Self-amplifying RNA may reduce side effects associated with RNA vaccines

Back in pre-COVID-19 pandemic times, most scientists had never heard of mRNA vaccines. Behind the scenes, however, researchers had been tinkering with and improving the technology for decades. While mRNA vaccines were out of the limelight, RNA scientists like Anna Blakney, a bioengineer and RNA therapeutics researcher at the University of British Columbia, saw their potential.

“The first half of my talks used to be just telling people what mRNA vaccines were and trying to convince them that they could actually work,” said Blakney. “There were only a few clinical trials that had been done with RNA. Nobody had ever done a phase three clinical trial, so now it's incredible that we have two approved mRNA vaccines.”

     

The spotlight on the mRNA vaccines for SARS-CoV-2 ignited the RNA field with exciting new questions, but it also prompted apprehension among some members of the public about how exactly these vaccines work. In the fall of 2020, Blakney joined other vaccine scientists on TikTok to present short videos explaining the science behind the new mRNA vaccines and their effectiveness against the SARS-CoV-2 virus. Her TikTok account has since gained more than 272,000 followers.

When not explaining RNA vaccine science on social media, Blakney designs systems to deliver RNA more efficiently into cells and develops new models in which to test RNA vaccines and therapeutics. She also investigates the potential of a virally derived system called self-amplifying RNA (saRNA) to decrease the dose — and thus the side effects — of future RNA vaccines and therapies.

What is self-amplifying RNA?

Self-amplifying RNA is a type of messenger RNA. It has a lot of features similar to mRNA: a five prime untranslated region, a cap on the five-prime end, and a poly A tail on the three-prime end. The middle part is where it really varies. Here, the saRNA encodes a replicase derived from a virus, which is usually an alpha virus. Once the cell translates this replicase, it's able to go back to that original strand of RNA and make copies of it.

What are the advantages of using saRNA to make RNA vaccines or therapeutics?

For mRNA vaccines, the initial dose of RNA is the only RNA that’s there. But once saRNA gets into a cell, there is an amplification of that RNA. We can get much higher protein expression from saRNA than from mRNA, which is advantageous for many applications. It also means that we can give people a much lower dose of RNA. We know that the side effects of mRNA vaccines are directly proportional to the dose of RNA, so minimizing that is really a priority in the field.

saRNA also has a very different protein expression profile than mRNA. mRNA has a protein expression duration of about three to five days in vivo, but saRNA continues for about 30 to 60 days depending on the protein that we express. That's obviously a hugely different window of expression, so we think that saRNA will be advantageous for protein replacement therapy applications. We're also looking at encoding monoclonal antibodies in saRNA to express those for a much longer duration than is currently possible.

What are some of the limitations of saRNA?

Everything about it is more challenging than mRNA because it's so much bigger. Because saRNA encodes lots of different proteins for the replicase, for example, it's usually around 10,000 nucleotides, while mRNA is around 1000 nucleotides. Because it’s a bigger construct, there are many more opportunities for degradation. It's also harder to produce the longer RNA because we get a lot of shorter transcripts.

Another challenge is the immunogenicity of saRNA. One of the major discoveries with mRNA vaccines was that swapping the nucleotide uridine for the non-canonical nucleotide pseudouridine reduced the immunogenicity of these RNAs because it changed the secondary structure of the RNA, preventing cells from detecting the RNA as foreign. For saRNA, the secondary structure is important because it encodes a lot of elements that are required for its replication. Additionally, once the saRNA gets into the cell and replicates, it incorporates normal nucleotides, not the modified nucleotides. Because of that, saRNA is inherently more immunogenic than a modified mRNA.

How can scientists reduce the immunogenicity of saRNA?

Our saRNA system is a virally derived system, so we can actually use more tools from the viral toolkit to decrease the immunogenicity of saRNA. As soon as the cells detect the saRNA, a type one interferon response, which is part of the innate immune response, gets triggered. This leads to degradation of the RNA and inhibition of its translation. To prevent this, viruses with saRNA have evolved interferon inhibiting proteins. For one of my lab’s recent papers, we studied what would happen if we encoded those interferon inhibiting proteins in our saRNA (1). Would we see the same interferon inhibition? When we included two of those proteins in particular, we saw a tenfold increase in protein expression. It's a synthetic biology approach looking at how nature already gets around this and how we can do the same.

How do you plan to further improve your saRNA technology?

We're still looking at different ways to inhibit that interferon response, whether that's with proteins encoded in the RNA or adding different things to the formulation. Also, everybody working on saRNA has used essentially the same three viral replicases, but there are so many different viruses out there and many that infect and replicate in human tissues. We're really interested in doing a high-throughput barcoded screen of those different saRNAs so that we can see if there are even better designs than the three in use so far. The main limitation to this approach is that synthesizing these huge saRNA constructs is expensive, but in recent years, the cost of large-scale syntheses and sequencing has come down quite a bit. Now these projects to screen around 1000 different constructs are actually feasible.

What excites you most about developing new RNA therapies and vaccines?

It's a really exciting time to be in the field because there are so many big questions right now. One of the main goals is minimizing the dose of RNA given to people. Researchers are looking at saRNA and circular RNA. I think that RNA delivery also plays a huge part in this. There are estimates now that only 1-2% of the administered RNA is actually delivered into the cell. If that could change, that would really change the field.

You do a lot of science communication about RNA vaccines on TikTok. What motivated you to do that?

Before October 2020, I had never been on TikTok, so I didn't really know how to use it or know what it was all about. I started out on TikTok when I was invited to join Team Halo, which is a collaboration between the United Nations Verified Initiative and the Vaccine Confidence Project. The goal of Team Halo was to get scientists and clinicians who are working on COVID-19 on TikTok so that they could educate the general public about how we make vaccines, how we test them, and what it means to work in a lab and make these different vaccines.

What surprised you most about communicating vaccine science on TikTok?

I was surprised to find such an appetite for science and learning on TikTok. I've had so many good interactions with people who have questions and don't know who to ask. How many people in the world right now know an RNA vaccine scientist? It's really cool to have a conversation and say, “Here's where I got the data from.” I’m very transparent about the fact that these vaccines aren't perfect, but here’s the data that is convincing about what the vaccines do and why it's important to get them.

How has your experience on TikTok influenced your thoughts on how researchers communicate science?

My personal view is that part of the reason there was so much vaccine hesitancy during the pandemic was that the first two vaccines that were approved were RNA vaccines. Nobody had heard of an RNA vaccine, and prior to January 2020, very few scientists had heard about RNA vaccines either. Of course people were skeptical about a vaccine that they'd never heard of that was magically rolled out in the context of a pandemic. But that's such a shame because RNA vaccines have been studied since the early 1990s. There were decades and decades of work that nobody really knew was going on. That exemplifies a gap between the speed of research progress — which isn't like the speed of light — and how slowly we as scientists and educators communicate with the public. That gap is huge. 

Now with my science communication on TikTok, I’m trying to be less reactive and instead be more forward looking. I want to say, “Here's what we're doing now. Here are the advances that we're trying to make and how we're trying to make vaccines better.” That way, people can understand what's happening, as opposed to trying to catch up on practically 20 years of research.

TikTok is a great way to teach people about science because I can actually show them what I do in the lab, answer their questions, and have conversations about vaccines. It’s been a cool opportunity and such a privilege for me to be able to talk with people who are interested in vaccines and who want to learn more about them.

Reference

1. Blakney, A.K. et al. Innate Inhibiting Proteins Enhance Expression and Immunogenicity of Self-Amplifying RNA. Molecular Therapy  29, 1174-1185 (2021).