A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates

Abstract

The success of mRNA-based therapies depends on the availability of a safe and efficient delivery vehicle. Lipid nanoparticles have been identified as a viable option. However, there are concerns whether an acceptable tolerability profile for chronic dosing can be achieved. The efficiency and tolerability of lipid nanoparticles has been attributed to the amino lipid. Therefore, we developed a new series of amino lipids that address this concern. Clear structure-activity relationships were developed that resulted in a new amino lipid that affords efficient mRNA delivery in rodent and primate models with optimal pharmacokinetics. A 1-month toxicology evaluation in rat and non-human primate demonstrated no adverse events with the new lipid nanoparticle system. Mechanistic studies demonstrate that the improved efficiency can be attributed to increased endosomal escape. This effort has resulted in the first example of the ability to safely repeat dose mRNA-containing lipid nanoparticles in non-human primate at therapeutically relevant levels.

Keywords: delivery; lipid nanoparticle; mRNA; mRNA therapeutics; safe chronic dosing; tolerability.

Figure 1

Optimization of Efficiency and Clearance of Amino Lipid (A) Structures of amino lipids. (B) Whole-body luciferase bioluminescence AUC of novel LNPs versus MC3 LNPs, measured in CD-1 mice (n = 6 at 3 and 6 hr, n = 3 at 24 hr), 0.5 mg/kg dose firefly luciferase (ffLuc) mRNA, i.v. bolus, error bars indicate SD of the ratio of novel lipid AUC versus MC3 AUC. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. = not statistically significant. (C) Parent amino lipid levels measured in liver tissue from Sprague-Dawley rats (n = 3 per time point), 0.2 mg/kg dose hEPO mRNA, mean ± SD, p < 0.05 for lipids 5 and 8 AUC relative to MC3.

Figure 2

Pharmacokinetic and Expression Profile of Lipid 5 after Multiple Doses (A) Comparison of tissue distribution of MC3 and lipid 5 after three i.v. bolus doses, CD-1 mice (n = 3 per time point, 1, 4, and 24 hr), 0.05 mg/kg mRNA, weekly dosing, mean ± SD, lipid 5 AUC < MC3 AUC at p < 0.05 at all time points. (B) hEPO serum concentration, 6 hr, i.v. bolus, CD-1 mice (n = 8), 0.5 mg/kg dose mRNA, weekly dosing, mean ± SD. (C) Structure of the primary metabolite (11) of lipid 5. (D) Liver tissue clearance of lipid 5 and metabolite 11, i.v. bolus, CD-1 mice (n = 3 per time point), 0.25 mg/kg dose mRNA, weekly dosing, mean ± SD.

Figure 3

Expression Profile in Cynomolgus Monkey (A) hEPO serum concentrations after delivery of 0.01 mg/kg hEPO mRNA in MC3 or lipid 5 LNPs, i.v. 60 min infusion, n = 3, mean ± SD, p < 0.05 for lipid 5 AUC relative to MC3. (B) Human IgG influenza A antibody serum concentrations after delivery of 0.3 mg/kg antibody mRNA in MC3 or lipid 5 LNPs, i.v. 60 min infusion, n = 3, mean ± SD, p < 0.05 for lipid 5 AUC relative to MC3. (C) hEPO serum concentrations after delivery of 0.2 mg/kg hEPO mRNA in lipid 5 LNPs, weekly dosing, i.v. 60 min infusion, n = 4, mean ± SD.

Figure 4

One-Month Toxicology Evaluation of Lipid 5 LNPs in Rat and Non-human Primate (A) Serum alanine aminotransferase levels 24 hr post-fifth-dose, Sprague-Dawley rat, i.v. 10 min infusion, weekly dosing for 5 weeks, n = 10, mean ± SD, no statistical difference between PBS and any dose level. (B) Serum aspartate aminotransferase levels 24 hr post-fifth-dose, Sprague-Dawley rat, i.v. 10 min infusion, weekly dosing for 5 weeks, n = 10, mean ± SD, *p < 0.05, no statistical difference between PBS and 0.05 and 2.0 mg/kg dose. (C) Serum alanine aminotransferase levels 24 hr post-fifth-dose, cynomolgus monkey, 1 mg/kg mRNA, i.v. 60 min infusion, weekly dosing, n = 4, mean ± SD, no statistical difference between PBS pre-dose and day 30 levels with lipid 5 levels. (D) Serum aspartate aminotransferase levels 24 hr post-fifth-dose, cynomolgus monkey, 1 mg/kg mRNA, i.v. 60 min infusion, weekly dosing, n = 4, mean ± SD, no statistical difference between PBS pre-dose and day 30 levels with lipid 5 levels. (E) C5b9 serum concentration on day 1 and day 29, cynomolgus monkey, 1 mg/kg mRNA, i.v. 60 min infusion, weekly dosing, n = 4, mean ± SD, no statistical difference between PBS and lipid 5 at all time points. (F) MCP-1 serum concentration on day 1 and day 29, cynomolgus monkey, 1 mg/kg mRNA, i.v. 60 min infusion, n = 4, mean ± SD, *p < 0.05, no statistical difference between PBS and lipid 5 at any other time point.

Figure 5

Fixed Cell Imaging of Endosomal Escape Efficiency HeLa cells were transfected with ATTO 647 (magenta)-labeled MC3 and lipid 5 LNPs encapsulating luciferase mRNA, and processed for Stellaris single-molecule FISH (smFISH, Quasar 570, red) after 4 hr incubation, alongside cells electroporated with unformulated luciferase mRNA. The mRNA molecules that egressed the endocytic organelles into the cytosol are shown in green (image analysis overlay). Endosomal escape efficiency was evaluated by computing the ratio between the number of cytosolic mRNA (green) and the number of internalized LNPs per cell (magenta). (A) Single-particle imaging on glass, ATTO 647-labeled MC3, and lipid 5 LNPs (magenta). (B) ATTO 647-labeled LNP cellular uptake. (C) smFISH processing for intracellular delivered mRNA (red) and image analysis overlay (green).