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.
Key Wordsantisense oligonucleotides in vitro splice correction HeLa pLuc/705 cells lipidoids lipid-polymer hybrid nanoparticles (LPNs) quality-by-design statistical optimisation
ACKNOWLEDGMENTS AND DISCLOSURES
We gratefully acknowledge the support from the Innovative Medicines Initiative Joint Undertaking under grant agreement No. 115363 resources, which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007–2013) and EFPIA companies’ in kind contribution. This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 600207. We are also grateful to the Lundbeck Foundation – Denmark (R219–2016-908), the Novo Nordisk Foundation – Denmark (grant no. NNF17OC0026526) and Hørslev-Fonden – Denmark for financial support. We also acknowledge Stat-Ease Inc. for generously providing the evaluation license of Design Expert version 11 and ChemAxon for kindly providing the academic license of Marvin Suite including calculator plugins. The authors report no potential conflicts of interest.
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