A novel category of lipids is enhancing the efficiency with which lipid nanoparticles (LNPs) deliver RNA-based therapies and gene editing tools, potentially increasing the effectiveness of these treatments.
Whenever a shuttle docks with the International Space Station (ISS), a careful interaction occurs between the shuttle’s docking mechanism and that of the station. Thanks to international guidelines, these systems are universally compatible, allowing astronauts and cargo to safely and easily access the station.
A similar issue occurs at a microscopic level when lipid nanoparticles (LNPs) — the groundbreaking delivery systems behind the COVID-19 vaccines — work to deliver mRNA to cells. By optimizing the design and delivery of LNPs, we can significantly improve their capability to deliver mRNA, providing cells with the necessary instructions to combat diseases and revolutionize medical treatments.
Overcoming the Endosome Challenge
Unfortunately, even when LNPs successfully reach their target cells, these particles often become trapped within endosomes—small protective sacs inside the cell. If LNPs fail to escape, it’s akin to a shuttle being stuck during docking, leaving the safety of the station just beyond reach.
“If the endosomal escape doesn’t occur, LNPs become ensnared and cannot deliver their therapeutic contents,” explains Michael J. Mitchell, an Associate Professor of Bioengineering at the University of Pennsylvania School of Engineering and Applied Science (Penn Engineering). “They might navigate through the injection into the cell, but without overcoming that last hurdle, they’re ineffective.”
A New Solution
Years ago, scientists at Carnegie Mellon University made a fascinating discovery: by adding a branch to the ends of the typically linear lipid tails of LNPs, mRNA delivery improved significantly. This discovery inspired Marshall Padilla, a postdoctoral researcher in the Mitchell Lab, to explore whether this could lead to the development of enhanced lipids for mRNA delivery.
“Researchers are continuously creating new lipids to boost the effectiveness and safety of LNPs,” Padilla notes. “However, we are missing a clear framework for creating superior lipids.”
The majority of research in this area resembles a guessing game. Scientists test numerous lipid variations without completely understanding why some perform better than others. Padilla, who holds a Ph.D. in Chemistry from the University of Wisconsin-Madison, believed that it might be possible to step beyond experimentation and craft lipids with branched tails right from the beginning to enhance their ability to exit endosomes.
Unveiling BEND Lipids
Creating these improved lipids presented challenges, particularly with developing branched ionizable lipids—essential components of LNPs that modify their charge to facilitate endosomal escape. Such lipids are not available for commercial purchase in a branched form, so Padilla had to synthesize them himself.
“The main challenge was forming carbon-carbon bonds, which are notoriously challenging,” Padilla remarks. “I utilized a complex combination of lithium, copper, and magnesium to achieve the reaction.”
The outcome was a new class of lipids called branched endosomal disruptor (BEND) lipids. These specially designed branched molecules assist LNPs in penetrating the endosomal membrane, thus enhancing their ability to deliver mRNA and gene editing tools more effectively.
Enhancing mRNA Delivery
In a recent publication in Nature Communications, Mitchell, Padilla, and their team demonstrated that BEND lipids significantly enhance LNP delivery of mRNA and gene-editing tools, with some instances showing improvements by up to tenfold.
After a series of experiments—from gene editing in liver cells to executing complex biochemical models—the researchers concluded that BEND lipids consistently outperform the LNPs utilized by Moderna and Pfizer/BioNTech, the developers of the COVID-19 vaccines.
“We discovered that the branching groups enable the lipids to assist in transporting our payload out of the endosome, where most cargo is destroyed, and into the cytosol, where it can exert its intended therapeutic effect,” Padilla stated.
Advancing Therapeutic Development
The researchers aim for BEND lipids to not only boost LNP delivery but also inspire a novel methodology in lipid design, moving beyond the trial-and-error approach. With a deeper understanding of lipid functionality, scientists could better develop innovative delivery systems for advanced treatments.
“Testing hundreds or thousands of LNP variants to identify effective solutions can be an immense burden in terms of time, cost, and labor — many labs may not have the resources for this,” Mitchell observes. “We want to understand the design principles so that solutions can be created more efficiently and economically.”
This research was carried out at the University of Pennsylvania School of Engineering and Applied Science, with support from several organizations, including the National Institutes of Health and the National Science Foundation.
Additional co-authors include Kaitlin Mrksich, Yiming Wang, Rebecca M. Haley, Jacqueline J. Li, Emily L. Han, Rakan El-Mayta, Emily H. Kim, Ningqiang Gong, Sridatta V. Teerdhala, Xuexiang Han, Lulu Xue, Zain Siddiqui, Hannah M. Yamagata, Dongyoon Kim, Il-Chul Yoon, and Ravi Radhakrishnan from Penn Engineering; Sofia Dias from Penn Engineering and the University of Porto; and Vivek Chowdhary and James Wilson from Penn Medicine.
