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Nature Nanotechnology Apr 2020CRISPR-Cas gene editing and messenger RNA-based protein replacement therapy hold tremendous potential to effectively treat disease-causing mutations with diverse...
CRISPR-Cas gene editing and messenger RNA-based protein replacement therapy hold tremendous potential to effectively treat disease-causing mutations with diverse cellular origin. However, it is currently impossible to rationally design nanoparticles that selectively target specific tissues. Here, we report a strategy termed selective organ targeting (SORT) wherein multiple classes of lipid nanoparticles are systematically engineered to exclusively edit extrahepatic tissues via addition of a supplemental SORT molecule. Lung-, spleen- and liver-targeted SORT lipid nanoparticles were designed to selectively edit therapeutically relevant cell types including epithelial cells, endothelial cells, B cells, T cells and hepatocytes. SORT is compatible with multiple gene editing techniques, including mRNA, Cas9 mRNA/single guide RNA and Cas9 ribonucleoprotein complexes, and is envisioned to aid the development of protein replacement and gene correction therapeutics in targeted tissues.
Topics: Animals; CRISPR-Cas Systems; Drug Delivery Systems; Gene Editing; Mice; Nanoparticles; Organ Specificity; RNA, Messenger
PubMed: 32251383
DOI: 10.1038/s41565-020-0669-6 -
Cold Spring Harbor Perspectives in... Oct 20193' untranslated regions (3' UTRs) of messenger RNAs (mRNAs) are best known to regulate mRNA-based processes, such as mRNA localization, mRNA stability, and translation.... (Review)
Review
3' untranslated regions (3' UTRs) of messenger RNAs (mRNAs) are best known to regulate mRNA-based processes, such as mRNA localization, mRNA stability, and translation. In addition, 3' UTRs can establish 3' UTR-mediated protein-protein interactions (PPIs), and thus can transmit genetic information encoded in 3' UTRs to proteins. This function has been shown to regulate diverse protein features, including protein complex formation or posttranslational modifications, but is also expected to alter protein conformations. Therefore, 3' UTR-mediated information transfer can regulate protein features that are not encoded in the amino acid sequence. This review summarizes both 3' UTR functions-the regulation of mRNA and protein-based processes-and highlights how each 3' UTR function was discovered with a focus on experimental approaches used and the concepts that were learned. This review also discusses novel approaches to study 3' UTR functions in the future by taking advantage of recent advances in technology.
Topics: 3' Untranslated Regions; Protein Binding; Protein Biosynthesis; RNA Stability; RNA, Messenger; RNA-Binding Proteins
PubMed: 30181377
DOI: 10.1101/cshperspect.a034728 -
Cold Spring Harbor Perspectives in... Feb 2019Nonsense-mediated mRNA decay (NMD) is arguably the best-studied eukaryotic messenger RNA (mRNA) surveillance pathway, yet fundamental questions concerning the molecular... (Review)
Review
Nonsense-mediated mRNA decay (NMD) is arguably the best-studied eukaryotic messenger RNA (mRNA) surveillance pathway, yet fundamental questions concerning the molecular mechanism of target RNA selection remain unsolved. Besides degrading defective mRNAs harboring premature termination codons (PTCs), NMD also targets many mRNAs encoding functional full-length proteins. Thus, NMD impacts on a cell's transcriptome and is implicated in a range of biological processes that affect a broad spectrum of cellular homeostasis. Here, we focus on the steps involved in the recognition of NMD targets and the activation of NMD. We summarize the accumulating evidence that tightly links NMD to translation termination and we further discuss the recruitment and activation of the mRNA degradation machinery and the regulation of this complex series of events. Finally, we review emerging ideas concerning the mechanistic details of NMD activation and the potential role of NMD as a general surveyor of translation efficacy.
Topics: Codon, Nonsense; Eukaryota; Nonsense Mediated mRNA Decay; Peptide Chain Termination, Translational; Protein Biosynthesis; RNA, Messenger
PubMed: 29891560
DOI: 10.1101/cshperspect.a032862 -
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 -
Molecular Cancer May 2019N-methyladenosine (mA) is identified as the most common, abundant and conserved internal transcriptional modification, especially within eukaryotic messenger RNAs... (Review)
Review
N-methyladenosine (mA) is identified as the most common, abundant and conserved internal transcriptional modification, especially within eukaryotic messenger RNAs (mRNAs). MA modification is installed by the mA methyltransferases (METTL3/14, WTAP, RBM15/15B and KIAA1429, termed as "writers"), reverted by the demethylases (FTO and ALKBH5, termed as "erasers") and recognized by mA binding proteins (YTHDF1/2/3, IGF2BP1 and HNRNPA2B1, termed as "readers"). Acumulating evidence shows that, mA RNA methylation has an outsize effect on RNA production/metabolism and participates in the pathogenesis of multiple diseases including cancers. Until now, the molecular mechanisms underlying mA RNA methylation in various tumors have not been comprehensively clarified. In this review, we mainly summarize the recent advances in biological function of mA modifications in human cancer and discuss the potential therapeutic strategies.
Topics: Adenosine; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Neoplasms; Prognosis; RNA Splicing; RNA Stability; RNA, Messenger
PubMed: 31142332
DOI: 10.1186/s12943-019-1033-z -
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 -
RNA Biology Nov 2012mRNA vaccines combine desirable immunological properties with an outstanding safety profile and the unmet flexibility of genetic vaccines. Based on in situ protein... (Review)
Review
mRNA vaccines combine desirable immunological properties with an outstanding safety profile and the unmet flexibility of genetic vaccines. Based on in situ protein expression, mRNA vaccines are capable of inducing a balanced immune response comprising both cellular and humoral immunity while not subject to MHC haplotype restriction. In addition, mRNA is an intrinsically safe vector as it is a minimal and only transient carrier of information that does not interact with the genome. Because any protein can be expressed from mRNA without the need to adjust the production process, mRNA vaccines also offer maximum flexibility with respect to development. Taken together, mRNA presents a promising vector that may well become the basis of a game-changing vaccine technology platform. Here, we outline the current knowledge regarding different aspects that should be considered when developing an mRNA-based vaccine technology.
Topics: Adjuvants, Immunologic; Animals; Biological Transport; Gene Expression; Humans; RNA, Messenger; Vaccines, Synthetic
PubMed: 23064118
DOI: 10.4161/rna.22269 -
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 -
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 -
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