Blog | May 22, 2023

Where Fly-Fishing Meets mRNA: The Art Of mRNA Immunogenicity

Source: Cell & Gene Collaborative
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By Anna Rose Welch, Director, Cell & Gene Collaborative
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At the beginning of the year, I launched a new video series, ARW on RNA, to creatively explore the scientific and business-related evolutions taking shape in the mRNA therapeutics industry.    Though inspiration has rarely been akin to a “lightning strike” in my life as a poet, I have to admit that, in many cases, the inspiration for these videos often did come to me in a flash. This most recent article series on fly-fishing and mRNA (video forthcoming!) was no exception.

Though fishing — fly fishing or otherwise — is a hobby far outside my wheelhouse, a chance encounter with a fly-fisherman ultimately turned into a lengthy exploration on how fly fishing could be an apt metaphor for some of the mRNA industry’s current R&D efforts. As I spelled out at length in part one of this two-part article, fly-fishing is the perfect blend of art and science. Outside of the aesthetic considerations that go into making and choosing a fly (i.e., color, materials, shape, etc.), fly choice and casting technique are often informed by the careful study of the environment and a fish’s behavior, diet, and eating habits.

There were a lot of directions in which I could’ve taken the fly-fishing/mRNA metaphor — with successful delivery of the fly (i.e., the mRNA drug product) to different fish (i.e., cell types) being high up there. But as I thought about it, a much more nuanced discussion emerged. Rather than focusing on expanding our product’s reach beyond the liver, I was drawn instead to the careful balance a fly-fisherman must achieve between attraction and deception — a duality that is encompassed nicely in the industry’s ongoing considerations about mRNA vaccine and therapeutics immunogenicity. With fly-fishing, we are seemingly walking a tightrope between nurture and nature; the (quite artful) presentation of the fly is everything, but — to a fish — our artifice must also seem like nothing out of the ordinary. In many ways, we in the mRNA space are striving to maintain just as difficult a balance when it comes to the immunogenicity of our therapies.  

It goes without saying that immunology and mRNA immunogenicity are immensely complex topics that could barely be covered exhaustively in an entire book — let alone in a 2000-word article. However, here in part two of this series, I will attempt to summarize some of the basic immunology-centric underpinnings of our mRNA development efforts and the enduring immunogenicity questions and concerns shaping and re-shaping these efforts today.  

Understanding The Immune System — In Brief

The oxymoronic complexity of fly-fishing is beautifully mirrored by the fact that mRNA is being used in vaccines and therapeutics — both of which have different relationships with the immune system. With a prophylactic mRNA vaccine, we need to stimulate the immune system to train the body to recognize and mount an immune response against a certain antigen (ideally, without toxicity or reactogenicity/side effects following administration). For a protein replacement therapeutic, however, our goal is the complete opposite; we ultimately need to avoid detection by the immune system to ensure our therapy is uninhibited in the long-term.

While such black-and-white distinctions are helpful in a pinch, our understanding of how the immune system responds to an mRNA product is quite nuanced. After all, the immune system is broken into two equally important and complex parts: the innate immune system and the adaptive immune system. Though both systems fight pathogens to keep us healthy and safe, they go about it in two distinct ways. The innate immune system is the first line of defense against damage/injury and infection. Scavenger cells, acids, enzymes, mucus, and natural killer cells are all actors in destroying common danger associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs).

I like to characterize the adaptive immune system, on the other hand, as a more studious, discerning, and experienced fly-fisherman. A fly-fisherman may not catch a fish immediately, but a large part of that process is taking the time upfront to identify and experiment on the best ways to catch a specific fish. The same can be said for the adaptive immune system; though it may identify a pathogen relatively quickly, the clonal expansion process that’s necessary to thoroughly fight/defeat that pathogen takes a bit longer. Thanks to the overall thoughtfulness of its approach, the adaptive immune system — the agents of which are T cells and B cells — has the potential to remember an invader and fight it off much more quickly in the future.

Now, if you’ve sat through any presentations or read anything about mRNA, you’ll likely already know that in vivo-administered synthetic mRNA is inherently recognized as an intruder (a DAMP or PAMP) by the innate immune system. There are a variety of different immune response pathways that wake up when they detect single stranded (i.e., mRNA) and double stranded RNA and/or in vitro transcribed guide RNA (gRNAs). The innate immune system’s recognition and response to the RNA drug product can then go on to incite an adaptive immune response — the strength/durability of which will vary depending on how our unique drug products and the innate immune system speak to each other. Getting to the root of these individual product- and immune-/cell-specific response mechanisms is an inherently difficult task — one that currently raises more questions than answers. 

In turn, one of the biggest challenges we face as an industry is to ensure that our mRNA therapeutics and vaccines make a good “first impression” on the immune system. Otherwise, the important message we’re sending into the body via an mRNA vaccine or therapeutic will be akin to a message in a bottle that is obliterated on a rocky shore and remains unread.

Presentation Matters: Making The “Right” Impression On The Immune System

If my inquiries into fly-fishing have taught me anything, it’s that presentation of the fly into the fish’s environment is absolutely everything and that there are many controllable and uncontrollable factors that can affect that presentation. As I explained in part 1, the time of year and location; the type of insect you’re imitating; and the feeding habits of the fish you’re striving to catch are just some of the considerations a fisherman faces in approaching a fish. What works in one environment and for one type of fish might not work in a different region for a different species.

Likewise, in the context of mRNA, making a good “first impression” on the immune system will mean slightly different things depending on whether we’re working on a therapeutic or a prophylactic vaccine. On the one hand, our mRNA therapeutics must completely bore the innate immune system, in turn keeping the adaptive immune system at bay. However, our hopes and dreams for a prophylactic vaccine are a bit more complicated. Broadly speaking, it’s important that our mRNA vaccines, like a therapeutic, don’t “come in too hot,” as over-stimulation of innate immune response pathways can lead to translation and safety issues and a lack of efficacy. But innate immune system recognition is ultimately necessary for our vaccines to create a durable immune response. As such, our vaccine products must flirt just enough with the innate immune system to elicit a controlled — but still powerful and protective — adaptive immune response to the encoded antigen. As if that isn’t hard enough, we/our product must manage this feat without causing extreme side effects.

There have been several important steps the industry has taken or is continuing to perfect to reduce or avoid harmful innate immune detection of our (linear) mRNA therapeutics/vaccines. These include but certainly aren’t limited to:

  • “Decorating” our mRNA with a complete cap, as opposed to an incomplete cap;
  • Intensifying or exploring novel purification processes to eliminate immune-stimulating impurities, including double-stranded RNA;
  • Optimizing plasmid DNA sequences;
  • Reengineering certain IVT raw materials (i.e., T7 polymerase, in particular) to produce fewer immune-stimulating impurities;
  • Chemically modifying mRNA’s uridine base (i.e., replacing uridine with pseudouridine); and,
  • Exploring/characterizing additional modifications. For those particularly interested in a deeper understanding of the current “to modify or not to modify” debate in the mRNA R&D space, I’d refer you to this journal article and this [subscription-only] Nature article.

Of course, the physical presentation and chemical properties of the mRNA’s chosen delivery vehicle (i.e., the lipid nanoparticle [LNP]) will also play a role in a therapy’s immunogenicity profile (as well as dictate the impact of nucleoside modifications!) Just as the physical characteristics of a fisherman’s flies can be implicated in hooking a fish, an LNP’s components (i.e., the ionizable lipid, PEG, & cholesterol), and the overall size, charge, polydispersity, and molar ratio of the LNP all impact a vaccine’s or therapeutic’s relationship with the immune system. In fact, as this publication argues, the LNP should not just be thought of solely as a delivery vehicle for mRNA but as a necessary adjuvant enabling a vaccine product to create a much stronger immune response. Given LNPs’ inherent stimulation of the immune system, however, there are also ongoing experiments (including this one) to reduce the innate immune system’s recognition of LNPs when being used to deliver mRNA therapeutics.

“Tying” It All Together: The Curiosity Behind Fly-Fishing & mRNA Immunogenicity

Naturally, much of the research on mRNA immunogenicity is limited to our experiences with mRNA vaccine products, thanks to the pandemic and the nascency of mRNA therapeutic clinical development. Regardless of how much has been learned in the past three years, there remains much room for growth in our understanding of how the innate immune pathway informs adaptive immunity. In particular, the jury is still out on 1.,) which innate immune pathways are most influential in achieving adaptive immunity, 2.) which innate immune pathways are most responsible for reactogenicity, and 3.,) which combination of pathway responses will help an mRNA vaccine reach each disease-specificsweet spot of immune activation.”

Though recent research from Pfizer/BioNTech has given us more insight into how the innate immune system informed the adaptive immune response for the companies’ COVID vaccine, it’s still too early to make sweeping assertions across COVID vaccines. This indicates the need to gain greater understanding of how our unique mRNA cargoes and LNPs — individually and collectively — activate the innate immune system, and, in turn, stimulate an adaptive immune response. Such inquiries will not only be essential for finding “the sweet spot of immune activation” for vaccines in different diseases, but also for helping companies improve the immuno-silencing properties of their mRNA therapeutic products.

I particularly appreciated this quote from Verbeke et. al., which beautifully summarizes the industry’s vastly different approaches to immunogenicity across therapeutic types and the importance of “the proper presentation.” As they describe: 

“A wide variety of future mRNA therapeutics may eventually become possible as we learn how to tailor mRNA and carrier molecules to preferentially activate the B cell response (most relevant to acute infections) vs. the T cell response (e.g., for chronic infections and cancer) to become completely immune silent (e.g., for gene therapy), and even to induce immunological tolerance to treat autoimmune diseases.”

That ceaseless curiosity and experimentation will be the driving force behind accomplishing the future outlined above is a completely “duh-worthy” statement, I know. But when I began this project tying together fly-fishing and mRNA, it didn’t quite occur to me that this metaphor would be a two-way street. I didn’t anticipate that, with every research article I sifted through, I’d find the mRNA industry’s own biological curiosities reflected in a common past time like fly-fishing. In fact, I daresay the below quote from Mark Kurlansky’s book, The Unreasonable Virtue of Fly-Fishing, holds true for those of us striving to answer the many biological and immunology-related questions outlined in this article. As Kurlansky writes:

“Fly-fishing is for the curious. Every river is different; every beat on every river is different; every river has its own fish, shape, and bottom; and every river sings its own song…. Every time you fish, you learn something new. There is no end to it. The fisher who thinks he or she knows everything knows nothing.”

Miss part 1? Check it out here!

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Works Cited/Consulted

This paper took weeks of additional reading, research, and collaboration.

Special thanks go out to Ryan Murray, senior consultant at ValSource and fly-fisherman extraordinaire, for introducing me to the whacky — but beautiful — world of fly-fishing and for planting the seed for this project.  

Likewise, as immunology was (sadly) not a course requirement to receive my MFA in poetry, I’d also like to sincerely thank Natalie Fekete, manager of science and industry affairs for Alliance for Regenerative Medicines, for lending this article her time and immunologist eyes & brain to ensure its accuracy. 

Other works referenced and/or consulted in the creation of this article include: