(387d) Award Submission: Lipid Nanoparticle Ionization at Endosomal pH Is a Cell-Free Predictor of mRNA Delivery Efficacy In Vivo

Messenger RNA (mRNA) therapy has the potential to transform the lives of millions of people suffering from a wide range of diseases by augmenting protein expression or replacing missing or mutated proteins. The versatility of mRNA-based drugs has made it a leading gene therapy platform, with dozens of studies utilizing it for applications such as such as protein replacement, prophylactic vaccines, cancer immunotherapy, gene editing, and cellular reprogramming. However, the clinical translation of mRNA therapeutics is contingent upon their safe and effective delivery into target cells. Lipid nanoparticles (LNPs) have emerged as a promising class of delivery materials with the potential to facilitate mRNA delivery. Given the high cost of preclinical testing, most delivery systems are screened in cell culture before advancing into murine and non-human primate models. Unfortunately, cell culture testing is resource intensive and prone to high rates of false negatives and positives. Here, we describe an alternative, cell-free screening strategy that accurately predicts mRNA delivery potency in animals. This was accomplished through the synthesis of a small library of lipid-like (lipidoid) materials, a subset of which effectively delivered mRNA to the liver and spleen of mice following systemic injection of mRNA-LNPs at a 0.75 mg/kg dose. In particular, the lipidoid 306O_i10, which contains a branched tail, facilitated marked levels of firefly luciferase expression in mice. A structure-function analysis using our in vivo results revealed that in vitro efficacy, particle size, mRNA entrapment, and pKa did not correlate with efficacy in mice. However, the degree of lipid nanoparticle surface ionization at the late endosomal pH of 5.0 was a strong predictor of in vivo efficacy (R²= 0.95). The generation and testing of a secondary library of 33 lipidoids confirmed LNP surface ionization as an effective screening tool, correctly predicting materials with both high and low efficacy in mice. These data indicate that the measurement of nanoparticle surface ionization is an inexpensive, high throughput, and reliable method to identify mRNA delivery materials that are highly potent in vivo.