Cellular uptake of modified mRNA occurs via caveolae-mediated endocytosis, yielding high protein expression in slow-dividing cells

Claudia Del Toro Runzer, Shivesh Anand, Carlos Mota, Lorenzo Moroni, Christian Plank, Martijn van Griensven, Elizabeth R Balmayor*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Nucleic acids have clear clinical potential for gene therapy. Plasmid DNA (pDNA) was the first nucleic acid to be pursued as a therapeutic molecule. Recently, mRNA came into play as it offers improved safety and affordability. In this study, we investigated the uptake mechanisms and efficiencies of genetic material by cells. We focused on three main variables (1) the nucleic acid (pDNA, or chemically modified mRNA), (2) the delivery vector (Lipofectamine 3000 or 3DFect), and (3) human primary cells (mesenchymal stem cells, dermal fibroblasts, and osteoblasts). In addition, transfections were studied in a 3D environment using electrospun scaffolds. Cellular internalization and intracellular trafficking were assessed by using enhancers or inhibitors of endocytosis and endosomal escape. The polymeric vector TransIT-X2 was included for comparison purposes. While lipoplexes utilized several entry routes, uptake via caveolae served as the main route for gene delivery. pDNA yielded higher expression levels in fast-dividing fibroblasts, whereas, in slow-dividing osteoblasts, cmRNA was responsible for high protein production. In the case of mesenchymal stem cells, which presented an intermediate doubling time, the combination vector/nucleic acid seemed more relevant than the nucleic acid per se. In all cases, protein expression was higher when the cells were seeded on 3D scaffolds.

Original languageEnglish
Pages (from-to)960-979
Number of pages20
JournalMolecular Therapy - Nucleic Acids
Volume32
Issue number1
DOIs
Publication statusPublished - 13 Jun 2023

Fingerprint

Dive into the research topics of 'Cellular uptake of modified mRNA occurs via caveolae-mediated endocytosis, yielding high protein expression in slow-dividing cells'. Together they form a unique fingerprint.

Cite this