INTRODUCTION: Within the field of tissue engineering, biomaterial scaffolds augmented with gene therapeutics have emerged as a promising treatment strategy for tissue regeneration. To date, the majority of ‘gene-activated’ scaffolds in tissue engineering have utilised plasmid DNA as the gene therapeutic of choice. Recently, messenger RNA (mRNA) has emerged as an attractive alternative to DNA-based therapeutics due to its increased safety profile and faster protein expression. The aim of this study is to optimise the delivery of mRNA to difficult-to-transfect mesenchymal stem cells (MSCs) and incorporate optimised particles into collagen scaffolds to create a platform that can be used for multiple tissue engineering (TE) applications.
METHODS: A wide range of non-viral gene delivery vectors were screened for their ability to encapsulate and condense mRNA. The complexes were characterised in terms of physicochemical properties before being brought forward to transfection studies using rat MSCs. All mRNA complexes were compared in terms of transgene (luciferase) expression and cytotoxicity in MSCs grown both in 2D monolayer and in 3D on collagen-based scaffolds. In addition, three different types of mRNA – unmodified mRNA (uRNA), modified mRNA (modRNA) and self-amplifying mRNA (saRNA) (BioNTech) were screened to determine the effect of mRNA type on expression for TE applications.
RESULTS: Various polymeric and lipid-based vectors were found capable of successfully delivering mRNA to MSCs. It was found that both the vector and mRNA type used had a significant impact on transgene expression in our cell type. Overall, the base-modified mRNA achieved the highest levels of protein expression in MSCs demonstrating a 1.2-fold and 5.6-fold increase versus uRNA and saRNA respectively in 2D monolayer studies. When delivered from a collagen-based scaffold, lipid-based vectors (e.g. MessegerMax/RNAiMAX) outperformed polymeric vectors (e.g. jetPEI) and achieved high levels of protein expression in the MSCs ( 2.3x106 relative light units).
CONCLUSION: Messenger RNA represents a promising tool for tissue engineering applications. This study highlights the optimised transfection conditions for mRNA delivery to mesenchymal stem cells in 2D and 3D. It is hoped that this work will serve as a template for future translational research within the field.