Branched Lipids Increase Efficiency of mRNA Delivery

A research team in Japan reports that it has developed a novel branched ionizable lipid which, when included in lipid nanoparticles (LNPs), greatly increases the efficiency of mRNA delivery. The study (“Branching ionizable lipids can enhance the stability, fusogenicity, and functional delivery of mRNA”) appears in Small Science.

“Ionizable lipids with branched tails have been used in lipid nanoparticles (LNPs)-based messenger RNA (mRNA) therapeutics like COVID-19 vaccines. However, due to the limited commercial availability of branched ingredients, a systematic analysis of how the branched tails affect LNP quality has been lacking to date,” the investigators wrote.

“Herein, α-branched tail lipids are focused, as they can be synthesized from simple commercially available chemicals, and the length of each chain can be independently controlled. Furthermore, symmetry and total carbon number can be used to describe α-branched tails, facilitating the design of a systematic lipid library to elucidate ‘structure–property–function’ relationships.

“Consequently, a lipid library is developed containing 32 different types of α-branched tails. This library is used to demonstrate that branched chains increase LNP microviscosity and headgroup ionization ability in an acidic environment, which in turn enhances the stability and in vivo efficacy of mRNA-LNPs. Of the branched lipids, CL4F 8-6 LNPs carrying Cas9 mRNA and sgRNA could achieve 54% genome editing and 77% protein reduction with a single dose of 2.5 mg kg⁻¹. This mechanism-based data on branched lipids is expected to provide insights into rational lipid design and effective gene therapy in the future.”

Previous work has shown that ionizable lipids with branching tails increase the efficiency of mRNA delivery by LNPs. However, two major issues have prevented a systematic analysis of the effect of branching ionizable lipids.

Large diversity of chemicals

First, tail branching leads to an enormous diversity of chemicals; second, the number of commercially available branching ionizable lipids is limited. To overcome these hurdles, the researchers generated a systematic lipid library of branching ionizable lipids and limited this library to a specific subset of branching lipids which could be described with just two parameters: total carbon number and symmetry. They then tested the 32 lipids in this library for their effect on the stability of LNPs containing mRNA (LNP-RNA).

The most stable storage and most efficient delivery of the mRNA was achieved by the branching lipid CL4F 8-6. The authors demonstrated that this particular lipid could be used in LNPs designed for gene editing, achieving a 77% suppression of the target gene in mice with just one dose of LNPs.

This study revealed that branched lipids with a high level of symmetry contributed to optimal LNP properties for efficient intracellular delivery and stable formulations. Future work will focus on developing expanded lipid libraries to understand the properties of other branched lipids and may lead to the design of novel lipids.

Yusuke Sato, PhD, assistant professor, and Hideyoshi Harashima, PhD, professor, at the faculty of pharmaceutical sciences, Hokkaido University, and Kazuki Hashiba, at Nitto Denko, led the research group.