Strategies for simultaneous and successive delivery of RNA

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.

Chemical modification of uridine modulates mRNA-mediated proinflammatory and antiviral response in primary human macrophages

“…4,5 Clinical applications of mRNA include both, protein replacement therapies 6 and mRNA vaccines, 7,8 deployed not only for treatment of inherited and non-infectious acquired diseases such as cancer, 9 but also viral diseases, such as recently the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 10,11 The latter is a showcase example for the power of mRNA technology in tackling disease, outpacing other types of vaccines, with rather fast development from bench to market. 12 Despite progress in mRNA production technology by in vitro transcription (IVT) via bacteriophage enzymes such as SP6, T3, and T7 RNA polymerases, potential immunogenicity of transcripts remains a major issue for some mRNA-based medicines.…”

Co-delivery of genes can be confounded by bicistronic vector design

“…Herein, the former is referred to as “MonoCis (CoTF)”, while the latter is named “BiCis (2A-P)” throughout all experiments. As we previously validated the reliability of MonoCis (CoTF) approach, [ 26 ] this condition was mainly included as reference and control to determine the performance of BiCis (2A-P) method. Throughout this study a fluorescent marker protein, i.e.…”

“…Alternatively, two distinct monocistronic genes could be packaged within the same carrier, and being taken up and co-expressed by the same cell. [ 25 , 26 ]…”

“…Alternatively, two distinct monocistronic genes could be packaged within the same carrier, and being taken up and co-expressed by the same cell. [25,26] The aim of this study was to find the most reliable and robust gene co-delivery approach for simultaneous production of two proteins in the same cell, by comparing two commonly used strategies including delivery of a "bicistronic" gene versus co-delivery of two distinct "monocistronic" genes. We hypothesized that co-expression of two transgenes directly coupled by 2A-design should be most efficient to ensure predictable synthesis of both the corresponding proteins in a cell, due to the inherently equivalent molar ratio of the two genes encoded in the same open reading frame, as one transcription unit with continuous ribosomal protein synthesis.…”

Co-Delivery of mRNA and pDNA Using Thermally Stabilized Coacervate-Based Core-Shell Nanosystems

“…In the past decades, several nanocarriers have been developed for the delivery of either mRNA, siRNA, or pDNA. Often, the carrier would show success in discrete delivery of more than one NA species with minor modifications from one species to the other [9][10][11][12]. Yet very little research has so far been dedicated to the simultaneous delivery of more than one NA species on the same carrier [13][14][15].…”