4 RNA Engineering Trends To Watch

By Life Science Connect Editorial Staff

Cell & Gene Live recently hosted an engaging discussion on the major trends in next-gen RNA engineering, featuring experts Sam Deutsch, EVP research and early development at Nutcracker Therapeutics, and Nathaniel Wang, CEO and cofounder at Replicate Bioscience. Moderated by Erin Harris, editor of Cell & Gene, the conversation delved into the dynamic realm of RNA therapies, shedding light on tissue specificity, potency, and the evolving landscape of RNA engineering.

Tissue Specificity

The discussion kicked off with the intricacies of accurately targeting tissues to minimize off-target effects. Wang challenged common misconceptions, stating, “When people hear tissue specificity, they hear something like kidney specificity, and they just assume that all of the material goes into the kidney as soon as you inject it.”

Wang underscored the complexity of tissue-specific approaches, emphasizing that where the material ends up versus where it’s expressed can be significantly different. He noted, “The safety and the biodistribution of these tissue-specific approaches are a lot more complex than I think it sounds on the surface.”

Both Wang and Deutsch mentioned the benefits of changing the charge of nanoparticles to ensure distribution into the appropriate cells and avoid being filtered out by the liver. In addition, Duetsch noted that Nutcracker has been targeting through avidity rather than high-affinity interactions, leveraging a unique library of chimeric lipids to explore precision targeting. “We can conjugate a large number of different and diverse chemistries onto our scaffolds. And this allows us to explore the target in question by adding these low-affinity moieties,” he said.

Potency And Durability

The panel pivoted to improving the quantity, quality, and longevity of expressed proteins for expanded RNA applications. Wang brought up circular RNA, which is offering a marginal increase in expression time. “You end up having expression actively for about four to seven days, about a one- to three-day gain,” he said.

Self-replicating or self-amplifying RNAs, according to Wang, have much more potential on this front. “You can get a wide distribution looking at these technologies on the total amount of protein that can be produced on them and the durability for them,” he said. He envisions broader applications in personalized medicine and more cost-effective off-the-shelf approaches.

In terms of potency and durability of RNA, Deutsch emphasized the importance of considering the specific purpose, target profile, and competitive landscape when designing RNA therapeutics. He distinguished between acute settings, like late-stage oncology, requiring shorter treatment durations, and chronic indications, such as rare diseases, necessitating prolonged exposure.

Deutsch delved into Nutcracker Therapeutics’ proactive strategies, focusing on two key approaches: first, the development of a proprietary circular RNA platform, and second, precision optimization through intentional secondary and tertiary structure evolution. In particular, he noted the potential for super folding knots. “This new concept of RNA knots or super folding is of very high interest to us as we’re seeing that these are efficient and quite powerful ways of modulating the durability of the RNA,” he said. “So, we're seeing that even with linear RNAs, we can see pretty significant shifts in the duration of expression.”

Highlighting the nuances of potency and pharmacokinetics (PK), Deutsch articulated the significance of not just the duration of expression but the rate at which expression levels drop. “Because ultimately what we're most interested in is the total exposure of the drug,” he said. “So that area under the curve, we see that in many real-life applications, actually you want to treat the opposite. You want to have a very slow wrap rate of expression.”

Wang provided a tangible scale for the advancements in potency. He clarified that newer technologies enable dosing at significantly lower levels, potentially transforming manufacturing and deployment capabilities. “The preclinical or the animal models show that you can start to dose these things anywhere from 1,000th to 1 millionth of the dose that people have currently been using,” he said. To illustrate the significance of this, he added, “If you look at the COVID-19 vaccines that many of us got, if you went at 1,000th of the dose, then a one-liter bioreactor […] could basically produce 50 million doses of a vaccine candidate, which obviously would’ve been transformational.”

Challenges In Deployment

The panel examined some of the challenges in scaling up production for novel RNA engineering technologies. Deutsch emphasized the iterative nature of process development, highlighting the importance of minimizing run-to-run variation through the company’s microfluidic-based manufacturing platform (RNMU). “That's what we're seeing at Nutcracker, because every time you change some aspect of manufacturing or you change the format, we tend to see some unknowns that become manifested, such as, for example, side product formation that you weren’t necessarily anticipating,” he said.

The modularity of RNA manufacturing is both an asset and a challenge, according to Wang. For example, the process of mass producing an RNA vaccine for RSV, EBV, and the flu are essentially the same. But this can lull many into false confidence, particularly for products with a significantly greater number of base pairs. “That poses a bigger engineering challenge as you're doing production than I think people appreciate,” he said, adding, “You need to take the time for that process development to optimize each of the parameters and the input to it, and the field is just catching up in that sense.”

To this end, Wang stressed the necessity of strategic relationships with CDMOs, particularly when integrating new technologies like microfluidics into manufacturing processes. “Forming those relationships early on so that you can really build this end-to-end integration as you’re creating new manufacturing processes is pretty critical to the longer term,” he explained.

Moving Beyond “Plug-and-Play”

The panel shifted to dispelling the notion that RNA technologies are “plug-and-play.” Wang admitted that he once promoted the idea, but he now considers the characterization as inaccurate. He clarified that the different components of the drug product — specifically, the RNA version, the gene being encoded, and the delivery system (lipid or polymer) — are independent variables. Changing just one of these variables can significantly impact the final product.

Wang acknowledged the attraction of RNA in accelerating drug development remains strong nonetheless, as evident during the COVID-19 pandemic. “But as we start scaling the complexity of the targets that we're going after, I think what becomes critical is being honest about the fact that these technologies are not plug-and-play, so you build the end-to-end infrastructure so you can rapidly find a way to optimize the gene-of-interest or the proteins for whatever the specific RNA modality that you're using,” he said.

When it comes to specification and specialization, Deutsch stated that the engineering required depends on the mechanism of action and the specific therapeutic goals. He highlighted the importance of synergy and interplay between different components of mRNA therapeutics. While he wouldn’t call RNA a plug-and-play technology, Duetsch insisted it does have a plug-and-play component.

“When we’re manufacturing this RNA through cell-free systems, some aspects of these, like, for example, the yield, the purity profiles, and the formulation stability behaviors, can be quite reproducible,” he said. “But that's assuming that we stick to exactly the same excipients, exactly the same type of formulations, and exactly the same RNA for modalities.”

However, Duetsch pointed to the importance of an integrated platform, particularly when tweaking variables, as it accelerates process development and troubleshoots efficiently compared to working with CDMOs.

He provides an example related to subvisible particles in their product for cervical dysplasia/cervical cancer. Facing issues in late-stage development, they made changes to the excipients and experimented with microfluidic chips, quickly observing different patterns of subvisible particles. This rapid in-house experimentation allowed them to meet USP release guidelines within a month. Duetsch emphasized the value of having control over variables, understanding their importance, and utilizing a reproducible and software-controlled platform, which proved highly beneficial in this case.