Ionizable lipid nanoparticles for RAS protease delivery to inhibit cancer cell proliferation.

Mutations in RAS, a family of proteins found in all human cells, drive a third of cancers, including many pancreatic, colorectal, and lung cancers. However, there is a lack of clinical therapies that can effectively prevent RAS from causing tumor growth. Recently, a protease was engineered that specifically degrades active RAS, offering a promising new tool for treating these cancers. However, like many other intracellularly acting protein-based therapies, this protease requires a delivery vector to reach its site of action within the cell. In this study, we explored the incorporation of cationic lipids into ionizable lipid nanoparticles (LNPs) to develop a RAS protease delivery platform capable of inhibiting cancer cell proliferation in vitro and in vivo. A library of 13 LNPs encapsulating RAS protease was designed, and each formulation was evaluated for in vitro delivery efficiency and toxicity. A subset of four top-performing LNP formulations was identified and further evaluated for their impact on cancer cell proliferation in human colorectal cancer cells with mutated KRAS in vitro and in vivo, as well as their in vivo biodistribution and toxicity. In vivo, both the concentration of cationic lipid and type of cargo influenced LNP and cargo distribution. All lead candidate LNPs showed RAS protease functionality in vitro, and the top-performing formulation achieved effective intracellular RAS protease delivery in vivo, decreasing cancer cell proliferation in an in vivo xenograft model and significantly reducing tumor growth and size. Overall, this work demonstrates the use of LNPs as an effective delivery platform for RAS proteases, which could potentially be utilized for cancer therapies.