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Nano Letters Mar 2020Chimeric antigen receptor (CAR) T cell therapy relies on the manipulation of patient T cells to create potent, cancer-targeting therapies, shown to be capable of...
Chimeric antigen receptor (CAR) T cell therapy relies on the manipulation of patient T cells to create potent, cancer-targeting therapies, shown to be capable of inducing remission in patients with acute lymphoblastic leukemia and large B cell lymphoma. However, current CAR T cell engineering methods use viral delivery vectors, which induce permanent CAR expression and could lead to severe adverse effects. Messenger RNA (mRNA) has been explored as a promising strategy for inducing transient CAR expression in T cells to mitigate the adverse effects associated with viral vectors, but it most commonly requires electroporation for T cell mRNA delivery, which can be cytotoxic. Here, ionizable lipid nanoparticles (LNPs) were designed for mRNA delivery to human T cells. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for luciferase mRNA delivery to Jurkat cells, revealing seven formulations capable of enhanced mRNA delivery over lipofectamine. The top-performing LNP formulation, C14-4, was selected for CAR mRNA delivery to primary human T cells. This platform induced CAR expression at levels equivalent to electroporation, with substantially reduced cytotoxicity. CAR T cells engineered via C14-4 LNP treatment were then compared to electroporated CAR T cells in a coculture assay with Nalm-6 acute lymphoblastic leukemia cells, and both CAR T cell engineering methods elicited potent cancer-killing activity. These results demonstrate the ability of LNPs to deliver mRNA to primary human T cells to induce functional protein expression, and indicate the potential of LNPs to enhance mRNA-based CAR T cell engineering methods.
Topics: Cell Engineering; Cell Line, Tumor; Coculture Techniques; Drug Delivery Systems; Humans; Lipids; Nanoparticles; RNA, Messenger; Receptors, Chimeric Antigen; T-Lymphocytes
PubMed: 31951421
DOI: 10.1021/acs.nanolett.9b04246 -
Nature Reviews. Molecular Cell Biology Mar 2018Long intergenic non-coding RNA (lincRNA) genes have diverse features that distinguish them from mRNA-encoding genes and exercise functions such as remodelling chromatin... (Review)
Review
Long intergenic non-coding RNA (lincRNA) genes have diverse features that distinguish them from mRNA-encoding genes and exercise functions such as remodelling chromatin and genome architecture, RNA stabilization and transcription regulation, including enhancer-associated activity. Some genes currently annotated as encoding lincRNAs include small open reading frames (smORFs) and encode functional peptides and thus may be more properly classified as coding RNAs. lincRNAs may broadly serve to fine-tune the expression of neighbouring genes with remarkable tissue specificity through a diversity of mechanisms, highlighting our rapidly evolving understanding of the non-coding genome.
Topics: Animals; Conserved Sequence; Epigenesis, Genetic; Evolution, Molecular; Female; Gene Expression Regulation; Humans; Male; Models, Genetic; Open Reading Frames; Organ Specificity; RNA Stability; RNA, Long Noncoding; RNA, Messenger
PubMed: 29138516
DOI: 10.1038/nrm.2017.104 -
Cell Mar 2022In the last decade, the notion that mRNA modifications are involved in regulation of gene expression was demonstrated in thousands of studies. To date, new technologies... (Review)
Review
In the last decade, the notion that mRNA modifications are involved in regulation of gene expression was demonstrated in thousands of studies. To date, new technologies and methods allow accurate identification, transcriptome-wide mapping, and functional characterization of a growing number of RNA modifications, providing important insights into the biology of these marks. Most of the methods and approaches were developed for studying mA, the most prevalent internal mRNA modification. However, unique properties of other RNA modifications stimulated the development of additional approaches. In this technical primer, we will discuss the available tools and approaches for detecting and studying different RNA modifications.
Topics: Epigenesis, Genetic; RNA; RNA Processing, Post-Transcriptional; RNA, Messenger; Transcriptome
PubMed: 35245480
DOI: 10.1016/j.cell.2022.02.007 -
Molecular Pharmaceutics Nov 2022Lipid nanoparticles containing messenger RNA (mRNA-LNPs) have launched to the forefront of nonviral delivery systems with their realized potential during the COVID-19...
Lipid nanoparticles containing messenger RNA (mRNA-LNPs) have launched to the forefront of nonviral delivery systems with their realized potential during the COVID-19 pandemic. Here, we investigate the impact of commonly used biological buffers on the performance and durability of mRNA-LNPs. We tested the compatibility of three common buffers─HEPES, Tris, and phosphate-buffered saline─with a DLin-MC3-DMA mRNA-LNP formulation before and after a single controlled freeze-thaw cycle. We hypothesized that buffer composition would affect lipid-aqueous phase separation. Indeed, the buffers imposed structural changes in LNP morphology as indicated by electron microscopy, differential scanning calorimetry, and membrane fluidity assays. We employed in vitro and in vivo models to measure mRNA transfection and found that Tris or HEPES-buffered LNPs yielded better cryoprotection and transfection efficiency compared to PBS. Understanding the effects of various buffers on LNP morphology and efficacy provides valuable insights into maintaining the stability of LNPs after long-term storage.
Topics: Humans; RNA, Messenger; Lipids; Pandemics; COVID-19; Nanoparticles; Liposomes; RNA, Small Interfering
PubMed: 36129254
DOI: 10.1021/acs.molpharmaceut.2c00587 -
Expert Review of Vaccines Sep 2017The present review summarizes the growing body of work defining the mechanisms of action of this exciting new vaccine technology that should allow rational approaches in... (Review)
Review
The present review summarizes the growing body of work defining the mechanisms of action of this exciting new vaccine technology that should allow rational approaches in the design of next generation mRNA vaccines. Areas covered: Bio-distribution of mRNA, localization of antigen production, role of the innate immunity, priming of the adaptive immune response, route of administration and effects of mRNA delivery systems. Expert commentary: In the last few years, the development of RNA vaccines had a fast growth, the rising number of proof will enable rational approaches to improving the effectiveness and safety of this modern class of medicine.
Topics: Adaptive Immunity; Drug Delivery Systems; Humans; Immunity, Innate; RNA, Messenger; Vaccines
PubMed: 28701102
DOI: 10.1080/14760584.2017.1355245 -
Biophysical Journal Jul 2021Understanding the structure of messenger RNA (mRNA) lipid nanoparticles, and specifically the microenvironment of the mRNA molecules within these entities, is...
Understanding the structure of messenger RNA (mRNA) lipid nanoparticles, and specifically the microenvironment of the mRNA molecules within these entities, is fundamental to advancing their biomedical potential. Here, we show that a permeating cationic dye, thionine, can serve as a cryogenic electron microscopy contrasting agent by binding selectively to encapsulated mRNA without disturbing lipid nanoparticle morphology. Cryo-electron microscopy images identify the mRNA location, revealing that mRNA may exist within solvent-filled cavities or may be substantially lipid associated.
Topics: Cryoelectron Microscopy; Lipids; Nanoparticles; RNA, Messenger
PubMed: 33773963
DOI: 10.1016/j.bpj.2021.03.012 -
Current Protocols Feb 2021Synthetic messenger RNA (mRNA)-based therapeutics are an increasingly popular approach to gene and cell therapies, genome engineering, enzyme replacement therapy, and...
Synthetic messenger RNA (mRNA)-based therapeutics are an increasingly popular approach to gene and cell therapies, genome engineering, enzyme replacement therapy, and now, during the global SARS-CoV-2 pandemic, vaccine development. mRNA for such purposes can be synthesized through an enzymatic in vitro transcription (IVT) reaction and formulated for in vivo delivery. Mature mRNA requires a 5'-cap for gene expression and mRNA stability. There are two methods to add a cap in vitro: via a two-step multi-enzymatic reaction or co-transcriptionally. Co-transcriptional methods minimize reaction steps and enzymes needed to make mRNA when compared to enzymatic capping. CleanCap AG co-transcriptional capping results in 5 mg/ml of IVT with 94% 5'-cap 1 structure. This is highly efficient compared to first-generation cap analogs, such as mCap and ARCA, that incorporate cap 0 structures at lower efficiency and reaction yield. This article describes co-transcriptional capping using TriLink Biotechnology's CleanCap AG in IVT. © 2021 Wiley Periodicals LLC. Basic Protocol 1: IVT with CleanCap Basic Protocol 2: mRNA purification and analysis.
Topics: Humans; In Vitro Techniques; Protein Biosynthesis; RNA Cap Analogs; RNA Stability; RNA, Messenger
PubMed: 33524237
DOI: 10.1002/cpz1.39 -
Biochimica Et Biophysica Acta. Gene... 2019
Topics: Animals; Gene Expression Regulation; Humans; Nucleic Acid Conformation; RNA Splicing; RNA, Messenger
PubMed: 31730825
DOI: 10.1016/j.bbagrm.2019.194448 -
Nature Biotechnology Jul 2019The ability to predict the impact of cis-regulatory sequences on gene expression would facilitate discovery in fundamental and applied biology. Here we combine polysome...
The ability to predict the impact of cis-regulatory sequences on gene expression would facilitate discovery in fundamental and applied biology. Here we combine polysome profiling of a library of 280,000 randomized 5' untranslated regions (UTRs) with deep learning to build a predictive model that relates human 5' UTR sequence to translation. Together with a genetic algorithm, we use the model to engineer new 5' UTRs that accurately direct specified levels of ribosome loading, providing the ability to tune sequences for optimal protein expression. We show that the same approach can be extended to chemically modified RNA, an important feature for applications in mRNA therapeutics and synthetic biology. We test 35,212 truncated human 5' UTRs and 3,577 naturally occurring variants and show that the model predicts ribosome loading of these sequences. Finally, we provide evidence of 45 single-nucleotide variants (SNVs) associated with human diseases that substantially change ribosome loading and thus may represent a molecular basis for disease.
Topics: 5' Untranslated Regions; Base Sequence; Gene Expression Regulation; Humans; Models, Genetic; Protein Biosynthesis; Pseudouridine; RNA, Messenger; Reproducibility of Results; Ribosomes
PubMed: 31267113
DOI: 10.1038/s41587-019-0164-5 -
Science (New York, N.Y.) Feb 2023The circadian clock modulates human physiology. However, the organization of tissue-specific gene expression rhythms and how these depend on age and sex is not defined...
The circadian clock modulates human physiology. However, the organization of tissue-specific gene expression rhythms and how these depend on age and sex is not defined in humans. We combined data from the Genotype-Tissue Expression (GTEx) project with an algorithm that assigns circadian phases to 914 donors, by integrating temporal information from multiple tissues in each individual, to identify messenger RNA (mRNA) rhythms in 46 tissues. Clock transcripts showed conserved timing relationships and tight synchrony across the body. mRNA rhythms varied in breadth, covering global and tissue-specific functions, including metabolic pathways and systemic responses. The clock structure was conserved across sexes and age groups. However, overall gene expression rhythms were highly sex-dimorphic and more sustained in females. Rhythmic programs generally dampened with age across the body.
Topics: Female; Humans; Circadian Clocks; Circadian Rhythm; Gene Expression Regulation; RNA, Messenger; Sex Characteristics; Age Factors; Male; Organ Specificity
PubMed: 36730411
DOI: 10.1126/science.add0846