End to End with Circular RNA
RNA therapeutics are programmable medicines: their sequence encodes their biological function. Yet RNA is not purely a store of digital information. It is also a physical molecule that needs to be packaged and delivered to patients. By circularizing RNA, we can stabilize the physical molecule while preserving the therapeutic information it encodes. Ginkgo offers a range of assets and capabilities to support our partners in developing RNA therapeutics for the circular format.
Correction: At 3:30 I mention an SDS-PAGE gel. In fact, the gel is PAGE without SDS. I’ve run enough SDS-PAGE gels in my life that my brain just adds the “SDS” automatically. Apologies. - Jake
Transcript
Circular RNA is a particularly elegant piece of biotechnology. Somebody looked at the RNA molecule and said: "Love what you're doing here, except for the beginning and the end. What if we took those ends and just …didn't."
I love mRNA as a therapeutic modality. It's an incredibly versatile molecule that lives at the center of the central dogma. It can code for almost any protein, including engineered proteins. It can include regulatory elements to express that protein at a certain level, under certain conditions, or in certain cell types. The possibilities really are endless.
But the molecule itself is not endless. Anyone who has ever worked with RNA in the lab will know what I'm talking about. RNA is very fussy - it falls apart just from breathing on it. That instability is a big headache for manufacturers, who need to produce RNA at scale. And it might be the biggest challenge for developing RNA as a therapeutic - protecting that activity in patients.
RNA decay is mediated mainly through three enzyme activities. Endonucleases that cut an RNA strand in the middle and exonucleases that chew from either the 5' or the 3' end. Cells normally control the rate of RNA degradation by protecting the ends with a 5' cap and a 3' poly-A tail.RN
Circular RNA is a synthetic strategy for the same problem. No 5' end, no 3' end, no exonuclease activity. Here's some data to make that point. These cells were transfected with equal amounts of RNA expressing GFP, either conventional mRNA or circular. The GFP expression is similar on day 1, but only the circRNA keeps expressing on day 5.
Here's a quantitative measurement of the same idea using a high-turnover luciferase.The background signal is down here. The linear RNA construct starts strong but decays by day 3. The circular RNA starts off a little lower, but it is still going after 5 days. And this was true for three different sequence variations of the circRNA. And it was true for two different cell lines.
How do we make circular RNA? The molecular details vary but the general concept is simple. We start with linear mRNA and we add specific sequence elements to either end. These elements might be self-splicing, taking advantage of the power of RNA to act as its own enzyme. Or they might be recognized by an external enzyme. This sequence-specific activity takes the ends of the RNA, connects them together and cuts off the excess.
Ginkgo offers different ways to circle up your RNA. Depending on your RNA sequence and your application, one method might be preferred. We also find that some partners want to use novel elements that haven't been previously described. I'm not a lawyer and I can't make any promises about IP or patentability in a general presentation like this one. But of course novelty can be an important part of your IP strategy and we can help with that.
Here's an example of how we do discovery for RNA circularization elements. This is a scatter plot of some RNA sequences that are known to circularize. This plot is giving a sense of both the sequence and the structure of each RNA element. These red dots represent known and described circularizing introns, these blue dots are ones that we found in our in-house sequence databases.
We can pick sequences from a discovery effort like this and confirm that they really do circularize. Here's a good old fashioned PAGE gel showing the band that corresponds to the circular shape. And some basic sequence analysis shows that these are pretty distinct from what was known, in this case only about 50% sequence identity.
Working with RNA in the circular format usually requires changes in the design of your sequence. In particular, the control elements that you use to express a protein are different for circular RNA. For linear RNA, that 5' cap helps to guide the RNA to the ribosome and initiate translation. For circular RNA, we use Internal Ribosome Entry Sites that don't require the cap.
Gingko partners get access to our library of structural and non-structural elements that can vary expression strength across cell types. RNA design is tricky. You just don't have as much regulatory control as you would with, for example, a DNA construct. So starting from data cuts down on development time.
Once you've got a design that you like, you'll want to produce it for preclinical studies. In other words, with quantity and quality. Conventional mRNA production methods don't translate directly to the circular molecule, but we've put together a circRNA-specific process. Here I'm showing the capillary electrophoresis traces for some circRNA made with in-vitro transcription. We get good separation of the complete circle from the linear precursors.
Our RNA production setup works in either high throughput or high yield. We can generate lots of different RNA variants at the microgram scale - good for pooled or comparison testing. We can produce single variants at the milligram scale. The latest numbers I'm hearing from the foundry are that we can hit 10-20% recovery and up to 90% purity, depending on the details of the molecule.
And that's the state-of-the-art for circRNA at Ginkgo. I think a lot of next-gen RNA therapeutics are going to adopt this format. It just seems like such an elegant way to stabilize RNA. As usual in therapeutics, it isn't a silver bullet. Sometimes linear mRNA is going to be the right answer. Sometimes the biggest challenges are not in the RNA format but in other technologies for packaging or delivering that RNA. At Ginkgo we can do all those things for our partners, and I think we're likely to see RNA for therapeutics and vaccines come in all different shapes and sizes.
That's the versatility of RNA as a modality. There are so many different biological functions that are mediated by RNA and so many possibilities that open up when you can dial in that RNA sequence just right. Circular RNA, and the other RNA services available at Ginkgo, are about getting the most out of RNA as a programmable medicine and then delivery in a format that will do the most good for patients.