scholarly journals Successful reprogramming of cellular protein production through mRNA delivered by functionalized lipid nanoparticles

2018 ◽  
Vol 115 (15) ◽  
pp. E3351-E3360 ◽  
Author(s):  
Marianna Yanez Arteta ◽  
Tomas Kjellman ◽  
Stefano Bartesaghi ◽  
Simonetta Wallin ◽  
Xiaoqiu Wu ◽  
...  
Keyword(s):  
Small Angle ◽  
Messenger Rna ◽  
Rational Design ◽  
Protein Production ◽  
Surface Composition ◽  
Cationic Lipid ◽  
Lipid Nanoparticles ◽  
Cellular Protein ◽  
Human Adipocytes ◽  
Characteristic Distance

The development of safe and efficacious gene vectors has limited greatly the potential for therapeutic treatments based on messenger RNA (mRNA). Lipid nanoparticles (LNPs) formed by an ionizable cationic lipid (here DLin-MC3-DMA), helper lipids (distearoylphosphatidylcholine, DSPC, and cholesterol), and a poly(ethylene glycol) (PEG) lipid have been identified as very promising delivery vectors of short interfering RNA (siRNA) in different clinical phases; however, delivery of high-molecular weight RNA has been proven much more demanding. Herein we elucidate the structure of hEPO modified mRNA-containing LNPs of different sizes and show how structural differences affect transfection of human adipocytes and hepatocytes, two clinically relevant cell types. Employing small-angle scattering, we demonstrate that LNPs have a disordered inverse hexagonal internal structure with a characteristic distance around 6 nm in presence of mRNA, whereas LNPs containing no mRNA do not display this structure. Furthermore, using contrast variation small-angle neutron scattering, we show that one of the lipid components, DSPC, is localized mainly at the surface of mRNA-containing LNPs. By varying LNP size and surface composition we demonstrate that both size and structure have significant influence on intracellular protein production. As an example, in both human adipocytes and hepatocytes, protein expression levels for 130 nm LNPs can differ as much as 50-fold depending on their surface characteristics, likely due to a difference in the ability of LNP fusion with the early endosome membrane. We consider these discoveries to be fundamental and opening up new possibilities for rational design of synthetic nanoscopic vehicles for mRNA delivery.

scholarly journals Tuning the Immunostimulation Properties of Cationic Lipid Nanocarriers for Nucleic Acid Delivery

2021 ◽  
Vol 12 ◽  
Author(s):  
Arindam K. Dey ◽  
Adrien Nougarède ◽  
Flora Clément ◽  
Carole Fournier ◽  
Evelyne Jouvin-Marche ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Messenger Rna ◽  
Cationic Lipid ◽  
Lipid Nanoparticles ◽  
Cationic Lipids ◽  
Fine Tuning ◽  
Target Cells ◽  
Substantial Impact ◽  
Lipid Nanocarriers ◽  
The Impact

Nonviral systems, such as lipid nanoparticles, have emerged as reliable methods to enable nucleic acid intracellular delivery. The use of cationic lipids in various formulations of lipid nanoparticles enables the formation of complexes with nucleic acid cargo and facilitates their uptake by target cells. However, due to their small size and highly charged nature, these nanocarrier systems can interact in vivo with antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. As this might prove to be a safety concern for developing therapies based on lipid nanocarriers, we sought to understand how they could affect the physiology of APCs. In the present study, we investigate the cellular and metabolic response of primary macrophages or DCs exposed to the neutral or cationic variant of the same lipid nanoparticle formulation. We demonstrate that macrophages are the cells affected most significantly and that the cationic nanocarrier has a substantial impact on their physiology, depending on the positive surface charge. Our study provides a first model explaining the impact of charged lipid materials on immune cells and demonstrates that the primary adverse effects observed can be prevented by fine-tuning the load of nucleic acid cargo. Finally, we bring rationale to calibrate the nucleic acid load of cationic lipid nanocarriers depending on whether immunostimulation is desirable with the intended therapeutic application, for instance, gene delivery or messenger RNA vaccines.

scholarly journals Tuning the immunostimulation properties of cationic lipid nanocarriers for nucleic acid delivery

2021 ◽  
Author(s):  
Arindam K Dey ◽  
Adrien Nougarede ◽  
Flora Clement ◽  
Carole Fournier ◽  
Evelyne Jouvin-Marche ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Messenger Rna ◽  
Cationic Lipid ◽  
Lipid Nanoparticles ◽  
Cationic Lipids ◽  
Fine Tuning ◽  
Target Cells ◽  
Substantial Impact ◽  
Lipid Nanocarriers ◽  
The Impact

Nonviral systems, such as lipid nanoparticles, have emerged as reliable methods to enable nucleic acid intracellular delivery. The use of cationic lipids in various formulations of lipid nanoparticles enables the formation of complexes with nucleic acid cargo and facilitates their uptake by target cells. However, due to their small size and highly charged nature, these nanocarrier systems can interact in vivo with antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. As this might prove to be a safety concern for developing therapies based on lipid nanocarriers, we sought to understand how they could affect the physiology of APCs. In the present study, we investigate the cellular and metabolic response of primary macrophages or DCs exposed to the neutral or cationic variant of the same lipid nanoparticle formulation. We demonstrate that macrophages are the cells affected most significantly and that the cationic nanocarrier has a substantial impact on their physiology, depending on the positive surface charge. Our study provides a first model explaining the impact of charged lipid materials on immune cells and demonstrates that the primary adverse effects observed can be prevented by fine-tuning the load of nucleic acid cargo. Finally, we bring rationale to calibrate the nucleic acid load of cationic lipid nanocarriers depending on whether immunostimulation is desirable with the intended therapeutic application, for instance, gene delivery or messenger RNA vaccines.

scholarly journals Hybrid Biopolymer and Lipid Nanoparticles with Improved Transfection Efficacy for mRNA

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2034 ◽  
Author(s):  
Christian D. Siewert ◽  
Heinrich Haas ◽  
Vera Cornet ◽  
Sara S. Nogueira ◽  
Thomas Nawroth ◽  
...  
Keyword(s):  
Small Angle ◽  
Messenger Rna ◽  
Physicochemical Characterization ◽  
Single Step ◽  
Model Systems ◽  
Molecular Organization ◽  
Hybrid Nanoparticles ◽  
Polymer Mixture ◽  
Internal Organization ◽  
Transfection Efficacy

Hybrid nanoparticles from lipidic and polymeric components were assembled to serve as vehicles for the transfection of messenger RNA (mRNA) using different portions of the cationic lipid DOTAP (1,2-Dioleoyl-3-trimethylammonium-propane) and the cationic biopolymer protamine as model systems. Two different sequential assembly approaches in comparison with a direct single-step protocol were applied, and molecular organization in correlation with biological activity of the resulting nanoparticle systems was investigated. Differences in the structure of the nanoparticles were revealed by thorough physicochemical characterization including small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM). All hybrid systems, combining lipid and polymer, displayed significantly increased transfection in comparison to lipid/mRNA and polymer/mRNA particles alone. For the hybrid nanoparticles, characteristic differences regarding the internal organization, release characteristics, and activity were determined depending on the assembly route. The systems with the highest transfection efficacy were characterized by a heterogenous internal organization, accompanied by facilitated release. Such a system could be best obtained by the single step protocol, starting with a lipid and polymer mixture for nanoparticle formation.

scholarly journals Intracellular Routing and Recognition of Lipid-Based mRNA Nanoparticles

Pharmaceutics ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 945
Author(s):  
Christophe Delehedde ◽  
Luc Even ◽  
Patrick Midoux ◽  
Chantal Pichon ◽  
Federico Perche
Keyword(s):  
Gene Therapy ◽  
Immune Responses ◽  
Transient Expression ◽  
Messenger Rna ◽  
Lipid Nanoparticles ◽  
Delivery Systems ◽  
Clinical Applications ◽  
Cellular Proteins ◽  
Mrna Sequence ◽  
Cytoplasmic Expression

Messenger RNA (mRNA) is being extensively used in gene therapy and vaccination due to its safety over DNA, in the following ways: its lack of integration risk, cytoplasmic expression, and transient expression compatible with fine regulations. However, clinical applications of mRNA are limited by its fast degradation by nucleases, and the activation of detrimental immune responses. Advances in mRNA applications, with the recent approval of COVID-19 vaccines, were fueled by optimization of the mRNA sequence and the development of mRNA delivery systems. Although delivery systems and mRNA sequence optimization have been abundantly reviewed, understanding of the intracellular processing of mRNA is mandatory to improve its applications. We will focus on lipid nanoparticles (LNPs) as they are the most advanced nanocarriers for the delivery of mRNA. Here, we will review how mRNA therapeutic potency can be affected by its interactions with cellular proteins and intracellular distribution.

scholarly journals Ionizable lipid nanoparticles for in utero mRNA delivery

2021 ◽  
Vol 7 (3) ◽  
pp. eaba1028
Author(s):  
Rachel S. Riley ◽  
Meghana V. Kashyap ◽  
Margaret M. Billingsley ◽  
Brandon White ◽  
Mohamad-Gabriel Alameh ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Gene Editing ◽  
Genetic Diseases ◽  
Lipid Nanoparticles ◽  
In Utero ◽  
Luciferase Mrna ◽  
Fetal Size ◽  
Mouse Fetuses ◽  
Clinical Advances ◽  
Immature Immune System

Clinical advances enable the prenatal diagnosis of genetic diseases that are candidates for gene and enzyme therapies such as messenger RNA (mRNA)–mediated protein replacement. Prenatal mRNA therapies can treat disease before the onset of irreversible pathology with high therapeutic efficacy and safety due to the small fetal size, immature immune system, and abundance of progenitor cells. However, the development of nonviral platforms for prenatal delivery is nascent. We developed a library of ionizable lipid nanoparticles (LNPs) for in utero mRNA delivery to mouse fetuses. We screened LNPs for luciferase mRNA delivery and identified formulations that accumulate within fetal livers, lungs, and intestines with higher efficiency and safety compared to benchmark delivery systems, DLin-MC3-DMA and jetPEI. We demonstrate that LNPs can deliver mRNAs to induce hepatic production of therapeutic secreted proteins. These LNPs may provide a platform for in utero mRNA delivery for protein replacement and gene editing.

scholarly journals Systems Analysis of Transcriptomic and Proteomic Profiles Identifies Novel Regulation of Fibrotic Programs by miRNAs in Pulmonary Fibrosis Fibroblasts

Genes ◽  
2018 ◽  
Vol 9 (12) ◽  
pp. 588 ◽  
Author(s):  
Steven Mullenbrock ◽  
Fei Liu ◽  
Suzanne Szak ◽  
Xiaoping Hronowski ◽  
Benbo Gao ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Systems Analysis ◽  
Messenger Rna ◽  
Protein Production ◽  
Lung Fibroblasts ◽  
Effector Cells ◽  
Signature Genes ◽  
Gene And Protein Expression ◽  
Lung Transplants ◽  
Generation Sequencing

Fibroblasts/myofibroblasts are the key effector cells responsible for excessive extracellular matrix (ECM) deposition and fibrosis progression in both idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) patient lungs, thus it is critical to understand the transcriptomic and proteomic programs underlying their fibrogenic activity. We conducted the first integrative analysis of the fibrotic programming in these cells at the levels of gene and microRNA (miRNA) expression, as well as deposited ECM protein to gain insights into how fibrotic transcriptional programs culminate in aberrant ECM protein production/deposition. We identified messenger RNA (mRNA), miRNA, and deposited matrisome protein signatures for IPF and SSc fibroblasts obtained from lung transplants using next-generation sequencing and mass spectrometry. SSc and IPF fibroblast transcriptional signatures were remarkably similar, with enrichment of WNT, TGF-β, and ECM genes. miRNA-seq identified differentially regulated miRNAs, including downregulation of miR-29b-3p, miR-138-5p and miR-146b-5p in disease fibroblasts and transfection of their mimics decreased expression of distinct sets of fibrotic signature genes as assessed using a Nanostring fibrosis panel. Finally, proteomic analyses uncovered a distinct “fibrotic” matrisome profile deposited by IPF and SSc fibroblasts compared to controls that highlights the dysregulated ECM production underlying their fibrogenic activities. Our comprehensive analyses of mRNA, miRNA, and matrisome proteomic profiles in IPF and SSc lung fibroblasts revealed robust fibrotic signatures at both the gene and protein expression levels and identified novel fibrogenesis-associated miRNAs whose aberrant downregulation in disease fibroblasts likely contributes to their fibrotic and ECM gene expression.

scholarly journals Cationic and Biocompatible Polymer/Lipid Nanoparticles as Immunoadjuvants

Pharmaceutics ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 1859
Author(s):  
Yunys Pérez-Betancourt ◽  
Péricles Marques Araujo ◽  
Bianca de Carvalho Lins Fernandes Távora ◽  
Daniele Rodrigues Pereira ◽  
Eliana Lima Faquim-Mauro ◽  
...  
Keyword(s):  
Immune Responses ◽  
Methyl Methacrylate ◽  
Cationic Lipid ◽  
Lipid Nanoparticles ◽  
Hybrid Nanoparticles ◽  
Ammonium Bromide ◽  
The Novel ◽  
Biocompatible Polymer ◽  
Dimethyl Ammonium ◽  
Oppositely Charged

Nanostructures have been of paramount importance for developing immunoadjuvants. They must be cationic and non-cytotoxic, easily assembling with usually oppositely charged antigens such as proteins, haptens or nucleic acids for use in vaccines. We obtained optimal hybrid nanoparticles (NPs) from the biocompatible polymer poly(methyl methacrylate) (PMMA) and the cationic lipid dioctadecyl dimethyl ammonium bromide (DODAB) by emulsion polymerization of methyl methacrylate (MMA) in the presence of DODAB. NPs adsorbed ovalbumin (OVA) as a model antigen and we determined their adjuvant properties. Interestingly, they elicited high double immune responses of the cellular and humoral types overcoming the poor biocompatibility of DODAB-based adjuvants of the bilayer type. The results suggested that the novel adjuvant would be possibly of use in a variety of vaccines.

scholarly journals Two Modes of Toxicity of Lipid Nanoparticles Containing a pH-Sensitive Cationic Lipid on Human A375 and A375-SM Melanoma Cell Lines

BPB Reports ◽  
2019 ◽  
Vol 2 (4) ◽  
pp. 48-55
Author(s):  
Ahmed Y. AlBaloul ◽  
Yusuke Sato ◽  
Nako Maishi ◽  
Kyoko Hida ◽  
Hideyoshi Harashima
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Cationic Lipid ◽  
Lipid Nanoparticles ◽  
Ph Sensitive ◽  
Melanoma Cell Lines

scholarly journals Aerosolizable Lipid Nanoparticles for Pulmonary Delivery of mRNA through Design of Experiments

Pharmaceutics ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1042
Author(s):  
Hairui Zhang ◽  
Jasmim Leal ◽  
Melissa R. Soto ◽  
Hugh D. C. Smyth ◽  
Debadyuti Ghosh
Keyword(s):  
Design Of Experiments ◽  
Protein Expression ◽  
Messenger Rna ◽  
Pulmonary Delivery ◽  
Lipid Nanoparticles ◽  
In Vivo Studies ◽  
Molar Ratio ◽  
Mouse Lung ◽  
Intracellular Protein ◽  
Before And After

Messenger RNA is a class of promising nucleic acid therapeutics to treat a variety of diseases, including genetic diseases. The development of a stable and efficacious mRNA pulmonary delivery system would enable high therapeutic concentrations locally in the lungs to improve efficacy and limit potential toxicities. In this study, we employed a Design of Experiments (DOE) strategy to screen a library of lipid nanoparticle compositions to identify formulations possessing high potency both before and after aerosolization. Lipid nanoparticles (LNPs) showed stable physicochemical properties for at least 14 days of storage at 4 °C, and most formulations exhibited high encapsulation efficiencies greater than 80%. Generally, upon nebulization, LNP formulations showed increased particle size and decreased encapsulation efficiencies. An increasing molar ratio of poly-(ethylene) glycol (PEG)-lipid significantly decreased size but also intracellular protein expression of mRNA. We identified four formulations possessing higher intracellular protein expression ability in vitro even after aerosolization which were then assessed in in vivo studies. It was found that luciferase protein was predominately expressed in the mouse lung for the four lead formulations before and after nebulization. This study demonstrated that LNPs hold promise to be applied for aerosolization-mediated pulmonary mRNA delivery.

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