Drug delivery

the first RNA drug to reach the market in 2018. The process Cullis and his colleagues developed for combining ionisable lipids with nucleic acids also laid the blueprints for today’s LNP manufacturing processes. In “cartoon form”, they found that rapidly mixing lipids in ethanol with nucleotides in water at roughly pH 4 causes the ingredients to bind as they fall out of the solution. “At the end of it, all that’s left are these little globules that are stabilised by the PEG lipids,” explains Cullis. “You just dialyse away the ethanol and raise the pH to physiological pH, and you have your system that’s nice and stable.” The other two lipid ingredients in that microfluidic whirlpool are phospholipids and cholesterol molecules, which can also be swapped out and tweaked to alter how LNPs behave and interact with the body. Thousands of different combinations have been engineered, tested and abandoned on the way to the LNPs in today’s vaccines. That said, as these LNPs come from a lineage originally optimised for expression in the liver, Cullis notes that there is plenty of scope to further improve their potency, stability and even cost. He points to the differing cold chain requirements of the Pfizer-BioNTech and Moderna vaccines to show that slight changes in formulation, or even just the time to do stability studies, can make profound differences. “If you look at the lipid constituents and the mRNA, they’re pretty much identical,” he notes. “And there’s no intrinsic reason why they can’t be more stable or more potent.” The CureVac candidate, for instance, which uses unmodified mRNA with similar LNPs, can be stored at 5°C.

Peer makes a similar point regarding manufacturing, noting that one of the systems he works with could create enough for the entire country of Israel in just 12 days. That’s still about 25 years for the world’s population, though – more systems are clearly needed than there were when mRNA drugs were a niche interest. Equally, he believes the more pressing bottlenecks in raw material supplies for synthesising lipids and generating RNAs are primarily a result of moving “from zero to 100 in a very short time”.

Open the door

Once they’re optimised for expression in a particular location, LNPs allow developers to simply swap out the genetic material they would like to deliver. That already makes them perfect for building vaccine platforms, as Moderna’s rapid responses to Covid-19 variants attest, but most LNPs are quickly removed from circulation and trapped in the liver, making it difficult to use them to address issues elsewhere in the body. Solving that might eventually mean sending the entire PEG clan to join their unnumbered cousins in the lipid family cemetery. In the same way they initially help LNPs sneak past

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the immune system, pegylated lipids inhibit their interactions with cells and can raise the risk of adverse reactions.

Still, PEG has a few more whirls through the bloodstream in it yet. In 2020, Peer’s lab made a breakthrough by using LNPs studded with targeting molecules to deliver mRNA encoding the CRISPR enzyme Cas9 to tumours in vivo, where, directed by guide RNAs also contained in the LNPs, it cut cancer cells’ DNA strands, effectively destroying them from the inside. It’s a similar ‘plug-and-play’ approach to the wider idea of LNP platforms, with a lipoprotein ‘universal adapter’ that binds on the lipid side to the LNP and on the protein side to specific regions of cells of the same isotype.

In mouse models, injected CRISPR-LNPs were selectively taken up by disseminated ovarian tumours, enabling up to ~80% gene editing in vivo, inhibiting tumour growth and increasing survival by 80%. Perhaps more impressively, intracerebral injection of cLNPs targeting the most aggressive types of brain cancer enabled up to ~70% gene editing in vivo, which caused tumour cell apoptosis, inhibited tumour growth by 50%, and improved survival by 30%.

Nonetheless, Cullis warns that more needs to be done for LNPs to effectively reach new locations in human bodies. The leaky vasculature of tumours means drug delivery systems diffuse into them more easily than other tissues, but sophisticated targeting systems count for nothing if the LNPs they decorate are quickly taken out of the bloodstream by the liver. “To get to other tissues, you have to have a very long circulation lifetime,” Cullis explains. GPS systems don’t compensate for a lack of fuel.

“It’s all about timing in life. You need to be in the right location at the right time.”

Dan Peer

Though he’s wary of “simplistic thinking” about solving everything through targeting, Cullis believes the work that will open the rest of the body up to LNPs is already happening. “The door has been pried open a bit, we’ve got the crowbar in our hands,” he says. Neither he nor Langer imagined anything like what’s happened when they first tried putting drugs in nanocarriers, but, if anything, the next step sounds relatively simple for a technology that began by walking through walls. The work done by Peer’s lab to synchronise the expression of mRNA and guide RNA within cells in 2020 was a milestone for selective targeting in a year full of them. “It’s all about timing in life,” he observes wryly. “You need to be in the right location at the right time.” It might just take generations to get you there. ●

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Survival increase in mouse models with ovarian tumours treated with CRISPR- LNP injections, which enabled up ~80% gene editing in vivo.

Survival improvement due to intracerebral injection of cLNPs targeting the most aggressive brain cancers, which enabled ~70% gene editing in vivo.

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