Single-cell mRNA transfection studies: Delivery, kinetics and statistics by numbers

Leonhardt, Carolin; Schwake, Gerlinde; Stögbauer, Tobias; Rappl, Susanne; Kuhr, Jan-Timm; Ligon, Thomas; Rädler, Joachim O.

doi:10.1016/j.nano.2013.11.008

Cited by 107 publications

(99 citation statements)

References 44 publications

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“…Our measurements of physical mRNA stability via qRT-PCR include the entire population of translated and untranslated mRNA whereas any measurement of improvements in total protein amounts can be viewed as increases in therapeutic potential or “functional half-life” (a measure of productivity). Micro-pattern based single-cell arrays allow the estimation of the functional half-life in a high-throughput manner3536. Using these arrays, similar productivity enhancing effect of CYBA UTRs in A549 and Huh7 cells has been previously reported35.…”

Section: Discussionmentioning

confidence: 72%

Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts

Ferizi

Aneja

Balmayor

et al. 2016

Sci Rep

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Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5′- and 3′-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5′-UTR and/or downstream 3′-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs.

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Section: Discussionmentioning

confidence: 72%

Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts

Ferizi

Aneja

Balmayor

et al. 2016

Sci Rep

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“…(a) Sketch and mathematical formulation of the mathematical model of mRNA transfection presented by [36]. The intracellular release of mRNA at time point t r is modeled using the Dirac delta distribution δ .…”

Section: Methodsmentioning

confidence: 99%

Scalable Parameter Estimation for Genome-Scale Biochemical Reaction Networks

et al. 2017

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Mechanistic mathematical modeling of biochemical reaction networks using ordinary differential equation (ODE) models has improved our understanding of small- and medium-scale biological processes. While the same should in principle hold for large- and genome-scale processes, the computational methods for the analysis of ODE models which describe hundreds or thousands of biochemical species and reactions are missing so far. While individual simulations are feasible, the inference of the model parameters from experimental data is computationally too intensive. In this manuscript, we evaluate adjoint sensitivity analysis for parameter estimation in large scale biochemical reaction networks. We present the approach for time-discrete measurement and compare it to state-of-the-art methods used in systems and computational biology. Our comparison reveals a significantly improved computational efficiency and a superior scalability of adjoint sensitivity analysis. The computational complexity is effectively independent of the number of parameters, enabling the analysis of large- and genome-scale models. Our study of a comprehensive kinetic model of ErbB signaling shows that parameter estimation using adjoint sensitivity analysis requires a fraction of the computation time of established methods. The proposed method will facilitate mechanistic modeling of genome-scale cellular processes, as required in the age of omics.

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“…Unlike plasmid DNA and viral vectors, mRNA do not integrate into the host genome, avoiding aberrant transcription and insertional mutagenesis. 15 Its expression kinetics is predictable and consistent, 14,16,17 while nuclear localization of mRNA is not required before rapid protein expression even in nondividing and hard-to-transfect cells. 18,19 Moreover, mRNA is only transiently active and is completely biodegradable via metabolic pathways.…”

Section: 12-14mentioning

confidence: 95%

Delivery of modified mRNA encoding vesicular stomatitis virus matrix protein for colon cancer gene therapy

Men

Zhang

et al. 2018

RSC Adv.

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Plasmid DNA based gene delivery has been widely utilized among both pre-clinical and clinical gene therapy studies. However, therapeutic efficiency is usually limited by the size and potential immunestimulation issue of plasmid backbone. As an alternative form of genetic material, chemically modified messenger RNA (mRNA) provides a promising alternative to plasmid DNA. In this work, an in vitro transcription mRNA encoding vesicular stomatitis virus matrix protein (VSVMP) was delivered by a cationic liposome-protamine complex, resulting in high mRNA transporting and expression efficiency.The liposome-protamine complex delivered VSVMP mRNA strongly inhibits the growth of C26 tumor cells through inducing apoptosis, while obvious tumor regressions were achieved on both abdominal cavity metastatic and subcutaneous xenograft models in vivo with high safety. Our results also demonstrated that the liposome-protamine-mRNA complex was as potent as its plasmid DNA counterpart, showing strong potential in further colon cancer therapy.

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Single-cell mRNA transfection studies: Delivery, kinetics and statistics by numbers

Cited by 107 publications

References 44 publications

Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts

Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts

Scalable Parameter Estimation for Genome-Scale Biochemical Reaction Networks

Delivery of modified mRNA encoding vesicular stomatitis virus matrix protein for colon cancer gene therapy

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