still, amid the hype and stock market giddiness, valuations in many of these companies have run ahead of themselves and were ripe for a pull-back.

Next time you hear so much Happy Talk around a biotech subsector leader as has been the case for gene therapy, watch out for a correction.   

What about RNAi stocks?

RNAi stocks have also benefited from the overall bullishness in the biotech sector with ARWR and DRNA having run up by 300% or so in a matter of months.  While I still hold shares in these companies, I have written calls (largely in-the-money) on essentially all of them meaning that I am fairly insulated should they fall another 10% or so.   

Since I still like ARWR and DRNA ahead of their first clinical GalNAc knockdown data, and in the case of Dicerna the potential removal of the overhang from the Alnylam litigation, I am eyeing (and have been) selling out-of-the money puts on these for 10+% premiums or so.

If the market turns back up, you pocket the premium. 10% or so in a matter of 2 months is not bad in my mind.  If, however, the stocks decline and the puts are in-the-money at expiration, you get handed a stock that you like anyway.  Say Arrowhead for a price of $4.5 instead of $5.45 yesterday.  

But before you place any such options bets, consult with your investment advisor first to familiarize you with the risks of writing options!

On the simple, naked long side, I like Ionis Pharmaceuticals which, as a biotech stock that has essentially missed out on the post-Trump rally and with the upcoming Huntington’s data catalyst, has the potential to become a new biotech bellwether following subsector rotations that usually occur following corrections (remember the 2015 correction following which oligonucleotide stocks turned from sector leaders to laggards?).  Alnylam, meanwhile, looks tired here with a lot of the upside priced in, at least regarding the TTR story.

Happy trading and be mindful.