Design, synthesis, and biological evaluation of diketopiperazine based ionizable lipids for the in vivo delivery of messenger RNA

Abstract

Thousands of human diseases could be treated by selectively increasing the intracellular concentration of specific proteins. The successful delivery of messenger RNA (mRNA) to target cells in the body could accomplish this goal, but serious limitations with its systemic delivery must still be overcome. Recently, lipid nanoparticles (LNPs) have shown promise for mRNA delivery in vivo, however, current leads are limited in terms of their efficacy, biodistribution, and toxicity. Here, we synthesize novel LNP delivery materials (i.e. ionizable lipids) that, when formulated into LNPs, both outperform current leads for mRNA delivery and elucidate key relationships between chemical structure and biological function. Drawing inspiration from naturally occurring components of cellular membranes, we have designed and synthesized a new series of alkenyl amino alcohol (AAA) ionizable lipids for mRNA delivery to the liver. When formulated into LNPs, these AAA ionizable lipids induce high concentrations of human erythropoietin (EPO) protein at therapeutically relevant doses of mRNA. Notably, LNPs derived from our lead compound OF-02 are the most potent mRNA delivery vehicle yet reported in the scientific literature. While the liver is implicated in many diseases, targeting other tissues could drastically improve the clinical generality of mRNA based therapeutics. Towards this end, we have designed and synthesized ionizable lipid OF-77. Unlike other materials that afford more than 99% of total protein production in the liver, OF-77 mRNA LNPs promote more than 85% of total protein production in the spleen. Notably, OF-77 mRNA LNPs also demonstrate the first example of functional protein production within B lymphocytes, with current levels of protein production only limited by the administered dose. Finally, we synthesize a novel series of ionizable lipids by varying three key structural parameters within OF-77; tail length, linker spacing, and total degrees of unsaturation. Both in vitro and in vivo experiments are explored to glean crucial information relating LNP structure to biological response. We also demonstrate that these compounds, including OF-77, are capable of both complexing and delivering siRNA to reduce specific intracellular protein concentrations.