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.

Antibody-RNAi Trigger Conjugates Show Signs of Life

Despite jettisoning RNAi Therapeutics 4 years ago, the more innovative arm of Roche, Genentech, has continued to dabble in the technology.  In particular, it has been interested in applying its monoclonal antibody know-how, including antibody-drug conjugates (ADCs) to the delivery of RNAi Therapeutics.  A recent publication by Cuellar and colleagues provides insights into these efforts.

THIOMABs for the creation of drug-like conjugates

While antibodies have a relatively long history in the delivery of RNAi Therapeutics, some of the early findings were generally quite difficult to replicate.  Part of the problem may have been the fact the early efforts involved structurally ill-defined non-covalent protein-nucleic acid complexes held together by charge-charge interactions.

To get around this issue, Genentech applied their THIOMAB platform which allows for the covalent addition of therapeutic payloads at defined cysteine residues.  This process yielded THIOMAB-siRNA conjugates with one, or more often two RNAi triggers per monoclonal antibody.  To help visualize them, also for pharmacokinetic considerations, think of the monoclonal antibody part being ~13x bigger/heavier than each RNAi trigger.

Of note, the RNAi triggers were partially (~50% of residues) modified with 2’-O-methyls and 2’-F for stability (siSTABLE from Dharmacon).

Not all receptors are created equal

One of the reasons why I am picking out this paper for discussion is the thoroughness of the research presented.  For example, the Genentech researchers selected not just one, but seven distinct cell surface receptors for which various THIOMAB-siRNA were created which in turn were be tested in a number of settings.  The receptors were partly chosen to capture a range of cellular trafficking behaviors such as rapid lysosomal uptake, recycling receptors, and slow-turnover receptors.

Importantly, the research validated a critical rationale for the use of antibody-RNAi trigger conjugates namely uptake of the RNAi triggers that is dependent on the presence of the cognate receptor and covalent linkage to the antibody.

For all the conjugates (ARCs), the bulk was shown to accumulate in lysosomes regardless of presumed uptake kinetics.  Interestingly, despite the seemingly shared uptake pathway, if not dynamics, only two of the seven receptors were associated with gene silencing (TENB2 and NaPi2b, but not e.g. Her2). 

The degree of silencing was much weaker compared to when the same conjugates were lipofected with ~50% silencing starting to be seen at 10nM.  This was followed by a shallow dose-response plateauing at 70-80% silencing around 500nM.  Based on the data presented, it seems that limiting amounts of receptors were responsible for this. 

Selective accumulation in mouse tumor model

A highlight of the publication to me was the investigation of THIOMAB-siRNAs in a mouse tumor model.  Although you may consider a ~30% silencing (i.e. 70% expression of normal remaining) when you cherrypick tumor areas of most efficient delivery a somewhat disappointing outcome as was reported in this case, important lessons can be learned from that.

Firstly, THIOMAB-siRNAs only accumulated in the tumor when the tumor expressed the cognate receptor.  This is unlike nanoparticulate RNAi delivery to tumors which relies on a passive process of accumulation (the EPR effect).  This opens up the prospect of RNAi drugs with ‘cleaner’ delivery profiles with an increased margin of safety.

Selective accumulation for these conjugates also suggests that the ~180kDa macromolecules were able to relatively rapidly exchange between tumor interstitium and blood circulation despite their size.  Consequently, other conjugates in a similar size range or below, including Arrowhead’s DPCs, should be amenable to such ‘active targeting’ as well.

Nevertheless, despite this apparent agility, imaging techniques showed that tumor delivery was largely restricted to areas next to the vasculature.  This limitation in fact is what is also seen with nanoparticles which rely on the EPR effect.  So while the active targeting capability is an important step forward, tumor penetration issues remain to be solved.

Path forward

Despite the arguably underwhelming in vitro and in vivo knockdown results if you just look at the numbers, I am optimistic that the studies have further supported that systemic RNAi delivery can be applied beyond the liver, vascular endothelial cells, certain cells in the kidney and phagocytes.

What is missing in that particular piece of research making the conjugates that make it into the cells count.  Given the lysosomal accumulation, an obvious strategy to unlocking the true potential of ARCs would be to apply endosomal escape chemistries, especially masked chemistries such as in the DPCs by Arrowhead Research (actually specifically referenced by the authors).

In almost the same vein, the sparse chemical modification used mean that potency improvements will be gained if heavier modification is applied even without adding endosomal escape chemistries.  This is also because some of the data are consistent with a model whereby the THIOMAB-siRNA conjugates get broken apart in late endolysosomal compartments and it is from that population of freed RNAi triggers that escape into the cytoplasm may occur (note: this does not exclude spontaneous endolysosomal rupture as an additional escape pathway).  If RNAi triggers were made metabolically more stable through more extensive modification, the amount of RNAi triggers available for such escape would obviously be larger.

All that is lacking then is the issue of tumor penetration.  I could imagine, however, that some self-delivering and/or lipophilic strategies could be quite useful here.  The Genentech researchers actually speculate that the RNAi trigger may have been the culprit in limiting the claimed natural ability of monoclonal antibodies to more deeply penetrate tumors.  This suggests that charge masking may overcome this limitation.  

I am open to this idea, but based on my understanding of the literature, tumor penetration is a challenging issue for basically all therapeutic modalities, monoclonal antibodies and 'even' small molecules, so let's not blame double-strandedness for all pharmacologic problems.

With interest in RNA Therapeutics growing by the day, expect lines of delivery research such as this one to be picked up and pushed to the next level.

PS: for antibody-mediated RNA delivery, Avidity NanoMedicines and the Roche-ISIS collaboration on CNS delivery should also be worth watching.

January RNA Therapeutics Deal Frenzy Kicked Off

As is now tradition for Januaries in RNA Therapeutics, expect this month to be filled with mouthwatering business development announcements.

Without a doubt, privately held messenger RNA Therapeutics company Moderna Therapeutics stole the show today by announcing a $450M financing involving 'Viking Global Investors LP, Invus, RA Capital Management, and Wellington Management Company, LLP, as well as existing investors AstraZeneca and Alexion Pharmaceuticals'.

Biggest private financing in biotech history EVER.

With $800M in cash, close to a billion USD raised since starting up 2 years ago or so, the biggest challenge will be how to efficiently put the capital to good use and move into the clinic in the not-too-distant future.  Before that happens, we should be seeing one of the hottest biotech IPOs in 2015.

Before Moderna announced their financing, ISIS Pharmaceuticals was the talk of the day with their GI-related autoimmune deal with Johnson&Johnson.  Not only does the early development deal provide ISIS with another nice junk of upfront cash, this time $35M, and a gazillion in potential biotech milestone payments and royalties, jn practical terms I expect the research to be particularly useful to ISIS as it develops oral delivery for systemic applications (think lipid franchise for which the new CEO will be officially enthroned tomorrow).  

I believe that the deal was primarily driven by JNJ's interest in the space following the whopping $710M upfront deal by Celgene last year when it acquired rights to an antisense compound developed by oligonucleotide therapeutics nobody Nogra Pharma for IBD applications.  The high upfront payment indicates that competitive bidding was involved and it is likely that JNJ lost out.

Remember, Big Pharma moves in herds.

The good news for JNJ is that if the first-generation phosphorothioate DNA is indeed working by an on-target mechanism (which I doubt), then a competing compound employing much more advanced chemistry and design by ISIS Pharmaceuticals will be infinitely more potent and beat Celgene in the market.  And for $35M and $800M in milestones if everything goes according to plan, it's a comparative steal.

For ISIS Pharmaceuticals it is just another step to broaden their expertise and applications of their platform.  Moreover, the upfront payment will help the company to remain cashflow neutral to slightly positive until 2017, at which time I expect drug sales to be finally taking off.

Investor Appetite for Expanding the Nucleic Acid Therapeutics Tool Box

The last few days saw two more eye-catching examples of investors daring to dream big by committing significant early-stage capital to building new Nucleic Acid Therapeutics platforms. 

First, start-up mRNA Therapeutics company Moderna Therapeutics took advantage of the hype it was able to create in the wake of a deal earlier this year with AstraZeneca which involved $240M in upfront monies alone to raise another chunky $110M in a private round.  This brings the total raised by Moderna to over $400M in the last 2 years or so.  Then on Monday of Thanksgiving week, a consortium of VCscommitted up to $43M to start up Editas Medicine, a company built around genome editing harnessing the recently identified CRISPR adaptive immune system in bacteria.

Counting in IPOs and private rounds by nucleic acid therapeutics companies (e.g. Bluebird Bio in gene therapy, Prosensa in exon skipping), well over half a billion raised by RNAi Therapeutics companies in 2013, partnership monies earned by ISIS etc, we are talking here probably about approx. $1.5B raised by the industry so far this year.  And what is best about this in my opinion is that these fund raisings were not intended to help with commercializing a newly approved product or running a large phase III trial for a late-stage clinical candidate- no, they were mainly the result of broader interests in the platforms.

What a departure from the post-2008 world of biotech and oligonucleotide therapeutics when pipelines were commonly regarded as a financial liability and the platform concept was the ultimate biotech heresy belonging to the 2000 genomics bubble era.

If you like to see the glass half empty and secretly hope that the world is coming to an end soon (e.g. in the form of a stock market collapse), good luck to you!   While I expect some volatility as the industry moves towards bringing meaningful drugs onto the market and cash flow positivity (just witness the recent Prosensa and Sarepta stumbles) , looking at the nucleic acid therapeutics pipelines and a healthcare environment friendly towards targeted therapies that are the sweet spot of nucleic acid therapeutics, it is hard to come up with scenarios in which this nucleic acid therapeutics revolution can still be halted.  Moreover, the strong cash balances of some companies mean a virtuous cycle of value creation for companies like Arrowhead Research which are capital constraint, not technology constraint, and should also allow them and their investors to weather short-term volatility.

Having said that, the time for some healthy pruning will ultimately come, likely in the wake of the next major upheaval in the normal economic cycle, and I expect some investors to throw in the towel even before some of these technologies have had the time to prove themselves.  You think that making delivery work for RNAi Therapeutics strained some investor patience, then what about getting a protein, RNA, and DNA into the nuclei of target cells all at the right amount and time for the correction of single-gene disorders as appears to be the first therapeutic frontier of Editas?

   

But until then, the fact that investors dare to dream of medical breakthroughs again means that the future of biotechnology which lives off such bold concepts remains bright.   Nucleic acid therapeutics will play a critical part in that future.

Marina Biotech as a bet on the oligonucleotide therapeutics tool box

As the economy improves and interest rates remain low, I expect money continue to move into the higher risk end of the market.  In terms of the stock market this generally means that while the 'safe' companies were the first to recover following the collapse of the housing bubble, the more innovative and 'risky' groups such as social media and biotech followed with even larger returns.

Thinking of the oligonucleotide therapeutics space, the ultimate high-risk play that has yet to respond to the improved market conditions is Marina Bio.  In hindsight, the two main reasons for Marina Bio's spectacular share price erosion were (1) that it promulgated the idea of an oligonucleotide therapeutics supermarket concept when nobody wanted to hear of it, and (2) that it did so after it had lost its credibility in the markets after many broken promises (largely under previous leadership) and then tried to re-define itself by the year.

Most would consider it dead now, but with a number of licenses to companies like Novartis, Monsanto, and Tekmira, two products in the clinic that utilize its SMARTICLE delivery technology (one for a microRNA Rx mimic by Mirna Therapeutics, one for an DNAi Therapeutic by ProNAi) and an in-house clinical candidate for an orphan indication (transkingdom RNAi for FAP cancer) in a suspended (for financial reasons) phase I study that one ought to be able to revive, it should have more inherent value than the $3-4M current market cap suggests.   In addition, it retains access to usiRNAi triggers and high-affinity antisense chemistry for RNaseH gene knockdown and steric blocking applications that I see on par with the technologies by ISIS Pharmaceuticals, Santaris, and Regulus.

As appetite for all things oligonucleotide therapeutics grows, this could be an interesting play.

Whoa! AstraZeneca Pays RNA Therapeutics Start-Up Moderna $240M

In the age of rare/severe diseases in drug development and personalized medicine, RNA Therapeutics are enjoying broad interest like never before.  Following a series of RNaseH antisense, splice modulation, microRNAs, and RNAi Therapeutics deals with large pharmaceutical companies, the AstraZeneca-Moderna Therapeutics news today marks another high water mark in the deal-making.  According to their agreement, AstraZeneca will pay Moderna Therapeutics $240M in upfront alone for rights to Moderna’s technology in the cancer and cardiovascular/metabolic disease areas (40 targets).   

AstraZeneca’s Externalized RNA Therapeutics Efforts

The deal with an innovative biotechnology start-up while in the process of shedding yet another few thousand employees, particularly in internal R&D, illustrates AstraZeneca’s R&D externalization trend.  RNA Therapeutics here seem to play a key role as supported by additional recent multi-million dollar deals in the space with ISIS Pharmaceuticals (December 2012) and PTC Therapeutics (June 2011) in oncology.  Unfortunately for RNAi Therapeutics though, it has been left out so far from the AZ’s deal bonanza.  Only in 2011, it wrapped up a collaboration with UK-based Silence Therapeutics.  Still, as AstraZeneca faces the challenge of how to deliver messenger RNAs to the liver and cancer, I expect RNAi Therapeutics delivery companies to financially benefit from AZ's mRNA investment soon (not just for mRNA, but also for RNAi delivery).

Moderna’s Technology

Moderna’s approach is a gene therapy one.  However, while classical gene therapy involves the use of DNA vectors for expressing therapeutic proteins, Moderna aims to circumvent the need for DNA, which have certain regulatory and safety drawbacks, and deliver instead messenger RNAs encoding for the same proteins.  This, in fact, is not a new idea and particularly popular in the immunotherapy field (albeit delivered ex vivo here, by electroporation). Duke University for example had a clinical RNAi Therapeutics program that involved the transfection of mRNAs along with siRNAs (cancer vaccine).

A 2013 Nature Protocols paper by the company’s scientific co-founder Derrick Rossi also leaves me scratching my head as to why AstraZeneca concluded that Moderna’s IP was worth $240M to them.  According to the protocol, the mRNAs are generated by normal in vitro phage polymerase transcription as you would do in the lab using Life Technology’s MEGAscript kit.  The only difference from the standard protocol may be that modified CTPs and UTPs were included.  This is supposed to mitigate the immunostimulatory potential with RNAs just as in RNAi Therapeutics and also contribute to the stability of the long RNAs.  

Based on the fact that neither the concept of mRNA Therapeutics are novel nor the RNA modification strategy unexpected, I expect that Moderna has yet to come out with their secret sauce and that the Nature Protocol may be misleading.  I therefore look forward to studying the patent applications, two of which curiously just published today.  It must be the IP that explains why AZ took a $240M license, to get a sense of the secret sauce.  But still, given the hundreds of nucleotide modifications available, it seems hard to fathom that Moderna's a blocking IP position, and why pay $240M if not a blocking one?

Need for Delivery

In addition to AstraZeneca’s interest in cardiovascular/metabolic disease and cancer, the state of the RNA(i) delivery technologies explains their choice.  Compared to RNAi Therapeutics where small RNAs are involved, the longer mRNAs face an even steeper cytoplasmic delivery challenge.

The liver, of course, is a key target organ for metabolic and cardiovascular disease.  Among Arrowhead’s DPCs, Alnylam’s GalNAcs, and Tekmira’s SNALP, the most advanced RNAi delivery technologies for the liver, it is essentially only Tekmira’s SNALP technology which I regard to be readily applicable to mRNA delivery (also for cancer delivery).  Conjugate approaches such as DPCs and GalNAcs are disadvantaged for mRNA delivery because they would provide no extra protection to the long, fragile mRNAs.  Liposomes by contrast provide such protection by wrapping around the RNA.

I expect to hear more about Moderna’s and AstraZeneca’s mRNA delivery strategies soon.  I, for one, do not believe that Tekmira’s shares are trading up by 8% on strong volume on the back of a SeekingAlpha article.

Other evidence that RNA Therapeutics (and Gene Therapies) Are Hot

ISIS Pharmaceuticals yesterday presented phase I data for their spinal muscular atrophy (SMA) splice modulation drug candidate at the annual AAN meeting.  This compound is partnered with BiogenIdec.  The results from the single-dose PK-oriented study suggest that fully 2’-MOE phosphorothioate oligos are well tolerated in the CNS and that a once-a-year/once-every-half-year dosing regimen may be possible: phosphorothioate oligos sit like a rock in the CNS when intrathecally administered.  In addition to the hints of clinical efficacy at the highest dose level (9mg) presented at the conference, what makes me optimistic about this program is that relatively little (compared to RNaseH) phosphorothioate molecules seems to be required based on the pre-clinical results.

In other news, cancer drug developer Celgene will work with gene therapy company bluebird bio on cancer gene therapy.  Although financial details were not disclosed, they were probably substantial (wild guess: $20M) given the broad nature of the collaboration (multi-year, multiple targets).