To create A1 lipid nanoparticles for delivering mRNA to target cells, scientists at the University of Pennsylvania used A3 coupling. A3 refers to the amine–aldehyde–alkyne coupling reaction, which the Penn scientists leveraged to iteratively accelerate the structural optimization of propargylamine-based ionizable lipids. Naturally, these lipids are called A3 lipids. And when A3 lipids that have been properly optimized self-assemble into a lipid nanoparticle, the result is an mRNA delivery vehicle that can travel safely through the body to its target cells, efficiently release its contents, and fall apart via biodegradation.
So, how did the A3-lipid-incorporating lipid nanoparticles engineered by the Penn team qualify as A1? They performed better in preclinical models for two high-priority applications: the delivery of genes that could be used to treat hereditary amyloidosis, and the delivery of the COVID-19 mRNA vaccine. In both cases, the engineered lipids showed higher performance than current industry-standard lipids.
Details appeared recently in Nature Biomedical Engineering, in an article titled, “Optimization of the activity and biodegradability of ionizable lipids for mRNA delivery via directed chemical evolution.” The article described how the Penn team used A3 coupling reaction as an iterative chemical derivatization and combinatorial chemistry tool. With it, the Penn team achieved the stepwise optimization of ionizable lipid structure, a feat that hadn’t been achieved previously, despite the application of rational design and combinatorial synthesis to the development of potent and biodegradable ionizable lipids.
“Through five cycles of such directed chemical evolution, we identified dozens of biodegradable and asymmetric A3-lipids with delivery activity comparable to or better than a benchmark ionizable lipid,” the article’s authors wrote. “We then derived structure−activity relationships for the headgroup, ester linkage and tail.
“Compared with standard ionizable lipids, the lead A3-lipid improved the hepatic delivery of an mRNA-based genome editor and the intramuscular delivery of an mRNA vaccine against SARS-CoV-2. Structural criteria for ionizable lipids discovered via directed chemical evolution may accelerate the development of LNPs for mRNA delivery.”
This new method for designing ionizable lipids is expected to have broad implications for mRNA-based vaccines and therapeutics, which are poised to treat a range of conditions, from genetic disorders to infectious diseases. Also, the new approach has the potential to accelerate the development of mRNA therapies overall. While it can take years to develop an effective lipid using traditional methods, the team’s directed evolution process could reduce this timeline to just months or even weeks.
“Our hope is that this method will accelerate the pipeline for mRNA therapeutics and vaccines, bringing new treatments to patients faster than ever before,” said Michael J. Mitchell, an associate professor in bioengineering at Penn.
At the heart of the Penn team’s nanoparticles are ionizable lipids, that is, lipids that can switch between charged and neutral states depending on their surroundings. This switch is essential for the nanoparticle’s journey: In the bloodstream, ionizable lipids stay neutral, preventing toxicity. But once inside the target cell, they become positively charged, triggering the release of the mRNA payload.
To develop safer, more effective ionizable lipids, the Penn Engineers employed a unique approach that combines two prevailing methods: medicinal chemistry, which involves slowly and laboriously designing molecules one step at a time, and combinatorial chemistry, which involves generating many different molecules quickly through simple reactions. The former has high accuracy but low speed, while the latter has low accuracy and high speed.
“We thought it might be possible to achieve the best of both worlds—high speed and high accuracy,” said Xuexiang Han, the paper’s first author and, until recently, a postdoctoral fellow in the Mitchell Lab. “But we had to think outside the traditional confines of the field.”
“We found that the A3 reaction was not only efficient, but also flexible enough to allow for precise control over the lipids’ molecular structure,” Mitchell related. This flexibility was key to fine-tuning the ionizable lipid properties for safe and effective mRNA delivery.
