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Schematic overview of different transfection methods: integrated co-transfection a refers to mixing different IVT-mRNAs prior to complexation with carrier, whereas in parallel co-transfections b, IVT-mRNAs are complexed in particles and added to cells separately, and in successive transfections c, cells are transfected with two types of IVT-mRNA with a 24-h interval. d Cellular uptake of different ratios of the two types of mRNA (vertical axis) and of different doses of both IVT-mRNAs (horizontal axis) results in different color distribution and intensity and can be used as a key readout for this study
Source publication
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- a...
Citations
... 4,12 Moreover, transduction with multiple vectors proves significantly less efficient compared to single vectors, thereby restricting the potential applications of cell engineering that involve targeting multiple genes. 13 The application of cell-instructive materials to guide cellular responses, especially in delivery systems, represents a state-of-the-art approach. Typically designed at the nanoscale or microscale, these materials provide cues to cells through biochemical signals and physical attributes like stiffness and topography. ...
Efficient cellular uptake of biomolecules, including genetic material, mRNA, proteins, and nanoparticles, requires novel approaches to overcome inherent cellular barriers. This study investigates how nanotopographical cues from nanoporous surfaces impact the uptake efficiency of diverse molecules by cells. The results demonstrate that cellular uptake efficiency increases significantly on nanoporous surfaces compared to flat surfaces. Notably, this process is found to be dependent on the size and morphology of the nanopores, reaching its peak efficacy with blind pores of 400 nm in size. Enhanced genetic transduction on nanoporous surfaces were observed for multiple vectors, including lentiviruses, baculoviruses, and mRNA molecules. The versatile nature of this approach allows co-transfection of cells with multiple mRNA vectors. Moreover, the nanoporous platform was used for efficient and fast manufacturing of CAR-T cells through lentiviral transduction. Furthermore, we pinpoint macropinocytosis as the predominant mechanism driving increased cellular uptake induced by the nanoporous surfaces. The method introduced here for enhancing genetic transduction of cells has applications in immunotherapy research, drug delivery, and cell engineering.
... To this end, we relied on our previous observation of highly efficient co-delivery of nucleic acids (NA) via integrated co-transfection (iCo-TF) (i.e., premixing of different NA entities before complexation), which was experimentally validated by quantifying downstream protein production via flow cytometry and fluorescent microscopy. 30 Here, we identified similar encapsulation efficiencies for distinct IVT-mRNAs chemistries ( Figure 2b). Taken together, while the uptake was not directly measured in context of intracellular trafficking mechanism, we concluded that equal amounts of IVT-mRNA molecules of the same size can be delivered to individual cells by employing the iCo-TF method, independent of their chemistry or coding sequence. ...
... Taken together, while the uptake was not directly measured in context of intracellular trafficking mechanism, we concluded that equal amounts of IVT-mRNA molecules of the same size can be delivered to individual cells by employing the iCo-TF method, independent of their chemistry or coding sequence. 30 F I G U R E 1 Deconvolution of synthetic mRNA transfection and expression. (a) Distinct steps in cell-based IVT-mRNA expression. ...
Recent technological advances in the production of in vitro transcribed messenger RNA (IVT‐mRNA) facilitate its clinical use as well as its application in basic research. In this regard, numerous chemical modifications, which are not naturally observed in endogenous mRNA, have been implemented primarily to address the issue of immunogenicity and improve its biological performance. However, recent findings suggested pronounced differences between expression levels of IVT‐mRNAs with different nucleoside modifications in transfected cells. Given the multistep process of IVT‐mRNA delivery and subsequent intracellular expression, it is unclear which step is influenced by IVT‐mRNA chemistry. Here, we deconvolute this process and show that the nucleoside modification does not interfere with complexation of carriers, their physicochemical properties, and extracellular stability, as exemplified by selected modifications. The immediate effect of mRNA chemistry on the efficiency of ribosomal protein synthesis as a contributor to differences in expression was quantified by in vitro cell‐free translation. Our results demonstrate that for the nucleoside modifications tested, translatability was the decisive step in determining overall protein production. Also of special importance for future work on rational selection of tailored synthetic mRNA chemistries, our findings set a workflow to identify potentially limiting, modification‐dependent steps in the complex delivery process.
... 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. ...
... 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. enhanced green fluorescent protein (EGFP) was selected as the second protein, in order to enable facile and prompt monitoring of gene expression, investigated by fluorescent microscopy and quantified via flow cytometry. ...
Maximizing the efficiency of nanocarrier-mediated co-delivery of genes for co-expression in the same cell is critical for many applications. Strategies to maximize co-delivery of nucleic acids (NA) focused largely on carrier systems, with little attention towards payload composition itself. Here, we investigated the effects of different payload designs: co-delivery of two individual “monocistronic” NAs versus a single bicistronic NA comprising two genes separated by a 2A self-cleavage site. Unexpectedly, co-delivery via the monocistronic design resulted in a higher percentage of co-expressing cells, while predictive co-expression via the bicistronic design remained elusive. Our results will aid the application-dependent selection of the optimal methodology for co-delivery of genes.
Graphical abstract
... 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. ...
In vitro transcribed (IVT)-mRNA has been accepted as a promising therapeutic modality. Advances in facile and rapid production technologies make IVT-mRNA an appealing alternative to protein- or virus-based medicines. Robust expression levels, lack of genotoxicity and their manageable immunogenicity benefit its clinical applicability. We postulated that innate immune responses of therapeutically relevant human cells can be tailored or abrogated by combinations of 5’-end and internal IVT-mRNA modifications. Using primary human macrophages as targets, our data show the particular importance of uridine modifications for IVT-mRNA performance. Among five nucleotide modification schemes tested, 5-methoxy-uridine outperformed other modifications up to 4-fold increased transgene expression, triggering moderate proinflammatory and non-detectable antiviral responses. Macrophage responses against IVT-mRNAs exhibiting high immunogenicity (e.g., pseudouridine) could be minimized upon HPLC purification. Conversely, 5’-end modifications, had only modest effects on mRNA expression and immune responses. Our results revealed how the uptake of chemically modified IVT-mRNA impacts human macrophages, responding with distinct patterns of innate immune responses concomitant with increased transient transgene expression. We anticipate our findings are instrumental to predictively address specific cell responses required for wide range of therapeutic applications from eliciting controlled immunogenicity in mRNA vaccines to, e.g., completely abrogating cell activation in protein replacement therapies.
... There is also a low risk of human exposure to RNA during food consumption, as biologically there are multitudinous barriers to RNA exposure, such as the abundant presence of RNAse in saliva and the GI tract, as well as the low pH of the stomach [328]. For a commercial cultured fat process, the close to 100% transfection efficiency of RNA delivery also means that a majority of proliferated cells could be converted into adipocytes, which is advantageous for yield and cost [329][330][331][332]. There have been no reports of using mRNA to express adipogenic genes and generate adipocytes from precursor cells. ...
With rising global demand for food proteins and significant environmental impact associated with conventional animal agriculture, it is important to develop sustainable alternatives to supplement existing meat production. Since fat is an important contributor to meat flavor, recapitulating this component in meat alternatives such as plant based and cell cultured meats is important. Here, we discuss the topic of cell cultured or tissue engineered fat, growing adipocytes in vitro that could imbue meat alternatives with the complex flavor and aromas of animal meat. We outline potential paths for the large scale production of in vitro cultured fat, including adipogenic precursors during cell proliferation, methods to adipogenically differentiate cells at scale, as well as strategies for converting differentiated adipocytes into 3D cultured fat tissues. We showcase the maturation of knowledge and technology behind cell sourcing and scaled proliferation, while also highlighting that adipogenic differentiation and 3D adipose tissue formation at scale need further research. We also provide some potential solutions for achieving adipose cell differentiation and tissue formation at scale based on contemporary research and the state of the field.
Efficient cellular uptake of biomolecules, including genetic material, mRNA, proteins, and nanoparticles, requires novel approaches to overcome inherent cellular barriers. Here, the study investigates how nanotopographical cues from nanoporous surfaces impact the uptake efficiency by cells. The results demonstrate notable enhancements in cellular uptake efficiency across a range of vectors when cells are exposed to nanoporous surfaces. The uptake process is found to be dependent on the size and morphology of the nanopores, reaching a peak efficacy with blind pores of 400 nm in size. Enhanced genetic transduction on nanoporous surfaces are observed for multiple vectors, including lentiviruses, baculoviruses, and mRNA molecules. The versatile nature of this approach allows co‐transfection of cells with multiple mRNA vectors. Moreover, the nanoporous platform is used for efficient and fast manufacturing of Chimeric Antigen Receptor (CAR)‐T cells through lentiviral transduction. Furthermore, the study pinpoints macropinocytosis as the predominant mechanism driving increased cellular uptake induced by the nanoporous surfaces. The introduced method for enhancing genetic transduction of cells has applications in immunotherapy research, drug delivery, and cell engineering.
Liposomes can efficiently deliver messenger RNA (mRNA) into cells. When mRNA cocktails encoding different proteins are needed, a considerable challenge is to efficiently deliver all mRNAs into the cytosol of each individual cell. In this work, two methods are explored to co-deliver varying ratiometric doses of mRNA encoding red (R) or green (G) fluorescent proteins and it is found that packaging mRNAs into the same lipoplexes (mingle-lipoplexes) is crucial to efficiently deliver multiple mRNA types into the cytosol of individual cells according to the pre-defined ratio. A mixture of lipoplexes containing only one mRNA type (single-lipoplexes), however, seem to follow the “first come – first serve” principle, resulting in a large variation of R/G uptake and expression levels for individual cells leading to ratiometric dosing only on the population level, but rarely on the single-cell level. These experimental observations are quantitatively explained by a theoretical framework based on the stochasticity of mRNA uptake in cells and endosomal escape of mingle- and single-lipoplexes, respectively. Furthermore, the findings are confirmed in 3D retinal organoids and zebrafish embryos, where mingle-lipoplexes outperformed single-lipoplexes to reliably bring both mRNA types into single cells. This benefits applications that require a strict control of protein expression in individual cells.
Co-delivery of different species of protein-encoding polynucleotides, e.g., messenger RNA (mRNA) and plasmid DNA (pDNA), using the same nanocarrier is an interesting topic that remains scarcely researched in the field of nucleic acid delivery. The current study hence aims to explore the possibility of the simultaneous delivery of mRNA (mCherry) and pDNA (pAmCyan) using a single nanocarrier. The latter is based on gelatin type A, a biocompatible, and biodegradable biopolymer of broad pharmaceutical application. A core-shell nanostructure is designed with a thermally stabilized gelatin-pDNA coacervate in its center. Thermal stabilization enhances the core's colloidal stability and pDNA shielding effect against nucleases as confirmed by nanoparticle tracking analysis and gel electrophoresis, respectively. The stabilized, pDNA-loaded core is coated with the cationic peptide protamine sulfate to enable additional surface-loading with mRNA. The dual-loaded core-shell system transfects murine dendritic cell line DC2.4 with both fluorescent reporter mRNA and pDNA simultaneously, showing a transfection efficiency of 61.4 ± 21.6% for mRNA and 37.6 ± 19.45% for pDNA, 48 h post-treatment, whereas established commercial, experimental, and clinical transfection reagents fail. Hence, the unique co-transfectional capacity and the negligible cytotoxicity of the reported system may hold prospects for vaccination among other downstream applications.
