Characterizing exogenous mRNA delivery, trafficking, cytoplasmic release and RNA–protein correlations at the level of single cells

Kirschman, Jonathan L.; Bhosle, Sushma M.; Vanover, Daryll; Blanchard, Emmeline L.; Loomis, Kristin; Zurla, Chiara; Murray, Kathryn; Lam, Blaine C; Santangelo, Philip J.

doi:10.1093/nar/gkx290

Cited by 61 publications

(70 citation statements)

References 47 publications

(62 reference statements)

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“…MTRIPs were also utilized to study the RSV genome, its organization in viral particles, its localization in infected cells and interactions with viral and host cells [37,38]. Recently, we applied this single RNA sensitive probes to visualize synthetic mRNA uptake in vitro and in ex vivo tissue sections, as well as to characterize the release kinetics from endocytic compartments, and mRNA-protein expression correlations [39].…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Unifying in vitro and in vivo IVT mRNA expression discrepancies in skeletal muscle via mechanotransduction

et al. 2018

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The translational efficiency of an in vitro transcribed (IVT) mRNA was measured upon delivery to primary skeletal muscle cells and to a mouse model system, towards the development of a predictive in vitro assay for the screening and validation of intramuscular mRNA-based vaccines. When IVT mRNA was delivered either naked or complexed with novel aminoglycoside-based delivery vehicles, significant differences in protein expression in vitro and in vivo were observed. We hypothesized that this previously anticipated discrepancy was due to differences in the mechanism of IVT mRNA endosomal entry and release following delivery. To address this, IVT mRNA was fluorescently labeled prior to delivery, to visualize its distribution. Colocalization with endosomal markers indicated that different entry pathways were utilized in vivo and in vitro, depending on the delivery vehicle, resulting in variations in protein expression levels. Since extracellular matrix stiffness (ECM) influences mRNA entry, trafficking and release, the effect of mechanotransduction on mRNA expression was investigated in vitro upon delivery of IVT mRNA alone, and complexed with delivery vehicles to skeletal muscle cells grown on ∼10 kPa hydrogels. This in vitro hydrogel model more accurately recapitulated the results obtained in vivo upon IM injection, indicating that this approach may assist in the characterization of mRNA based vaccines.

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Section: Accepted Manuscriptmentioning

confidence: 99%

Unifying in vitro and in vivo IVT mRNA expression discrepancies in skeletal muscle via mechanotransduction

et al. 2018

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“…It appears however, that nucleic acids nanocarriers are largely trapped inside the endosomes or lysosomes and that delivery by lipid nanoparticles is limited by endocytic recycling (8). In timeresolved fluorescence microscopy studies, the uptake and fate of delivery particles has been visualized at the single-cell level (9)(10)(11)(12)(13). Recently, it was shown that siRNA release occurred during a narrow 'window of opportunity' showing that timing and efficiency are linked (12).…”

Section: Introductionmentioning

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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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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“…[1–2] Nevertheless, inefficient transfection of exogenous mRNA remains a key barrier to broad applications of mRNA-based drugs. [10] …”

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confidence: 99%

Polyamine‐Mediated Stoichiometric Assembly of Ribonucleoproteins for Enhanced mRNA Delivery

Wang

et al. 2017

Angew Chem Int Ed

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Messenger RNA (mRNA) represents a promising class of nucleic acid drugs. Although numerous carriers have been developed for mRNA delivery, the inefficient mRNA expression inside cells remains a major challenge. Inspired by the dependence of mRNA on 3′ terminal poly adenosine nucleotides (poly A) and poly A binding proteins (PABPs) for optimal expression, we complex synthetic mRNA containing a poly A tail with PABPs in a stoichiometric manner and stabilize the ribonucleoproteins (RNPs) via a family of polypeptides bearing different arrangements of cationic side groups. We find that the molecular structure of these polypeptides modulates the degree of PABP-mediated enhancement of mRNA expression. This strategy elicits an up to 20-fold increase in mRNA expression in vitro and ~4-fold increase in mice. These findings suggest a set of new design principles for gene delivery via synergistic co-assembly of mRNA with helper proteins.

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Characterizing exogenous mRNA delivery, trafficking, cytoplasmic release and RNA–protein correlations at the level of single cells

Cited by 61 publications

References 47 publications

Unifying in vitro and in vivo IVT mRNA expression discrepancies in skeletal muscle via mechanotransduction

Unifying in vitro and in vivo IVT mRNA expression discrepancies in skeletal muscle via mechanotransduction

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

Polyamine‐Mediated Stoichiometric Assembly of Ribonucleoproteins for Enhanced mRNA Delivery

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