Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

Yasar, Hanzey; Biehl, Alexander; Rossi, Chiara; Koch, Marcus; Murgia, Xabi; Loretz, Brigitta; Lehr, Claus‐Michael

doi:10.1186/s12951-018-0401-y

Cited by 53 publications

(38 citation statements)

References 41 publications

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“…Other studies also used multiple fluorescent signals in single cell time lapse measurement to exploit signal correlations (36,37). Our findings are in agreement with a study by Yasar et al showing that the kinetics of mRNA nanocarrier binding on the cell surface is the same for transfected and nontransfected cells without giving further insights on transfection efficiency (26). In future work, using fluorescent markers, which label endosomes or lysosomes, might reveal intermediate delivery steps and enable further correlation studies with endocytolytic release events.…”

Section: Discussionsupporting

confidence: 90%

“…In our approach, we used the expression rate as a measure for transfection efficiency. We showed that the expression rate constant m0kTL, the product of released mRNA molecules and the kinetic translation rate, is proportional to the protein level at a defined time point after transfection, which is more frequently used as the measure for transfection efficiency (25,26). The proportionality can be explained because the expression rate corresponds to the slope of a straight line between the expression onset and a certain protein level i.e.…”

Section: Discussionmentioning

confidence: 99%

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Correlation of mRNA delivery timing and protein expression in lipid-based transfection

Reiser

Woschée

Mehrotra

et al. 2019

Preprint

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Non-viral gene delivery is constrained by the dwell time that most synthetic nucleic acid nanocarriers spend inside endosomal compartments. In order to overcome this endosomalrelease bottleneck, methods are required that measure nanocarrier uptake kinetics and transfection efficiency simultaneously. Here, we employ live-cell imaging on single-cell arrays (LISCA) to study the delivery-time distribution of lipid-based mRNA complexes under varied serum conditions. By fitting a translation-maturation model to hundreds of individual eGFP reporter fluorescence time courses, the protein expression onset times and the expression rates after transfection are determined. Using this approach, we find that delivery timing and protein expression rates are not intrinsically correlated at the single-cell level, even though population-averaged values of both parameters conjointly change as a function of increasing external serum protein fraction. Lipofectamine mediated delivery showed decreased transfection efficiency and longer delivery times with increasing serum protein concentration. This is in contrast to ionizable lipid nanoparticles (LNPs) mediated transfer, which showed increased efficiency and faster uptake in the presence of serum. In conclusion, the interdependences of single-cell expression rates and onset timing provide additional clues on uptake and release mechanisms, which are useful for improving nucleic acid delivery. author/funder. All rights reserved. No reuse allowed without permission.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Correlation of mRNA delivery timing and protein expression in lipid-based transfection

Reiser

Woschée

Mehrotra

et al. 2019

Preprint

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“…Many achievements have been reached in the field of gene delivery of a variety of nucleic acids such as pDNA (Baoum et al 2010), mRNA (Yasar et al 2018), siRNA, and micro-RNA. These nucleic acids can both introduce genes that encode a functional protein that is vital in preventing disease progression or block the translation of specific mRNAs to prevent a toxic effect.…”

Section: Pla and Plga Based Mps And Nps Systems For Gene Deliverymentioning

confidence: 99%

Biocompatibility, biodegradation and biomedical applications of poly(lactic acid)/poly(lactic-co-glycolic acid) micro and nanoparticles

Elmowafy

Tiboni

Soliman

2019

J. Pharm. Investig.

359

208

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Background Poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) are among the well-documented FDAapproved polymers used for the preparation of safe and effective vaccine, drug and gene delivery systems using well-described reproducible methods of fabrication. Various nano and microparticulates are fabricated using these polymers. Their successful performance relies on PLA and PLGA biocompatibility and degradability characteristics. Area covered This review provides an overview of the biocompatibility and biodegradation of PLA, PLGA and their copolymers, with a special emphasis on tissue responses for these polymers as well as their degradation pathways and drug release models. Moreover, the potential of PLA and PLGA based nano and microparticulates in various advanced biomedical applications is highlighted. Expert opinion PLA and PLGA based delivery systems show promises of releasing different drugs, proteins and nucleic acids in a stable and controlled manner and greatly ameliorating their therapeutic efficacy. In addition, advancement in surface modification and targeting of nanoparticles has extended the scope of their utility.

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“…Aside from Flow Cytometry, fluorescence Microscopy has proven to be a valuable tool to monitor transfection and the time course of antigen mRNA expression and to evaluate different delivery systems in vitro [61][62][63][64][65][66]. More recently, live cell video microscopy has also been used to track the uptake of fluorescently labeled mRNA cell line [67,68].…”

Section: Evaluation Using Professional Antigen Presenting Cellsmentioning

confidence: 99%

Establishing Preferred Product Characterization for the Evaluation of RNA Vaccine Antigens

et al. 2019

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The preferred product characteristics (for chemistry, control, and manufacture), in addition to safety and efficacy, are quintessential requirements for any successful therapeutic. Messenger RNA vaccines constitute a relatively new alternative to traditional vaccine development platforms, and thus there is less clarity regarding the criteria needed to ensure regulatory compliance and acceptance. Generally, to identify the ideal product characteristics, a series of assays needs to be developed, qualified and ultimately validated to determine the integrity, purity, stability, and reproducibility of a vaccine target. Here, using the available literature, we provide a summary of the array of biophysical and biochemical assays currently used in the field to characterize mRNA vaccine antigen candidates. Moreover, we review various in vitro functional cell-based assays that have been employed to facilitate the early assessment of the biological activity of these molecules, including the predictive immune response triggered in the host cell. Messenger RNA vaccines can be produced rapidly and at large scale, and thus will particularly benefit from well-defined and well-characterized assays ultimately to be used for in-process, release and stability-indications, which will allow equally rapid screening of immunogenicity, efficacy, and safety without the need to conduct often lengthy and costly in vivo experiments.

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Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

Cited by 53 publications

References 41 publications

Correlation of mRNA delivery timing and protein expression in lipid-based transfection

Correlation of mRNA delivery timing and protein expression in lipid-based transfection

Biocompatibility, biodegradation and biomedical applications of poly(lactic acid)/poly(lactic-co-glycolic acid) micro and nanoparticles

Establishing Preferred Product Characterization for the Evaluation of RNA Vaccine Antigens

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