Correlation of mRNA delivery timing and protein expression in lipid-based transfection

Abstract

Non-viral gene delivery is constrained by the dwell time that most synthetic nucleic acid nanocarriers spend inside endosomal compartments. In order to overcome this endosomalrelease bottleneck, methods are required that measure nanocarrier uptake kinetics and transfection efficiency simultaneously. Here, we employ live-cell imaging on single-cell arrays (LISCA) to study the delivery-time distribution of lipid-based mRNA complexes under varied serum conditions. By fitting a translation-maturation model to hundreds of individual eGFP reporter fluorescence time courses, the protein expression onset times and the expression rates after transfection are determined. Using this approach, we find that delivery timing and protein expression rates are not intrinsically correlated at the single-cell level, even though population-averaged values of both parameters conjointly change as a function of increasing external serum protein fraction. Lipofectamine mediated delivery showed decreased transfection efficiency and longer delivery times with increasing serum protein concentration. This is in contrast to ionizable lipid nanoparticles (LNPs) mediated transfer, which showed increased efficiency and faster uptake in the presence of serum. In conclusion, the interdependences of single-cell expression rates and onset timing provide additional clues on uptake and release mechanisms, which are useful for improving nucleic acid delivery. author/funder. All rights reserved. No reuse allowed without permission.

Polyplex-Loaded Hydrogels for Local Gene Delivery to Human Dermal Fibroblasts

“…At the time of fluorescence measurement, not all released nanoparticles have been uptaken by cells and not all uptaken nanoparticles have been translated into green fluorescence protein. Moreover, previous studies report that GFP protein expression progressively increases in the 24 hours following mRNA transfection, and that there is an inherently large cellto-cell variability in the expression levels of GFP of transfected cells 58 . Hence, only those cells transfected in the first few hours are expected to emit a significant amount of fluorescence at the time of measurement.…”

Identifiability analysis for models of the translation kinetics after mRNA transfection

“…Single-cell fluorescence data from transfection experiments has been analyzed based on ODE modeling in several previous studies e. g. Ligon et al (2014), Leonhardt et al (2014), Fröhlich et al (2018), and Reiser et al (2019). Yet, several parameters of the considered ODE models for the translation kinetics after mRNA transfection are not identifiable from the experimental data.…”