Beyond mRNA: how advances in analytical techniques are enabling a revolution in the RNA drug landscape

Currently, there are 3 main types of in vitro transcribed (IVT) RNA drugs, encapsulated in lipid nanoparticles (LNPs). Two of these—conventional messenger RNA (mRNA) and base-modified mRNA (bmRNA), which incorporates chemically modified nucleotides—are non-replicating. The third type is self-replicating RNA (srRNA), which is based on an engineered viral genome but devoid of viral structural protein genes. Its self-replicating ability makes srRNA a promising tool for new therapeutic drugs.

The ability of a drug to copy itself after administration enables lower dosing and expands the range of potential indications. However, srRNAs are often much larger in size (9,000-16,000 bases) than other RNA types, and traditional biophysical analytical techniques have struggled with these large molecules.

We are fortunate to be able to post an expert session with Andrew Geall, PhD, Chief Development Officer and Co-Founder at Replicate Bioscience where he answers questions about how to achieve improved characterization of srRNAs using the newest advances in capillary electrophoresis (CE). The questions and answers are below.

A general question about terminology: is there any difference between self-amplifying RNA and self-replicating RNA?

Great question. I published for several years on self-amplifying RNA, and we are hoping that people will appreciate the distinction. We are characterizing the RNAs that are based on the V vector systems as “first-generation” systems that we refer to as self-amplifying RNA (saRNA) or sometimes self-amplifying mRNA (SAM). We have a variety of different species of alphaviruses that we’re using and have implemented synthetic optimization. We’re calling these “second-generation” vectors and designating them as self-replicating RNA (srRNA). That is the way we distinguish these terminologies. We believe there are advantages to this second generation that we’ll validate clinically going forward.

Have you seen that terminology being adopted generally?

Not yet. Certainly, we see both terms being used. Historically, the field has been dominated for many years by the viral replicon particle approach. And prior to the synthetic approaches that we have now, “self-amplifying,” “self-replicating” and “the replicon” were being used. We prefer to use the term “self-replicating RNA” for the next-generation approach—a “replica,” we’re being deliberate and saying this is different. There is a paradigm shift in terms of potency, and we think that will have huge therapeutic benefit in the future.

What is the viral origin of your amplicon?

We have a range of self-replicating RNAs. Most of them are novel and not derived from the prototypical alphavirus vector: Venezuelan equine encephalitis virus (VEEV). We have some candidates that are non-alphavirus-derived, but the majority are from different alphavirus subtypes with different non-structural proteins. We found various activities, which is empirical information at the moment. For a particular project, we’ll run a vector library with the desired protein cassette and figure out which vector is best.

We’ve also implemented synthetic optimization to help in manufacturing and to maintain potency. Overall, we’re seeing about a 100- to 1,000-fold improvement over the first-generation self-amplifying systems. With that, we can dose much lower than before.

Can you comment on the requirements for capping and poly-A tails for self-replicating RNA compared to mRNA? Is there a difference of acceptable heterogeneity for these critical quality attributes compared to mRNA?

Like for any mRNA, there is a need to have a capping structure to enable bioactivity. It’s therefore critical to have a 5-prime cap for self-replicating RNA as well. To achieve bioactivity, there might be a little bit more wiggle room with regards to the percentage of G1 cap, because 1 active construct delivered to a cell will copy itself into approximately 20,000 copies.

For the poly-A tail, it’s a similar answer: The poly-A tail imparts stability to the construct, and we have absolutely optimized the length of the poly-A tail of our products to suit the potency activities. We’re following a similar path that others have taken with shorter linear RNA, and we are characterizing poly-A tail and 5’caps to understand our product and be prepared for regulatory requests.

Can you run the RNA integrity assay and the poly-A tail assessment on the same capillary electrophoresis instrument? What adjustments would you need to do?

Yes, you can run both assays on the same CE instrument. Typically, what you’ll do if you’re characterizing the poly-A tail or the 5-prime end is enzymatically clip off that section. You’ll end up with a fragment that is much shorter in length than the mRNA or srRNA. Compared to the integrity assay, you’re therefore going to need a different gel setup for achieving optimal resolution. For further information, I suggest reaching out to the technical team at SCIEX. They can give recommendations to get you going. I know you can get single base resolution up to about 100 nucleotides using SCIEX systems. So, it’s very powerful for assessing the ends of IVT RNA as well. It’s very flexible.

And what we’re seeing from the regulators now is that they want to know more and more. They’re no longer accepting an agarose gel. They want you to provide more details and characterize what is at the 5-prime end, the 3-prime end, and what the heterogeneity of your product is. So, there’s lots of characterization to come.

Is your gel matrix proprietary or a catalog item from SCIEX?

They are all catalog items from SCIEX. We will be publishing our methods later in the year in 2023. We want to keep the exact method details proprietary for now, but I can say that we collaborated with the scientific team at SCIEX to enable this. They provided us with information, and we then optimized the systems. There’s nothing here that isn’t available through SCIEX.

What is the acceptable percentage integrity of self-replicating RNA?

The truth is we don’t know yet. We are at phase 1 and set specifications that are very wide. For the linear RNA for the COVID vaccines from Pfizer/BioNTech and Moderna, we were seeing specifications set at >45%—but then the regulators might ask what the other 55% is.

Not all self-replicating RNA is going to be full length and homogenous. And that’s not the first time that’s occurred for a product going forward. In polymer systems, there’s heterogeneity like in many other biological systems. I think we just need to keep improving the analytical techniques so we can characterize that heterogeneity within the system and ensure consistency batch to batch as we scale up. CE can certainly help us achieve that understanding.

Do you foresee the CE method being able to replace some of the in vitro potency assays? Why or why not?

We’re certainly hoping that we’re able to. The main benefit of the in vitro potency assay is it answers the questions: Does it express the protein? Does it replicate? I don’t think we’re going to be able to answer all questions with CE, but we can certainly start to drill down into the details of what’s in the material. With the ability to clip off the ends and characterize them with CE, in addition to the integrity of the srRNA product, we can provide a lot of information for regulators.

Will we completely move away from the in vitro system? Probably not. But the FDA wants to know what is in that non-full-length material, and I think our CE assays are going to help us in the development cycle as we move forward to be able to confidently say it is consistently the same.

How does the stability of mRNA compare at self-replicating RNA? Does it still require deep-freeze storage?

Great question. There was work coming out of Stanford University 3–4 years ago looking at the stability profile of linear RNA of approximately 5 kb compared to the longer self-replicating, self-amplifying systems. The scientists showed about a 3-fold decrease in stability with the longer constructs. The longer RNAs have historically been difficult to manufacture and the rate of hydrolysis is significantly higher and faster. However, a lot is known about how RNA hydrolyzes: it’s time- and temperature-dependent. So, you can modulate both those parameters during your manufacturing process. And if you have the assays that tell you when things are going wrong, you can adapt the process appropriately. Currently, most self-amplifying, self-replicating systems are stored at -80°C.

We have plenty of data suggesting that formulated RNA can be stored at +4°C to +8°C for several months with no degradation, that it can go through a lyophilization process without impact on the stability. We’re exploring higher temperatures as well. I do not think storage temperature is going to be a limitation. We’re already starting to see temperature-stable RNA vaccines making their way through development.

Could you elaborate on analyzing RNA material that’s 16,000 bases long with a ladder of 9,000 bases? What’s the strategy to convince the regulatory bodies that there is sufficient resolution for the 16 kb srRNA sample to identify impurities?

All we can do right now is correlate the migration time of the analyte with that of the 9 kb single-stranded ladder material. We would love to have a molecular weight ladder that is much longer, but there is nothing available at this time. We’re working with vendors to generate reference material that the field can use.

While still in earlier stages, our analytical strategy of using CE combined with in vitro potency assays has been performing as expected and informing productive initial discussions with the FDA. During the development cycle, we’ll work on improvements. Nobody has ever been to the regulators with a construct that long. The analytics are exceptionally challenging. Right now, we want to show the field that it’s possible to manufacture and analyze a 16 kb construct. This was inconceivable a few years ago. We’re pioneering in the space.

Is there a capacity limit for lipid nanoparticles to encapsulate self-replicating RNA?

No, I don’t think so. Historically, I’ve worked on encapsulating self-amplifying RNA at Novartis. Now, at Replicate, we observed little difference in encapsulation between a 9 kb and a 16 kb RNA in terms of the efficiency of packaging, the robustness of the process. The srRNAs are maybe 5–10 nanometers bigger than mRNA, but they’re pretty small anyway— about 70–80 nanometers—so I doubt that encapsulation is a limitation. I think we would be able to go bigger.

As we go bigger, we’ll run into other problems with those particles. The lipid nanoparticles ultimately have to be processed through a tangential flow filtration through a 0.2 µm filter. With an increase in size, the particles may start to loosen up and we may start to see processing problems, but right now, we’re not seeing any in the 9–16 kb size range.

The encapsulation efficiencies we achieve are very high, up in the +95% range, and we’re not seeing any difference between those 2 cargo sizes.

When would you estimate we might see this type of technology making its way into COVID-19 vaccines or other commonly used vaccines?

There are already first-generation saRNAs in COVID vaccines. The Imperial College London pushed a candidate forward, which didn’t gain traction. Arcturus Therapeutics in San Diego just licensed a product in Japan as a COVID booster vaccine. So, they’re already out there and being used. CSL is starting to look at the first-generation saRNA technology as well. We’re starting to see the implementation of it in seasonal flu vaccines.

At Replicate, we’re expecting to have clinical data for our second-generation srRNA towards the end of this year in 2023. If we’re able to dose 1.0 µg or 0.1 µg and get a robust and durable immune response in naive patient populations, that would be significant. The related production capacity would be meaningful. We’re looking to partner with people in the infectious disease space. We’ll see—we’re going to be data-driven.

What about the viscosity of self-replicating RNA solutions compared to mRNA solutions?

We’re at very low doses of RNA, therefore viscosity isn’t a problem. Yes, certainly as the concentrations go up, material becomes quite viscous, but we’re working at very low dilutions and we’re not seeing any viscosity issues.

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