Understanding Intracellular Biology to Improve mRNA Delivery by Lipid Nanoparticles

Area of Science:

• Nanomedicine
• Molecular Biology
• Cellular Biology

Background:

• Intracellular delivery and targeting of nucleic acid therapeutics by nanoparticles remain poorly understood.
• Developing effective delivery systems is crucial for advancing nucleic acid-based therapies.

Purpose of the Study:

• To elucidate the mechanism of lipid nanoparticle (MC3-LNP) delivery of messenger RNA (mRNA).
• To develop and apply a novel workflow, Advanced Cellular and Endocytic profiling for Intracellular Delivery (ACE-ID), for optimizing nanomedicine delivery.
• To re-engineer MC3-LNP for enhanced intracellular delivery.

Main Methods:

• A cell-based imaging assay was combined with perturbation of 178 intracellular trafficking targets.
• Advanced image analysis algorithms extracted phenotypic fingerprints from cellular images.
• Machine learning identified key features correlating with enhanced mRNA delivery, pinpointing fluid-phase endocytosis as a key entry route.

Main Results:

• The ACE-ID workflow provided biological insights into MC3-LNP delivery mechanisms.
• Fluid-phase endocytosis was identified as a productive cellular entry route for mRNA delivery.
• Re-engineering MC3-LNP to target macropinocytosis significantly improved mRNA delivery both in vitro and in vivo.

Conclusions:

• The ACE-ID approach offers a powerful strategy for understanding and optimizing nanomedicine intracellular delivery.
• Targeting macropinocytosis represents a promising strategy for enhancing mRNA delivery via lipid nanoparticles.
• This work has the potential to accelerate the development of nucleic acid-based therapeutics.