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Advanced Materials (Deerfield Beach,... Feb 2019Noninvasive aerosol inhalation is an established method of drug delivery to the lung, and remains a desirable route for nucleic-acid-based therapeutics. In vitro...
Noninvasive aerosol inhalation is an established method of drug delivery to the lung, and remains a desirable route for nucleic-acid-based therapeutics. In vitro transcribed (IVT) mRNA has broad therapeutic applicability as it permits temporal and dose-dependent control of encoded protein expression. Inhaled delivery of IVT-mRNA has not yet been demonstrated and requires development of safe and effective materials. To meet this need, hyperbranched poly(beta amino esters) (hPBAEs) are synthesized to enable nanoformulation of stable and concentrated polyplexes suitable for inhalation. This strategy achieves uniform distribution of luciferase mRNA throughout all five lobes of the lung and produces 101.2 ng g of luciferase protein 24 h after inhalation of hPBAE polyplexes. Importantly, delivery is localized to the lung, and no luminescence is observed in other tissues. Furthermore, using an Ai14 reporter mouse model it is identified that 24.6% of the total lung epithelial cell population is transfected after a single dose. Repeat dosing of inhaled hPBAE-mRNA generates consistent protein production in the lung, without local or systemic toxicity. The results indicate that nebulized delivery of IVT-mRNA facilitated by hPBAE vectors may provide a clinically relevant delivery system to lung epithelium.
Topics: Administration, Inhalation; Animals; Drug Compounding; Drug Liberation; Epithelial Cells; Female; Gene Transfer Techniques; Genetic Therapy; Hydrogen-Ion Concentration; Luciferases; Lung; Mice; Mice, Inbred C57BL; Models, Animal; Nanoparticles; Polymers; RNA, Messenger; Tissue Distribution; Transfection
PubMed: 30609147
DOI: 10.1002/adma.201805116 -
Cell Nov 2023A fundamental feature of cellular growth is that total protein and RNA amounts increase with cell size to keep concentrations approximately constant. A key component of...
A fundamental feature of cellular growth is that total protein and RNA amounts increase with cell size to keep concentrations approximately constant. A key component of this is that global transcription rates increase in larger cells. Here, we identify RNA polymerase II (RNAPII) as the limiting factor scaling mRNA transcription with cell size in budding yeast, as transcription is highly sensitive to the dosage of RNAPII but not to other components of the transcriptional machinery. Our experiments support a dynamic equilibrium model where global RNAPII transcription at a given size is set by the mass action recruitment kinetics of unengaged nucleoplasmic RNAPII to the genome. However, this only drives a sub-linear increase in transcription with size, which is then partially compensated for by a decrease in mRNA decay rates as cells enlarge. Thus, limiting RNAPII and feedback on mRNA stability work in concert to scale mRNA amounts with cell size.
Topics: Cell Size; Feedback; RNA Polymerase II; RNA Stability; RNA, Messenger; Transcription, Genetic
PubMed: 37944513
DOI: 10.1016/j.cell.2023.10.012 -
Nature Structural & Molecular Biology Sep 2020Precise control of protein synthesis by engineering sequence elements in 5' untranslated regions (5' UTRs) remains a fundamental challenge. To accelerate our...
Precise control of protein synthesis by engineering sequence elements in 5' untranslated regions (5' UTRs) remains a fundamental challenge. To accelerate our understanding of the cis-regulatory code embedded in 5' UTRs, we devised massively parallel reporter assays from a synthetic messenger RNA library composed of over one million 5' UTR variants. A completely randomized 10-nucleotide sequence preceding an upstream open reading frame (uORF) and downstream GFP drives a broad range of translational outputs and mRNA stability in mammalian cells. While efficient translation protects mRNA from degradation, uORF translation triggers mRNA decay in a UPF1-dependent manner. We also identified translational inhibitory elements with G-quadruplexes as marks for mRNA decay in P-bodies. Unexpectedly, an unstructured A-rich element in 5' UTRs destabilizes mRNAs in the absence of translation, although it enables cap-independent translation. Our results not only identify diverse sequence features of 5' UTRs that control mRNA translatability, but they also reveal ribosome-dependent and ribosome-independent mRNA-surveillance pathways.
Topics: 5' Untranslated Regions; HEK293 Cells; Humans; Open Reading Frames; Protein Biosynthesis; RNA Stability; RNA, Messenger
PubMed: 32719458
DOI: 10.1038/s41594-020-0465-x -
Molecular Therapy : the Journal of the... Jun 2018The success of mRNA-based therapies depends on the availability of a safe and efficient delivery vehicle. Lipid nanoparticles have been identified as a viable option....
The success of mRNA-based therapies depends on the availability of a safe and efficient delivery vehicle. Lipid nanoparticles have been identified as a viable option. However, there are concerns whether an acceptable tolerability profile for chronic dosing can be achieved. The efficiency and tolerability of lipid nanoparticles has been attributed to the amino lipid. Therefore, we developed a new series of amino lipids that address this concern. Clear structure-activity relationships were developed that resulted in a new amino lipid that affords efficient mRNA delivery in rodent and primate models with optimal pharmacokinetics. A 1-month toxicology evaluation in rat and non-human primate demonstrated no adverse events with the new lipid nanoparticle system. Mechanistic studies demonstrate that the improved efficiency can be attributed to increased endosomal escape. This effort has resulted in the first example of the ability to safely repeat dose mRNA-containing lipid nanoparticles in non-human primate at therapeutically relevant levels.
Topics: Animals; Lipids; Nanoparticles; Primates; RNA, Messenger; Rats
PubMed: 29653760
DOI: 10.1016/j.ymthe.2018.03.010 -
Wiley Interdisciplinary Reviews. RNA Jan 2017RNA modifications are indispensable for the translation machinery to provide accurate and efficient protein synthesis. Whereas the importance of transfer RNA (tRNA) and... (Review)
Review
RNA modifications are indispensable for the translation machinery to provide accurate and efficient protein synthesis. Whereas the importance of transfer RNA (tRNA) and ribosomal RNA (rRNA) modifications has been well described and is unquestioned for decades, the significance of internal messenger RNA (mRNA) modifications has only recently been revealed. Novel experimental methods have enabled the identification of thousands of modified sites within the untranslated and translated regions of mRNAs. Thus far, N -methyladenosine (m A), pseudouridine (Ψ), 5-methylcytosine (m C) and N -methyladenosine (m A) were identified in eukaryal, and to some extent in prokaryal mRNAs. Several of the functions of these mRNA modifications have previously been reported, but many aspects remain elusive. Modifications can be important factors for the direct regulation of protein synthesis. The potential diversification of genomic information and regulation of RNA expression through editing and modifying mRNAs is versatile and many questions need to be addressed to completely elucidate the role of mRNA modifications. Herein, we summarize and highlight some recent findings on various co- and post-transcriptional modifications, describing the impact of these processes on gene expression, with emphasis on protein synthesis. WIREs RNA 2017, 8:e1375. doi: 10.1002/wrna.1375 For further resources related to this article, please visit the WIREs website.
Topics: 5-Methylcytosine; Animals; Humans; Methylation; RNA Processing, Post-Transcriptional; RNA, Messenger; Transcriptome
PubMed: 27345446
DOI: 10.1002/wrna.1375 -
Nature Nanotechnology Aug 2022Cells that were previously described as homogeneous are composed of subsets with distinct transcriptional states. However, it remains unclear whether this cell...
Cells that were previously described as homogeneous are composed of subsets with distinct transcriptional states. However, it remains unclear whether this cell heterogeneity influences the efficiency with which lipid nanoparticles (LNPs) deliver messenger RNA therapies in vivo. To test the hypothesis that cell heterogeneity influences LNP-mediated mRNA delivery, we report here a new multiomic nanoparticle delivery system called single-cell nanoparticle targeting-sequencing (SENT-seq). SENT-seq quantifies how dozens of LNPs deliver DNA barcodes and mRNA into cells, the subsequent protein production and the transcriptome, with single-cell resolution. Using SENT-seq, we have identified cell subtypes that exhibit particularly high or low LNP uptake as well as genes associated with those subtypes. The data suggest that cell subsets have distinct responses to LNPs that may affect mRNA therapies.
Topics: Lipids; Liposomes; Nanoparticles; RNA, Messenger; RNA, Small Interfering
PubMed: 35768613
DOI: 10.1038/s41565-022-01146-9 -
The AAPS Journal Oct 2023Delivery of RNA using nanomaterials has emerged as a new modality to expand therapeutic applications in biomedical research. However, the delivery of RNA presents unique... (Review)
Review
Delivery of RNA using nanomaterials has emerged as a new modality to expand therapeutic applications in biomedical research. However, the delivery of RNA presents unique challenges due to its susceptibility to degradation and the requirement for efficient intracellular delivery. The integration of nanotechnologies with RNA delivery has addressed many of these challenges. In this review, we discuss different strategies employed in the design and development of nanomaterials for RNA delivery. We also highlight recent advances in the pharmaceutical applications of RNA delivered via nanomaterials. Various nanomaterials, such as lipids, polymers, peptides, nucleic acids, and inorganic nanomaterials, have been utilized for delivering functional RNAs, including messenger RNA (mRNA), small interfering RNA, single guide RNA, and microRNA. Furthermore, the utilization of nanomaterials has expanded the applications of functional RNA as active pharmaceutical ingredients. For instance, the delivery of antigen-encoding mRNA using nanomaterials enables the transient expression of vaccine antigens, leading to immunogenicity and prevention against infectious diseases. Additionally, nanomaterial-mediated RNA delivery has been investigated for engineering cells to express exogenous functional proteins. Nanomaterials have also been employed for co-delivering single guide RNA and mRNA to facilitate gene editing of genetic diseases. Apart from the progress made in RNA medicine, we discuss the current challenges and future directions in this field.
Topics: Nanomedicine; Nanotechnology; Pharmaceutical Preparations; RNA, Small Interfering; RNA, Messenger
PubMed: 37784005
DOI: 10.1208/s12248-023-00860-z -
FASEB Journal : Official Publication of... Oct 2020
Topics: Animals; Humans; RNA, Messenger; Transfection; Vaccines, Synthetic; mRNA Vaccines
PubMed: 33373163
DOI: 10.1096/fj.202001874 -
Advanced Science (Weinheim,... Jun 2023Mammalian oogenesis features reliance on the mRNAs produced and stored during early growth phase. These are essential for producing an oocyte competent to undergo...
Mammalian oogenesis features reliance on the mRNAs produced and stored during early growth phase. These are essential for producing an oocyte competent to undergo meiotic maturation and embryogenesis later when oocytes are transcriptionally silent. The fate of maternal mRNAs hence ensures the success of oogenesis and the quality of the resulting eggs. Nevertheless, how the fate of maternal mRNAs is determined remains largely elusive. RNA-binding proteins (RBPs) are crucial regulators of oogenesis, yet the identity of the full complement of RBPs expressed in oocytes is unknown. Here, a global view of oocyte-expressed RBPs is presented: mRNA-interactome capture identifies 1396 RBPs in mouse oocytes. An analysis of one of these RBPs, LSM family member 14 (LSM14B), demonstrates that this RBP is specific to oocytes and associated with many networks essential for oogenesis. Deletion of Lsm14b results in female-specific infertility and a phenotype characterized by oocytes incompetent to complete meiosis and early embryogenesis. LSM14B serves as an interaction hub for proteins and mRNAs throughout oocyte development and regulates translation of a subset of its bound mRNAs. Therefore, RNP complexes tethered by LSM14B are found exclusively in oocytes and are essential for the control of maternal mRNA fate and oocyte development.
Topics: Female; Animals; Mice; RNA, Messenger, Stored; Oocytes; Oogenesis; RNA-Binding Proteins; RNA, Messenger; Mammals
PubMed: 37083226
DOI: 10.1002/advs.202300043 -
Biochimica Et Biophysica Acta. Gene... Oct 2018RNA localization serves numerous purposes from controlling development and differentiation to supporting the physiological activities of cells and organisms. After a... (Review)
Review
RNA localization serves numerous purposes from controlling development and differentiation to supporting the physiological activities of cells and organisms. After a brief introduction into the history of the study of mRNA localization I will focus on animal systems, describing in which cellular compartments and in which cell types mRNA localization was observed and studied. In recent years numerous novel localization patterns have been described, and countless mRNAs have been documented to accumulate in specific subcellular compartments. These fascinating revelations prompted speculations about the purpose of localizing all these mRNAs. In recent years experimental evidence for an unexpected variety of different functions has started to emerge. Aside from focusing on the functional aspects, I will discuss various ways of localizing mRNAs with a focus on the mechanism of active and directed transport on cytoskeletal tracks. Structural studies combined with imaging of transport and biochemical studies have contributed to the enormous recent progress, particularly in understanding how dynein/dynactin/BicD (DDB) dependent transport on microtubules works. This transport process actively localizes diverse cargo in similar ways to the minus end of microtubules and, at least in flies, also individual mRNA molecules. A sophisticated mechanism ensures that cargo loading licenses processive transport.
Topics: Animals; In Situ Hybridization, Fluorescence; RNA Transport; RNA, Messenger
PubMed: 30496039
DOI: 10.1016/j.bbagrm.2018.08.004