Application of a Quality-By-Design Approach to Optimise Lipid-Polymer Hybrid Nanoparticles Loaded with a Splice-Correction Antisense Oligonucleotide: Maximising Loading and Intracellular Delivery.

Antisense oligonucleotides (ASOs) are promising therapeutics for specific modulation of cellular RNA function. However, ASO efficacy is compromised by inefficient intracellular delivery. Lipid-polymer hybrid nanoparticles (LPNs) are attractive mediators of intracellular ASO delivery due to favorable colloidal stability and sustained release properties. LPNs composed of cationic lipidoid 5 (L5) and poly(DL-lactic-co-glycolic acid) were studied for delivery of an ASO mediating splice correction of a luciferase gene transcript (Luc-ASO). Specific purposes were: (i) to increase the mechanistic understanding of factors determining the loading of ASO in LPNs, and (ii) to optimise the LPNs and customise them for Luc-ASO delivery in HeLa pLuc/705 cells containing an aberrant luciferase gene by using a quality-by-design approach. Critical formulation variables were linked to critical quality attributes (CQAs) using risk assessment and design of experiments, followed by delineation of an optimal operating space (OOS). A series of CQAs were identified based on the quality target product profile. The L5 content and L5:Luc-ASO ratio (w/w) were determined as critical formulation variables, which were optimised systematically. The optimised Luc-ASO-loaded LPNs, defined from the OOS, displayed high loading and mediated splice correction at well-tolerated, lower doses as compared to those required for reference L5-based lipoplexes, L5-modified stable nucleic acid lipid nanoparticles or LPNs modified with dioleoyltrimethylammonium propane (conventional cationic lipid). The optimal Luc-ASO-loaded LPNs represent a robust formulation that mediates efficient intracellular delivery of Luc-ASO. This opens new avenues for further development of LPNs as a broadly applicable technology platform for delivering nucleic acid cargos intracellularly.