"Holy Deliverability, Batman!": Next-Gen "Heroism" For The mRNA-[X] Space

By Anna Rose Welch, Editorial & Community Director, Advancing RNA

In the closing scenes of the 2022 Batman movie, Batman leaves us with the following words as he rescues the citizens of Gotham City from the Riddler’s chaos:

“Vengeance won’t change the past… I have to become more. People need hope. To know someone’s out there for them.”

I’ll be the first to admit that the relationship between this quote and the mRNA space is tenuous at best. But Batman’s realization about the importance of hope is what came to mind as I started to organize my thoughts following a fantastic conversation on the future of mRNA development with Michael Koeris, associate professor of bioprocessing, Keck Graduate Institute.

I think it’s safe to say the mRNA COVID vaccines were our real-life Batman — the hope we needed — during a particularly difficult time in the world. Today, as we set our sights on next-generation vaccines and mRNA therapeutics, we have dreams of how our companies and therapies can continue to save patients in a variety of indications. But the transition from vaccines to therapeutic development — and particularly the targeted delivery of these nonprophylactic therapies — is shaping up to be more difficult than the “comic books” may have made it seem. To be sure, innovation and scientific learnings are happening incrementally every day and year. But as my constructive conversation with Koeris revealed, to achieve several of our current therapeutic dreams, we will most likely need a few new heroes and/or sidekicks to support both our processes and our products.

In the first- of this two-part article, Koeris and I ford through some of the most prominent delivery-vehicle development pain-points that don’t get nearly as much conversation as they should today. Though Koeris did not sugarcoat his concerns with some of our current mRNA delivery/LNP development limitations, the questions he posed and considerations he offered give us clearer direction around where we must pursue more scientific “heroism” in the mRNA space. There is a lot of work ahead of us; but his thoughts also prove that there’s just as much about which we can be hopeful, so long as we continue to investigate the many yet-to-be thoroughly explored scientific and collaborative opportunities that exist to support our industry.

“And Deliver Us From Evil [The Liver]:” LNP Formulation Woes

Just as the flooding overtook Gotham city from all angles and met “in the middle” at “Gotham Square Garden,” Koeris sees the RNA therapeutics space with its many different therapeutic approaches (e.g., cell therapy, gene therapy, protein replacement) as a series of rippling, overlapping Venn diagrams. At the intersection of each of these diagrams is the most important area for the RNA therapeutics industry’s general advancement and ultimate success: Delivery.

“As a space, we are going to have to do a lot better with LNP development — and we’re going to have to do a lot more work on drug delivery vehicle exploration beyond LNPs — before we even start addressing some of the other technical and scientific challenges facing RNA therapeutics,” Koeris explained.

Based on data from RNA Beacon research, 70-plus percent of the (disclosed) mRNA programs in development rely on LNP delivery today. This is likely no surprise to anyone reading this article. On the one hand, the approval of Onpattro in 2018 and the two mRNA COVID vaccines did some heavy lifting for us by “derisking” the mRNA-LNP “dynamic duo” with regulators. Likewise, seeing as most investments in mRNA today remain in the prophylactic vaccine sphere, LNP’s inherent immunogenicity serves us well on our quest to increase the immunogenicity of our vaccines.

“This is a feature, not a bug of LNPs,” Koeris clarified. “We’re using LNP biology to our own benefit in the case of prophylactic vaccines.” However, as most of us already know, this immunogenicity profile is not a helpful biological feature for those of us with next-generation mRNA therapeutic goals — especially if we’ll be chronically dosing.

Beyond their inherent immunogenicity, Koeris identified a few challenges of developing/formulating LNPs that deserve much more attention in the upcoming year(s) if we’re going to see success with LNPs as a delivery system for ex-hepatic therapeutic indications. First things first, it behooves us to converse more frequently and transparently around the inefficiencies of LNP formulation today — the challenges of which don’t often get enough bandwidth during our drug-substance-heavy discussions. Not to mention, our successes in scaling LNPs to meet global vaccine demand can make it easy to underestimate the sheer amount of artistry necessary for controlled LNP development.

“LNP formation is a complex molecular assembly that needs to be appropriately managed,” Koeris defined. “Despite the fact we have several approved LNP products, we’re only at the beginning of the encapsulation and formulation journey for LNPs today.

As he went on to argue, we lack the engineering precision of other delivery approaches within the nanoparticle class (e.g., gold nanoparticles, drug-loaded microspheres, or quantum dots). Look no farther than the variety of creative ways folks are currently characterizing the mRNA-LNP formation process; some of my favorites refer to mRNA-LNP formation as a “brute-force” exercise and/or one “comparable to that of cocktail mixing.” (And as a friendly reminder, Batman taught us earlier that vengeance/brute force is not exactly the best method for getting ^%&# done.)

As Koeris described, our mRNA and lipids are currently forced to co-mingle in a solution which we then subject to any number of physical perturbations, such as turbulent mixing (whether in macro- or micro-scale environments), or sonication. It’s during this process that the LNP forms around the mRNA. However, arriving at the appropriate ratio of lipids and mRNA remains imprecise and hard to model today. In particular, the mRNA tends to be overpowered by the amount of lipids, predominantly because we dilute the mRNA to ensure we don’t encapsulate too much, Koeris explained.

“That this assembly happens at all is fascinating,” he admitted. “But the long-term question for us as a space becomes: How can we better control the concurrent formation of the LNP while it’s encapsulating our mRNA? Right now, all we know is that there is a wide amount of variability in terms of lipid nanoparticle sizes and the amount of mRNA each includes.” (And based on a presentation I heard from Moderna in recent months, demonstrating the homogeneity of our mRNA-LNPs matters quite a bit to regulators today.)

Much like our brethren in the AAV space with their empty-full capsid travails, we in the RNA space are also finding ourselves facing a highly similar challenge: many of the LNPs formed using current processes are “empty.” Such an attribute demands much more of our analytical platforms, especially seeing as the fragility of LNPs makes separating out “empty” LNPs via chromatography problematic, Koeris added.

Of course, it goes without saying our goal is to deliver as much RNA as is necessary to provide a safe and clinically meaningful benefit to patients. Not to mention we need to clearly understand and justify what “enough mRNA/RNA” means for each individual product and indication. But our current conversations about mRNA-LNP formulation in general and about homogeneity specifically become more complicated as companies start/continue to explore therapies comprising multiple mRNAs encoding multiple proteins. On the one hand, we need to understand how the overall combination/ratio of different mRNAs-LNPs team up to single-handedly tackle diseases. But we can also expect questions to be raised about the translation efficiency of each mRNA-LNP within the drug product cocktail — the individual compositions of which could be important factors to understand in relation to the translation efficiency of each individual protein. (And, by the way, a presentation I recently heard from an expert at Sanofi also revealed that some mRNAs — even those packaged in comparable LNPs — get preferential translation in vivo.)

Not All LNPs Wear Capes: Next-Gen Considerations for Delivery With & Beyond LNPs

When we talk about next-generation mRNA-LNP delivery, our conversations cover some familiar ground, whether it be the use of microfluidics for improved/more controlled formulation or next-gen lipid libraries. Koeris confirmed that we’ve barely scratched the surface in terms of the lipids/combinations we may be able to use to improve our therapies’ deliverability and targeting. Thanks to the fact that lipids are defined by their sequences, there are an increasing number of high-throughput and low-risk machine-learning tools that companies are using to build diversified lipid libraries.

However, he remains uncertain that simply mixing and matching novel lipids/combinations will be the silver bullet to broadly shuttling LNPs throughout the body — especially given our bodies’ biological proclivities to metabolize lipids via the liver.

“I don’t want to sell both our industry’s creativity and nature’s willingness to evolve short, but we start to run into some hard limits when we try to make nature do something it really doesn’t want to do,” he said.

In turn, beyond lipid libraries/diversification, he argues one of the most untapped areas for creativity we should be exploring is how we can “decorate” (or design a superhero costume?) for our LNPs. In the viral delivery space, for example, the proteins decorating their outer shells enable a bit more specificity in terms of targeting. This raises questions for Koeris about our industry’s ability to couple our lipids with proteins to improve their specificity.

To be clear, when Koeris says “coupling” our LNPs with proteins, he is thinking beyond conjugating our products to antibodies, for example. Though conjugation is one important approach being explored today, he is predominantly interested in the proteins that naturally incorporate themselves into lipid membranes. “There are plenty of proteins that naturally like to work with lipids,” he explained. Carbohydrates remain another promising element to explore, either as an LNP surface “decoration” or as an alternative form of polymer nanoparticle.

Ultimately, Koeris didn’t sugar coat the fact that our explorations to better “dress up” our LNPs could throw our already-imprecise formulation methods for a loop. Not only are we still striving to understand the product that emerges following the combining of mRNA and lipids, but the addition of another element into the mix, for example a protein, will demand greater understanding of the physiochemical properties of each element and how/if they work naturally together using current formulation methods.

BatmRNA & ?: Maybe We Haven’t Found Our “Robin” Yet?

Though many in the RNA space are dedicated to “characterizing” what our superhero mRNA-LNPs will be able to do in the future, Koeris and several other experts I’ve spoken to recently have begun emphasizing the importance of greater “delivery vehicle agnosticism” for the RNA space.

“We can’t just bet on one horse,” Koeris beautifully summarized. “In the world of soccer, kicking one good free kick doesn’t mean that you’re going to win the World Cup. This is a team sport and the team — in this case, our industry, and its RNA delivery approach — has to be multidisciplinary.”

Just as we have two comic book universes (Marvel & D.C.), the characters from each of which occasionally universe hop, Koeris envisions a world in which we see teamwork in fields that typically reside in separate universes. Though we are heavily invested in LNP delivery (i.e., chemistry), there is a lot we could learn about how to better make LNPs from alternative delivery vehicles, like exosomes (i.e., biology). An exosome’s ability to package great amounts of material and their sophisticated surface decorations offer potential learning opportunities for those of us trying to make our LNPs more specific delivery vehicles. In the same way, LNPs can be made relatively cleanly, compared to exosomes whose current manufacturing and purification process leave a lot to be desired in terms of exosome purity.

“Both scientific fields and delivery approaches have their merits, and I would like to see biologists and chemists work more closely together,” Koeris said. “Society — and humanity in general — would benefit from the learnings each could glean from the other.”

Given the pressures of commercialization facing companies, he admits such fundamental exchanges are more likely to happen/start in academia — especially since we no longer live in the days of industrial labs, like Bell Labs. But this is a fundamental shift that needs to happen across academic departments and institutions, and which could be the subject of federal funding beyond the grants for which academics often have to compete.

“It took a couple of decades of work and a Nobel Prize to get us to where we are today,” Koeris concluded. “There will be more Nobel Prizes in this field. In fact, I’d argue that cracking precise, systemic delivery of RNA is, hands down, a Nobel Prize in and of itself, given how many difficult biological and technical challenges we will need to overcome.”