Publication - Abstract
Feb 04, 2020
Gene delivery for the purpose of protein expression has been traditionally performed by electroporation or viral transduction of plasmid DNA. Plasmid delivery has numerous challenges as they could potentially integrate into host genome and require nuclear localization. They are also laborious and time consuming to construct and propagate and are not practical options for clinical applications. Hence, mRNA delivery is a desirable alternative for gene expression that overcomes some of the challenges of plasmid delivery and can be chemically synthesized and modified. mRNA however, is highly unstable in biological fluids and needs a transfection vehicle. Lipid nanoparticles (LNP) are known as an efficient delivery vehicle for nucleic acids and can deliver mRNA both in vitro and in vivo. Lipid nanoparticles inhibit mRNA degradation in biological fluids and facilitate cellular uptake. Following uptake and delivery via lipid nanoparticles, mRNA is released intracellularly by endosomal uptake and maturation which then leads to mRNA translation and expression of protein. However, it is speculated that only a very small percentage of lipid nanoparticle cargo (1-2%) can escape the endosome with the rest of the cargo degraded in the lysosome. The Sahay group from Oregon State University, in collaboration with Moderna Therapeutics, Massachusetts, have developed a method through which the intracellular delivery of mRNA lipid nanoparticles is enhanced. The Sahay group used the NanoAssemblrTM Benchtop instrument to formulate lipid nanoparticles containing mRNA which were tested both in vitro and in vivo to study the effect of endosomal/ lysosomal trafficking proteins and their modulators on mRNA uptake.
To study the intracellular trafficking of lipid nanoparticle-associated mRNA, the authors applied CRISPR-Cas technology to perturb the lysosomal pathway in haploid cells, omitting Rab5A, Rab4A or Rab7A proteins which are involved in early, recycling, and late endosome biogenesis. Treatment of the genetically modified cells with mRNA-lipid nanoparticles indicated that absence of Rab7A caused the most pronounced effect with a significant decrease of gene expression. Moreover, mRNA uptake was not greatly affected in the none of the cells lacking Rab proteins. These results indicate that late endosome formation, which is associated with Rab7A, is critical for exogenous gene expression. mRNA expression and regulation has also been associated with mechanistic target of rapamycin complex 1 (mTORC1), a complex that resides on the lysosome surface; it was hypothesized that deletion of Rab7A prevents mTORC1 from triggering expression. The authors therefore, treated cells with mTORC1 inhibitors and found reduced expression of mRNA-lipoplex. Conversely, constitutive activation of mTORC1 led to enhanced mRNA expression through sonication. Overall, these experiments indicated mTORC1 signaling plays a crucial role in exogenous mRNA expression.
In search for molecules that increase nucleic acid transfection, the authors screened a lipid library by pre-treating cells with a library of compounds prior to transfection with lipoplexes and found that MK-571, a leukotriene inhibitor enhanced transfection by 200%. Interestingly Leukotriene inhibitors are currently used in the clinic for treatment of asthma and other allergic reactions. The authors also tested three other clinically approved leukotriene antagonists, two of which (Pranlukast and Zafirlukast) had comparable activity to MK-571. MK-571 also enhanced gene delivery of mRNA-lipid nanoparticles, therefore, the authors developed formulations with MK-571 incorporated into the lipid nanoparticles (LNP-MK-571). LNP-MK-571 significantly outperformed the LNP in vitro and in vivo with 2-fold increase in gene delivery in mice for the LNP-MK-571, suggesting that including bioactive lipids in lipid nanoparticle composition can promote mRNA expression.
This article investigates the cellular uptake and trafficking of synthetically delivered mRNA concluding that biogenesis of the late endosome is potentially the most crucial step in endosomal escape and gene delivery. Additionally, the authors showed a correlation between the mTORC1 signaling and exogenous mRNA expression, indicating that mTORC1 activators can potentially be incorporated into lipid nanoparticles to improve mRNA delivery in vitro and in vivo. A better understanding of intracellular transport mechanisms for nucleic acids can lead to more potent formulations for gene delivery.
Intracellular delivery of mRNA holds great potential for vaccine1−3 and therapeutic4 discovery and development. Despite increasing recognition of the utility of lipid-based nanoparticles (LNPs) for intracellular delivery of mRNA, particle engineering is hindered by insufficient understanding of endosomal escape, which is believed to be a main limiter of cytosolic availability and activity of the nucleic acid inside the cell. Using a series of CRISPR-based genetic perturbations of the lysosomal pathway, we have identified that late endosome/lysosome (LE/Ly) formation is essential for functional delivery of exogenously presented mRNA. Lysosomes provide a spatiotemporal hub to orchestrate mTOR signaling and are known to control cell proliferation, nutrient sensing, ribosomal biogenesis, and mRNA translation. Through modulation of the mTOR pathway we were able to enhance or inhibit LNP-mediated mRNA delivery. To further boost intracellular delivery of mRNA, we screened 212 bioactive lipid-like molecules that are either enriched in vesicular compartments or modulate cell signaling. Surprisingly, we have discovered that leukotriene-antagonists, clinically approved for treatment of asthma and other lung diseases, enhance intracellular mRNA delivery in vitro (over 3-fold, p < 0.005) and in vivo (over 2-fold, p < 0.005). Understanding LNP-mediated intracellular delivery will inspire the next generation of RNA therapeutics that have high potency and limited toxicity.
Publication - Abstract
Feb 04, 2020
Publication - Abstract
Feb 17, 2016
Journal of Bone and Mineral Research
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