Strategies for simultaneous and successive delivery of RNA

Hanieh Moradian,Andreas Lendlein,Manfred Gossen

doi:10.1007/s00109-020-01956-1

Abstract

Advanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the second transfection round. All these differences proved to be more pronounced for low mRNA doses. Concurrent delivery of siRNA with mRNA also indicated the highest co-transfection efficiency for integrated method. However, the maximum efficacy was shown for successive delivery, due to the kinetically different peak output for the two discretely operating entities. Our findings provide guidance for selection of the co-delivery method best suited to accommodate experimental requirements, highlighting in particular the nucleic acid dose-response dependence on co-delivery on the single-cell level.

Highlights

Key messages Quantitative analysis of methods for co-delivery of distinct nucleic acids. Dose dependence of co-delivery efficacy. Successive delivery of mRNA revealed a lower efficiency in the second transfection. Simultaneous compared with successive transfection of cells with small interfering RNA (siRNA) and IVT-mRNA. Selection criteria for co-delivery method defined.Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Delivery of genes and other functional nucleic acids as a powerful tool for basic research [1,2,3], in biomedical/therapeutic applications and biotechnology [4,5,6,7], has been practiced intensely for decades
Results integrated cotransfection” (iCoTF), lipoplexes were prepared by premixing of enhanced green fluorescent protein (EGFP) and mCherry IVT-mRNAs before adding to the transfection reagent, whereas for parallel co-transfection” (pCoTF), the independently formed complexes were added to the same well (Fig. 1a, b)
PCoTF results in heterogeneous population of cells expressing different levels of each reporter gene, observed as different color spectrum in merged fluorescent image (Fig. 3a) and the wide distribution of cells within double-positive gate (Fig. 3b). pCoTF resulted in slightly higher level of fluorescent protein expression compared with iCoTF, measured via mean fluorescent intensity (MFI) of the peak on the adjacent histograms

Summary

Introduction

The simultaneous delivery of more than a single nucleic acid is advantageous or even mandatory. This includes co-delivery of multiple nucleic acids of the same type [16], multiple types of nucleic acids [14, 17], and a nucleic acid coordinated with another entity such as drug [18, 19] or protein [20]. Robust reliable codelivery methods are, of critical importance in many gene transfer studies. Few of these studies, focused on the applied co-delivery strategies and analyzed their impact on study’s outcomes, for IVT-mRNA. Exemplary applications are the parallel transient overexpression of genes when

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