Oligonucleotides Break Through to the Lung

It is days like today that I live for.  Days when new platform technology data is revealed that will change the practice of medicine and benefit patients for a number of diseases of high unmet need. In this case asthma, IPF, COPD etc. 

Almost a decade after GalNAc started to revolutionize oligonucleotide therapeutics delivery to the liver (hepatocytes) and turned oligonucleotides into the important therapeutic modality it has become today, Arrowhead Pharmaceuticals just reported the equivalent for the lung (lung epithelial cells to be precise).

Employing inhaled delivery of αvβ6 integrin-targeted stabilized RNAi triggers in healthy volunteers, the company found robust, -80% mean maximum target gene (RAGE) knockdown after 2 doses spaced a month apart. 

Since the knockdown reading was based on RAGE protein in serum (sRAGE), the true knockdown in the desired lung epithelium is likely higher.  This is also supported by the observation that more direct bronchoalveolar lavage measurements revealed -75% knockdown after just a single 92mg dose when the corresponding reading in the serum indicated -56%.  Further dose escalation to 184mg is ongoing and there are first indications that the long-lived pharmacodynamic response observed in animals will hold up in the clinic.

RAGE is a key player in pro-inflammatory signaling in the lung and thought to play a central role in related pulmonary disorders such as asthma.

In addition to clearing the efficacy hurdle, safety seemed excellent, or in the words of the company ‘no patterns of adverse changes in any clinical safety parameters’.

As some may remember, an earlier RNAi candidate targeting the lung (ENaC for Cystic Fibrosis) was shelved by Arrowhead due to preclinical findings in chronic tox studies in the rat.  The reason is thought to be that the sheer amount of material delivered to rat lungs overwhelmed and inflamed the macrophage-based particle clearance system.

What is different this time is that ARO-RAGE utilizes improved stabilization chemistries and therefore only a fraction of the overall tissue exposure is required to achieve the same knockdown. 

This is reminiscent of the early days in GalNAc conjugate-based delivery to the liver when a first-generation GalNAc-TTR RNAi trigger had to be discontinued by Alnylam due to adverse safety in the clinic.  Improved GalNAc RNAi drugs of increased metabolic stability (and reduced 3'-fluoro content) are now well established medicines.

Beyond RNAi Therapeutics, today’s results have important implications for oligonucleotide therapeutics applications in the lung in general, including RNA Editing. 

Most importantly, they establish αvβ6 integrin as a valid target receptor for oligo conjugates.  Moreover, some of the chemistries should be directly translatable for stabilization purposes and together with ARO-ENAC Arrowhead should now have good insights into the chemistry-safety relationship. 

RNAi Therapeutics and mRNA Vaccines for COVID19

This is a time where the biotech industry has to mobilize resources to mitigate the impact of pandemic SARS-CoV-2. 

Nucleic acid-based therapeutics are prime candidates in this battle because of the speed with which drug candidates can be designed and their specificity.  This blog provides an overview of efforts in this area with an emphasis on my two favorite (and IMO most promising) nucleic acid approaches: RNAi Therapeutics and mRNA vaccines.

RNAi Therapeutics

RNAi Therapeutics (and by extension the competing antisense, ASO approach), knocking down genes for therapy, can be used in two ways to address the current pandemic.

First, the RNAi triggers could target the virus itself in an existing infection.  The hope would be that by doing so the damage, mainly lung inflammation leading to respiratory distress, can be mitigated sufficiently for the patient’s immune system to gain time and strength to successfully overcome the infection.  The question then would be how early would be early enough.

The second approach would be to target a host factor critical for viral replication.  The cellular entry receptor ACE2 is the most promising candidate target gene here.  This strategy seems particularly promising for prophylactically protecting those at high-risk of an infection, e.g. medical personnel, as protection may last for a month or so based on the very long durations of efficacy seen in human studies targeting genes in the liver and early studies in the lungs of sheep.

Application in a prophylactic setting would probably have the added delivery advantage in that the lung would be less congested and thus more accessible.  Also, delivery may not have to reach so deep into the lung as it would need to during later stages.   

Delivery

Theory is one thing, but getting the RNAi triggers to where they are needed in the body is and has always been the main challenge for RNAi Therapeutics.  Unlike targeting genes in the liver which is now well-established, RNAi in the lung is a re-emerging area of RNAi development.

Given that COVID19 is a respiratory illness where the virus intrudes the body via the respiratory epithelium, a local, inhaled delivery approach should be adequate and also happens to be the most promising route for RNAi Therapeutics in addressing pulmonary disease.

In the early days of RNAi Therapeutics development, the world was facing the SARS outbreak. Seemingly attesting to the promise of speed and specificity against emerging pathogens, Sirnaomics (based in both the US and China) soon published a high-profile paper on the efficacy of RNAi in a monkey model of SARS coronavirus.  

In hindsight and with the benefit of 15 more years of RNAi Therapeutics development up-and-downs, however, these results were probably based on innate immunostimulatory artefacts given the use of unmodified Tuschl-type RNAi triggers and a nasal instillation route of administration making employing sugar water as a carrier.

Today, however, RNAi Therapeutics is much more advanced and an approach that utilizes aerosolized highly modified and thus stabilized RNAi triggers seems most appropriate. 

Arrowhead Pharmaceuticals has emerged as the front-runner in lung RNAi and is close to filing for clinical study approval for addressing cystic fibrosis.  Their approach combines the two most promising elements of today’s RNAi Therapeutics.  In addition to RNAi trigger stabilization, Arrowhead is adding targeting ligands to their agents, in this case small moieties targeting integrin on lung epithelial cells.

While Arrowhead has not announced their entry into the COVID19 race, Sirnaomics and Alnylam Pharmaceuticals (along with partnered Vir Biotechnology) have.  While it is unclear what particular approach Sirnaomics is using 15 years after their SARS work, Alnylam will be using highly modified RNAi triggers.  Whether targeting ligands will be utilized or not is unknown to me (update: the press release refers to 'conjugates of siRNA' so a targeting ligand is likely). 

Of note, Alnylam had suffered an anti-viral innate immunostimulatory fiasco with their first commercial RNAi development program around the same time that Sirnamoics was working on SARS.  That program was for the respiratory syncytial virus (RSV) and Alnylam should be able to capitalize on the the lung RNAi development experience back then.

Other RNAi-related companies with experience in inhaled lung delivery are miRNA Therapeutics company Miragen (miRNAs are structurally quite similar to RNAi triggers), Genevant/Arbutus (descendants of Tekmira which became famous for their Ebola efforts), and Arcturus Therapeutics.

mRNA vaccines

Arcturus Therapeutics and Genevant are now focused on developing messenger RNA (mRNA) therapeutics and vaccines, including for lung disease cystic fibrosis.  The most known name in this area is Moderna Therapeutics which has a high-profile, government-sponsored vaccine effort against SARS-CoV-2.  Ultimately, it is vaccines that will allow the world to fully recover from the COVID19 scare while therapeutics should be used in a much more focused manner.

The delivery challenge for vaccines is also lessened by the fact that it is a gain-of-function approach and that the immune system itself is expert at spotting foreign, in this case viral antigen expression.  The challenge here is that this is has to be achieved in the right immunological context so that a fruitful immune response is formed.  

The fact that Moderna were the first to ship mRNA for imminent trials shows how easy it is for mRNA to go from viral sequence to product candidate and drug material production.  While I used to poke fun at Moderna for building factories and installing robots without having much science behind it to fill the production halls, for emerging biothreats like SARS-CoV-2 this has proven to be prescient.

CureVac, an mRNA competitor of Moderna, has also announced their entree into COVID19.  While they may lag behind Moderna in terms of robotics and manufacturing capacity, they should be more expert in vaccine development (‘a little innate immune stimulation by the mRNA agent itself may go a long way’).

The most intriguing and differentiated entrant in the mRNA vaccine area to me, however, is Arcturus Therapeutics (disclosure: no stock position, but considering taking one) to me.  This is because they are using a self-replicating RNA ('STARR') that they claim to require ~40-fold less RNA to be intramuscularly injected. Formulation into an LNP 'LUNAR' particle may further lower the required dose.  Given that vaccinating the world will in the end also be a manufacturing challenge, being able to start with 40-times or thereabouts less material is a serious practical advantage.

COVID19 shall pass and therapeutics and vaccines will play an important role here.  Even more important, however, is that everybody does their part in minimizing and slowing the spread of the virus ('flatten the curve', #SocialDistancing), particularly to protect those most at risk without having to resort to draconian measures.

Silence Therapeutics Signs Lung Delivery Deal with Mystery Partner

As Tekmira is struggling to regain possession over its delivery technology from mighty Alnylam, it is foremost Silence Therapeutics' business development that is benefitting from having one of the most advanced, clinically tested, and commercially uncontested systemic delivery technologies in RNAi Therapeutics. Less than a month after closing a deal with Dutch company InteRNA under which Silence’s AtuPLEX lipoplex delivery technology will be evaluated for the delivery of microRNA cancer therapeutics, the company has just announced another delivery collaboration, this time for the related DACC delivery system. Like AtuPLEX, DACC targets vascular endothelial cells, but unlike AtuPLEX it does so with a high preference for the vascular endothelia of the lung.

Curiously, the identity of the partner was not disclosed, although it was mentioned that it was a ‘Top 10 Pharma Company’. 'Top 10 Pharma Company'....didn't Takeda just become Number 10 with the acquisition of Nycomed? Moreover, the Nycomed acquisition included a fresh COPD drug, a pulmonary disease with an inflammatory component for which the DACC delivery system could very well be useful.

You can imagine that Takeda is a bit hesitant to talk about RNAi Therapeutics, maybe having overpaid a bit for what Alnylam really sold them, but with their significant investment in RNAi already it has every incentive to get things moving while Tekmira and Alnylam are fighting it out. Maybe following this rationale, Takeda and South Korean company Samyang announced earlier this year a somewhat surprising delivery collaboration.

The press release also noted that it is the partner that will provide the RNAi triggers. This pretty much excludes Silence’s existing Big Pharma partners AstraZeneca and Dainippon Sumitomo, the latter of which is not a Top 10 Pharma anyway. On the other hand, Takeda licensed RNAi triggers from Alnylam only for the metabolic and oncology fields, something that would probably add to the apparent sensitivities. I highly doubt that Takeda will hand over another $50M to Alnylam for pulmonary.

Of course, it is always possible that RNAi Therapeutics has gone so much out of fashion that Big Pharma companies are embarrassed to be publicly associated with it. Not only that, their investors shun the word ‘Research’ like the plague.

More seriously, the data that Silence reported for the DACC system has been quite impressive. Working on a model for acute lung injury, it has demonstrated potent and persistent gene knockdown in the pulmonary vascular endothelium following a single intravenous administration. While less is known about the clinical safety profile of DACC, it can be considered somewhat de-risked by the fact that its chemistry seems to be very similar to the clinically proven AtuPLEX system (see the highly encouraging safety profile of their phase I Atu027 clinical candidate so far).

2008: The Year of the Liver

It is true that next year could bring the first human proof-of-concept for an RNAi therapeutic. But since results from the experimental infection studies for ALN-RSV01 were originally expected in late 2007 and the results would be an overhang of work done in ’07, I find it more appropriate to pick a fresh candidate. It is also true that the lung may rival the liver in the number of programs moving into the clinic in ‘08 given recent data that suggest delivery of siRNAs to the lung by nebulizer to be fairly innocuous, and the fact that AstraZeneca and GSK should be busy working in that area following their agreements with Silence and Sirna Therapeutics, respectively.

Nevertheless, I feel that most systematic progress has been made in delivering RNAi to the liver with more progress likely to follow. There have now been a fair number of publications that clearly show target-specific RNAi knockdown in the liver, unlike maybe some of the lung studies where there were occasional data interpretation issues. Of course, in declaring 2008 ‘The Year of the Liver’, I should disclose that I am somewhat biased due to the hepatotropic focus of the laboratory that I work in.

For siRNA-mediated RNAi in the liver, I currently see three front-runners, although it is possible that among the considerable work that is ongoing and not yet published, others have reached a similar stage of technological maturity. These are the in-famous SNALPs, Mirus Bio’s “Dynamic PolyConjugates”, and MIT’s lipidoids. SNALPs, being worked on by Protiva, Tekmira, Alnylam, and Sirna/Merck, are probably 12-18 months ahead of the game and best characterized, whereas the data on PolyConjugates and lipidoids are very promising, but still somewhat spotty.

SNALPs, stable nucleic acid lipid particles, are set to enter the clinic in 2008 with programs in hypercholesterolemia and liver cancer (both Alnylam) and possibly a clinical candidate chosen by Tekmira. The hypercholesterolemia program is also an opportunity to get an early measure of therapeutic efficacy, possibly in ‘08. Protiva’s and Sirna/Merck’s intentions are less clear and I believe that Big Pharma often chooses to keep phase I programs secret for competitive reasons, so that it is theoretically possible that Sirna has already entered the clinic or is about to do so with the long-anticipated SNALP RNAi for Hepatitis C.

It is obvious that there were some delays in bringing SNALP RNAi to the clinic, and I believe that this is largely due to dosing and safety issues. Both of these concerns may come down to the propensity of SNALP liposomes to be taken up by immune cells such as Kupffer cells in the liver and plasmacytoid dendritic cells which a) function like a sink for the liposomes when they enter the liver so that SNALPs become available for entering the desired hepatocytes only after the sink is saturated, something that complicates dosing; and b) increases the risk of triggering unwanted cytokine responses. I am optimistic, however, that by varying the composition of SNALPs, safe and efficacious formulations may be found, particularly if the liposome uptake mechanism by the professional immune surveillance system were to be different from that by the hepatocytes, which I think is quite reasonable to assume. I therefore hope that the fact that so many scientists are working on SNALP RNAi is a sign of its promise rather than desperation. As such, the number of R&D staff at Tekmira has more than doubled from 17 to 39 in the year ending September 2006 largely due to work on SNALPs.

As I have written before, I am much taken by Mirus’ PolyConjugate work, although this is based on only a single paper published in the middle of ‘07 (see 24 July 07 Blog: “Mirus Scientists Publish Elegant Paper on Targeted siRNA Delivery to Hepatocytes”). The neat aspect of that work was that it showed that it should be possible to avoid the Kupffer cells in the liver and specifically target hepatocytes for knockdown with the help of carbohydrate ligands (I am curious whether similar targeting ligands would also work in the context of other formulations). As we know, this work is partnered with Pfizer, and I wonder whether the upcoming loss of exclusive marketing rights for their wonder-drug Lipitor will spur them into action here.

I am still waiting for more data on lipidoid-mediated RNAi which hopefully will become available soon as was indicated in the footnotes of an October 2007 paper on the effect of siRNA delivery on microRNA function. From both a business and scientific perspective, it will be interesting to see whether Alnylam may choose lipidoids over SNALPs for its first liver RNAi programs and the overlap of SNALP and lipidoid both in IP and manufacturing terms.

The liver may get further attention from the targeting of microRNA-122 for the treatment of hypercholesterolemia and Hepatitis C, and other programs on targeting certain microRNAs for the treatment of hepatocellular carcinoma. There are various programs by Santaris, Regulus and others that have progressed into larger animals and we may even see a first IND being filed for one of these applications by the end of 2008. Development in the antisense field, particularly the partnering of ISIS’ mipomersen, should also generate heightened awareness for RNA-based therapeutics of liver disease.

Other predictions for 2008:

1) Unpredicted delivery technologies
2) Pfizer finally makes its move
3) News on RNAi for wet AMD- good and/or bad?
4) Protiva-Tekmira dispute resolved (wishful thinking)

For a nice presentation on the powers of SNALPs by Protiva chief scientist Ian MacLachlan, please visit: http://mms.technologynetworks.net/RNAi06Presentations/maclachlan/Player.html

RNAi and Bird Flu

Every year labs around the world are racing to get vaccines ready for next year’s flu season. Even if the logistical nightmare can be managed and complicated vaccines are manufactured in time, this is no guarantee that the vaccine protects against the actual flu strain that season. This is because even as we observe the new strains emerging from East Asia, once it has reached our shores, the virus is likely to have changed his outer surface making it more difficult to be recognised by the immune system. That’s what viruses have learned to do anyway. If seasonal flu is challenging enough to predict, then developing a vaccine for a pandemic flu strain that had been generated by considerable genetic drift, is a daunting task.

Here, using RNAi, and in principle other nucleic-acid based therapies for that matter, offers a number of considerable advantages. Significantly, rather than going after a moving target, RNAi allows us to aim at flu genes that are not as mutable given the structural constraints of their encoded proteins for viral replication. This means that even if a re-assorted bird flu might look quite different on the surface, many parts inside of the virus will look very similar between even divergent flu strains. These conserved parts, or better their underlying RNAs, can be computationally predicted, and it is then very straightforward to design small interfering RNAs (siRNAs) against these RNAs well before we even know that there will be a bird flu pandemic. This can be done with high statistical confidence.

Of course, this strategy is not only applicable to bird flu, but any other emerging viral threat. A number of high-quality scientific papers have been presented documenting the feasibility of this approach. Intradigm e.g. has shown in 2005 in a timely manner that siRNAs could protect monkeys from severe lung damage due to SARS. Research teams from Nastech and Alnylam have similarly documented the potential of RNAi for flu in animal models. The US government is funding much of the research and may eventually decide to stockpile siRNAs for pandemic bird flu. Concomitantly, manufacturing capabilities are also being developed.

It is further encouraging that the lung which has been exploited for eons by viruses to infect the host now emerges as a good organ for drug delivery. Specifically in the case of RNAi, it came as a rather pleasant surprise that even unformulated siRNAs could readily get into lung cells and silence their target genes. RNAi therefore not only holds promise as a rapid response to viruses, but also other diseases of the airways such as asthma and COPD. As I write, many of these conditions are the subject of pre-clinical RNAi programs and one program by Alnylam for RSV (respiratory syncytial virus) has already reached the clinic. Data from their viral challenge studies in the second half of this year are widely anticipated, and if successful, would present the first proof-of-concept for a human RNAi Therapeutic.