Summary
Lipid nanoparticles (LNPs) are promising for RNA delivery, yet their effectiveness is hindered by endosomal entrapment post cellular uptake, limiting their therapeutic potential. The challenge lies in the inadequate understanding of the endosomal escape mechanism. This project aims to comprehensively replicate endosomal escape in vitro, employing three objectives to explore LNP interactions with membranes and RNA translocation. Objective 1 focuses on understanding the impact of pH and LNP formulation on LNP/membrane interactions. Employing supported lipid bilayers (SLBs) mimicking endosomal membranes, the binding and fusion rates of distinct LNP formulations will be examined using total internal reflection fluorescence microscopy. This will highlight key factors influencing these interactions, which constitute the initial step of endosomal escape. Objective 2 aims to reconstitute RNA translocation across endosomal membranes using giant unilamellar vesicles (GUVs). These vesicles simulate endosomal conditions, enabling the visualisation of LNP-loaded RNA translocation across vesicle membranes via spinning disk microscopy and fluorescence correlation spectroscopy. This will offer direct evidence of the success of the second step of endosomal escape and insights into how various LNP formulations react to diverse pH conditions. Objective 3 introduces novel bifunctional phospholipids (BPs) into LNP formulations to facilitate visualising LNP fusion with endosomes in live cells through confocal microscopy. This technique also permits the visualisation of RNA release and subsequent protein expression. By addressing the endosomal escape challenge through innovative in vitro and cellular models, this project aims to unravel the complexities of LNP interactions with cellular barriers. The findings have the potential to enhance LNP design, optimising endosomal escape mechanisms and ultimately improving their efficacy in gene delivery applications.
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| Web resources: | https://cordis.europa.eu/project/id/101148549 |
| Start date: | 17-10-2024 |
| End date: | 16-10-2026 |
| Total budget - Public funding: | - 173 847,00 Euro |
Cordis data
Original description
Lipid nanoparticles (LNPs) are promising for RNA delivery, yet their effectiveness is hindered by endosomal entrapment post cellular uptake, limiting their therapeutic potential. The challenge lies in the inadequate understanding of the endosomal escape mechanism. This project aims to comprehensively replicate endosomal escape in vitro, employing three objectives to explore LNP interactions with membranes and RNA translocation. Objective 1 focuses on understanding the impact of pH and LNP formulation on LNP/membrane interactions. Employing supported lipid bilayers (SLBs) mimicking endosomal membranes, the binding and fusion rates of distinct LNP formulations will be examined using total internal reflection fluorescence microscopy. This will highlight key factors influencing these interactions, which constitute the initial step of endosomal escape. Objective 2 aims to reconstitute RNA translocation across endosomal membranes using giant unilamellar vesicles (GUVs). These vesicles simulate endosomal conditions, enabling the visualisation of LNP-loaded RNA translocation across vesicle membranes via spinning disk microscopy and fluorescence correlation spectroscopy. This will offer direct evidence of the success of the second step of endosomal escape and insights into how various LNP formulations react to diverse pH conditions. Objective 3 introduces novel bifunctional phospholipids (BPs) into LNP formulations to facilitate visualising LNP fusion with endosomes in live cells through confocal microscopy. This technique also permits the visualisation of RNA release and subsequent protein expression. By addressing the endosomal escape challenge through innovative in vitro and cellular models, this project aims to unravel the complexities of LNP interactions with cellular barriers. The findings have the potential to enhance LNP design, optimising endosomal escape mechanisms and ultimately improving their efficacy in gene delivery applications.Status
SIGNEDCall topic
HORIZON-MSCA-2023-PF-01-01Update Date
22-11-2024
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