Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for systemic delivery of RNAi therapeutics

Abstract

In recent years, RNA interference (RNAi) therapeutics, most notably with lipid nanoparticle-based delivery systems, have advanced into human clinical trials. The results from these early clinical trials suggest that lipid nanoparticles (LNPs), and the novel ionizable lipids that comprise them, will be important materials in this emerging field of medicine. A persistent theme in the use of materials for biomedical applications has been the incorporation of biodegradability as a means to improve biocompatibility and/or to facilitate elimination. Therefore, the aim of this work was to further advance the LNP platform through the development of novel, next-generation lipids that combine the excellent potency of the most advanced lipids currently available with biodegradable functionality. As a representative example of this novel class of biodegradable lipids, the lipid evaluated in this work displays rapid elimination from plasma and tissues, substantially improved tolerability in preclinical studies, while maintaining in vivo potency on par with that of the most advanced lipids currently available.

Figure 1

Design of lipids incorporating biocleavable ester functions within hydrophobic alkyl chains. positional variations of the ester linkage are indicated by numbers along the alkyl chain (see also Table 1).

Figure 2

Factor VII silencing activity of L319-lipid nanoparticle in mice after single intravenous administration. Serum was collected 48 hours postadministration and was analyzed for factor VII protein activity. Bars represent group mean ± SD (n = 3). PBS, phosphate-buffered saline.

Figure 3

Lipid pharmacokinetic profiles after single intravenous administration of lipid nanoparticle (0.3 mg/kg short interfering RNA) in mice. Concentration–time profiles of lipids L319, L343 and L322 in (a) plasma and (b) liver; concentration–time profiles of expected metabolites of L319 in (c) plasma and (d) liver; data points represent group mean ± SD (n = 2).

Figure 4

L319 is eliminated from intracellular compartments and is readily excreted in vivo. (a) Lipid nanoparticles (LNPs) containing BODIPY-labeled L319 were added at 20 nmol/l to HeLa cells co-stained with LysoTracker Red for 5 hours, green: BODIPY lipid, red: LysoTracker Red, blue: Hoechst. (b) Comparison of L319 lipid content per cell at 5 and 24 hours following a 5-hour pulse. (c) Excretion of L319 in urine and feces of rats after single intravenous administration of LNP-siRNA containing ¹⁴C-labeled cationic lipid. Urine and feces were collected 0–12, 12–24, 24–48, and 48–72 hours postadministration and analyzed for levels of ¹⁴C-label over each time period. Data show the cumulative amount of label detected over time and is expressed as % of injected dose and each data point indicates group mean ± SD (n = 4). RFU, relative fluorescence unit; siRNA, short interfering RNA.

Figure 5

Dose-dependent effect on rat serum chemistry (alanine transaminase (ALT) and aspartate transaminase (AST)) after intravenous infusion of L319-lipid nanoparticle (LNP) at 1, 3, 5, and 10 mg/kg dose and L343-LNP at 1, 3, and 5 mg/kg (based on short interfering RNA weight). Bars represent group mean ± SD (n = 5).

Figure 6

Efficacy and lipid pharmacokinetics in nonhuman primates after single intravenous administration of 0.3 mg/kg TTR short interfering RNA formulated in L319-LNPs. (a) Liver TTR mRNA levels in biopsy samples collected 48 hours postadministration. Bars represent group mean ± SD (n = 3). (b) Lipid-concentration-time profile in plasma in L319-LNP–treated animals. Data points represent group mean (n = 3). LNP, lipid nanoparticle; TTR, transthyretin.