Lisa Caproni: Inside the DNA factory
Image Courtesy of Lisa Caproni
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DNA and RNA vaccines have huge potential to save lives, not just from infections but from cancer and other diseases too. But, like any other medicine, their successful roll out to as many people as need them around the world depends on being able to make enough, in high enough quality - known as GMP.
Based in west London, Touchlight started out as a biotech company trying to make DNA vaccines. They soon realised that the key bottleneck wasn’t the vaccines themselves, but that there wasn’t really a good way to make large amounts of high quality DNA, not just for vaccines but for other medical applications like gene therapy too. So, they set about trying to develop one.
The result was a technology called doggybone DNA. To find out more I caught up with Lisa Caproni, Director of Platforms at Touchlight. But before we got our teeth into the Doggybone, I asked her to explain how DNA for medical applications is usually made, and what the problem is with it.
Lisa: Cells are very good at making DNA, and in particular E. coli, they can harbour what are called plasmids. So from an E. coli perspective, evolutionarily wise, these plasmids always contain some sort of benefit to the organism. Things like making them resistant to antibiotics, which means that when they live in the soil, they can have a much better selective advantage over their competitors.
Lisa: But in terms of biotechnology, what we've done is take those plasmids and make them fit our purpose, but they're always still manufactured inside a bacterial cell. So what that really means in an industrial setup is that you are growing vats of E. coli.
Lisa: And then the E. coli, of course, have their own genome, they have all of their own proteins and their cell walls, et cetera. You then have to purify that pDNA, that plasmid DNA, which obviously suits your therapeutic function from all of that bacterial soup. So that's the scale of that fermentation process - and then the complexity of that purification is really where a lot of the challenges for pDNA comes from.
Kat: One thing I know about bacteria is that they can be pretty nasty. They've got quite a lot of crap in them. I should imagine that if you want DNA for therapeutic purposes to inject into someone as a vaccine or to use as a gene therapy, it's gotta be pretty clean, and that can't be easy.
Lisa: It is. So E. coli, for example, have a bacterial cell wall, which contains endotoxin. It contains something called LPS, and we call it an endotoxin because in the context of delivering endotoxin, if you have a batch that isn't fully clean, that can cause real problems in the host organism. So in a patient, for example.
Lisa: The purification complexity from an E.coli, fermentation based, production of plasmid is complex - necessarily complex because you have to remove remove whole cell DNA, whole cell protein, all of that fermentation liquor. It's not actually as straightforward as you would imagine, and all the time you're doing that purification, you have to make sure that you don't damage the plasmid.
Lisa: Because if you damage the plasmid, you negatively impact functionality. So there's that fine balance all the way through that process of clearing everything else away, but maintaining the integrity of the final product. And that is where the complexity comes in.
Kat: So now let's come to the process that Touchlight is using this idea of doggybone DNA. Let's start with like, what is it? I am imagining something that looks like a dog bone, like a child might draw. What is it? What's its the structure?
Lisa: So the doggybone DNA. Yeah, it probably is a misnomer because it doesn't actually look like a bone. Even our early schematic drew it like a bone.
Lisa: It's not true, but what it is, it's a linear covalently closed piece of DNA and the covalently closed part is those ends. So if you understand what DNA is, being a double helix, you have two strands. In a linear molecule, normally those ends are free and they're open, so it's like two pieces of string and you twine them together.
Lisa: The doggybone DNA has just enough DNA to close off the ends. What those ends give you are stability, and they also are not available for exonuclease digestion. So that increased stability can help you in in intracellularly, when your DNA is functioning for whatever purpose you want it to function for.
Kat: So we've talked about how we make plasmids in E. coli bacteria. How do you make this doggybone DNA?
Lisa: In E. coli, you're amplifying the pDNA using the enzymes inside the E. coli. So that amplification is intracellular. We take enzymes and we do it essentially in a test tube. We create a very small amount of a circular template, and we use that template to amplify the material.
Lisa: We use this very specific enzyme called phi29 polymerase. It's highly processive. What that means is it can do really long DNA, but it's also strand displacing. What that means is that the phi29 amplifies, then rather than stop when it's reached the end of the circle, it displaces the newly synthesized strand, and then you make these long single stranded, then double stranded repeats of your original template.
Lisa: Sounds complex, but the video on the website really does help with this, I promise.
Kat: Certainly does.
Lisa: And that's the amplification part of the process. Then comes in the second main enzyme, and that's called protelomerase.
Lisa: So the protelomerase is really what creates these doggybone DNA units. It cuts through the double helix with a staggered six base pair overhang and closes the end.
Kat: So the way I'm kind of imagining this process - is almost like you are spooling string off a big spool and then just cutting it at regular intervals to give you these identical, almost like shoe laces that are, are sealed at the end. It does sound very clever.
Lisa: It is. It is very clever. And that's a great analogy that describes it really well.
Kat: So when we're thinking about using DNA as a therapeutic, using it as vaccines, using it in other kinds of treatments. If we're going to make this viable, we're gonna need large amounts of this DNA.
Kat: So how does this process scale up? I'm trying to imagine like how big a test tube do you need? How much can you make?
Lisa: It scales up beautifully. So unlike biologics processes and by biologics process, I mean something complex, it involves cells as part of that production. The perfect example, of course, is plasmid fermentation. Those don't necessarily always scale up linearly, and that's a really classic issue with biologics processes.
Lisa: Take an enzymatic process like ours. So far, we've gone from nanograms and we're multi-gram scale now, and it's been linearly scaling up. And that's not to say that we're not making massive improvements along the way, we never stay still in terms of process improvement, but we are currently at multi-gram scale.
Lisa: And actually our scale up process is aiming to take us beyond 40 grams, more like a hundred grams for a single batch. Now, for DNA, that is astonishing, but it's not just about scale up for us. We think that the flexibility for scale out is really, really important for some therapeutic indications.
Lisa: So take a neoantigen vaccine. Those are patient specific vaccine products. They still have to be GMP, but you don't need five grams per patient, you need maybe a hundred milligrams per patient. And that's really difficult to do with plasmid technology because all the GMP processes have been built for a larger scale. Whereas with our flexible manufacturing, we can do much lower scales, but we can also parallelize.
Lisa: In our new facility, like I mentioned, it's not a big building, but we believe it's possibly the largest DNA manufacturing facility in the world. And then you compare that to even a small fermentation plan for pDNA - it really is quite, quite a contrast.
Kat: It must be a bit of an odd feeling to be sitting there watching the production of this DNA and thinking, you know, that's going into people. Maybe one day you'll have a DNA vaccine or DNA treatment that's based on this stuff that you helped develop.
Lisa: That would be amazing.
Lisa: Yeah, so we have a few products going into the clinic, or in the clinic, and I think just greater adoption of this technology would just be my dream. It really is, like I said, it's that combination of the manufacturing benefits, but for me, what's really interesting as a scientist who's used this doggybone DNA in so many different applications - it’s the functionality benefits that you can get from it. And just being able to just keep pushing genetic therapies and nucleic acid vaccines.
Lisa: I really think we have something, something really quite special at Touchlight.
