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Cell Aug 2023A system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of...
A system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of delivering executable RNA programs to other cells. We developed genetically encoded cellular RNA exporters, inspired by viruses, that efficiently package and secrete cargo RNA molecules from mammalian cells within protective nanoparticles. Exporting and sequencing RNA barcodes enabled non-destructive monitoring of cell population dynamics with clonal resolution. Further, by incorporating fusogens into the nanoparticles, we demonstrated the delivery, expression, and functional activity of exported mRNA in recipient cells. We term these systems COURIER (controlled output and uptake of RNA for interrogation, expression, and regulation). COURIER enables measurement of cell dynamics and establishes a foundation for hybrid cell and gene therapies based on cell-to-cell delivery of RNA.
Topics: Animals; Biological Transport; Mammals; RNA; RNA, Messenger; Viruses; Molecular Typing; Sequence Analysis, RNA; Cytological Techniques; Genetic Techniques
PubMed: 37437570
DOI: 10.1016/j.cell.2023.06.013 -
Science (New York, N.Y.) Jun 2023The precise control of messenger RNA (mRNA) translation is a crucial step in posttranscriptional gene regulation of cellular physiology. However, it remains a challenge...
The precise control of messenger RNA (mRNA) translation is a crucial step in posttranscriptional gene regulation of cellular physiology. However, it remains a challenge to systematically study mRNA translation at the transcriptomic scale with spatial and single-cell resolution. Here, we report the development of ribosome-bound mRNA mapping (RIBOmap), a highly multiplexed three-dimensional in situ profiling method to detect cellular translatome. RIBOmap profiling of 981 genes in HeLa cells revealed cell cycle-dependent translational control and colocalized translation of functional gene modules. We mapped 5413 genes in mouse brain tissues, yielding spatially resolved single-cell translatomic profiles for 119,173 cells and revealing cell type-specific and brain region-specific translational regulation, including translation remodeling during oligodendrocyte maturation. Our method detected widespread patterns of localized translation in neuronal and glial cells in intact brain tissue networks.
Topics: Animals; Humans; Mice; Brain; HeLa Cells; Neuroglia; RNA, Messenger; Gene Expression Regulation; Chromosome Mapping; Protein Biosynthesis; Neurons; Single-Cell Gene Expression Analysis
PubMed: 37384709
DOI: 10.1126/science.add3067 -
Nature Reviews. Cancer Aug 2023Due to the fact that mRNA technology allows the production of diverse vaccines and treatments in a shorter time frame and with reduced expense compared to conventional... (Review)
Review
Due to the fact that mRNA technology allows the production of diverse vaccines and treatments in a shorter time frame and with reduced expense compared to conventional approaches, there has been a surge in the use of mRNA-based therapeutics in recent years. With the aim of encoding tumour antigens for cancer vaccines, cytokines for immunotherapy, tumour suppressors to inhibit tumour development, chimeric antigen receptors for engineered T cell therapy or genome-editing proteins for gene therapy, many of these therapeutics have shown promising efficacy in preclinical studies, and some have even entered clinical trials. Given the evidence supporting the effectiveness and safety of clinically approved mRNA vaccines, coupled with growing interest in mRNA-based therapeutics, mRNA technology is poised to become one of the major pillars in cancer drug development. In this Review, we present in vitro transcribed mRNA-based therapeutics for cancer treatment, including the characteristics of the various types of synthetic mRNA, the packaging systems for efficient mRNA delivery, preclinical and clinical studies, current challenges and future prospects in the field. We anticipate the translation of promising mRNA-based treatments into clinical applications, to ultimately benefit patients.
Topics: Humans; RNA, Messenger; Immunotherapy; Neoplasms; Receptors, Chimeric Antigen; Cytokines
PubMed: 37311817
DOI: 10.1038/s41568-023-00586-2 -
Cell Jul 2023The number of sequenced viral genomes has surged recently, presenting an opportunity to understand viral diversity and uncover unknown regulatory mechanisms. Here, we...
The number of sequenced viral genomes has surged recently, presenting an opportunity to understand viral diversity and uncover unknown regulatory mechanisms. Here, we conducted a screening of 30,367 viral segments from 143 species representing 96 genera and 37 families. Using a library of viral segments in 3' UTR, we identified hundreds of elements impacting RNA abundance, translation, and nucleocytoplasmic distribution. To illustrate the power of this approach, we investigated K5, an element conserved in kobuviruses, and found its potent ability to enhance mRNA stability and translation in various contexts, including adeno-associated viral vectors and synthetic mRNAs. Moreover, we identified a previously uncharacterized protein, ZCCHC2, as a critical host factor for K5. ZCCHC2 recruits the terminal nucleotidyl transferase TENT4 to elongate poly(A) tails with mixed sequences, delaying deadenylation. This study provides a unique resource for virus and RNA research and highlights the potential of the virosphere for biological discoveries.
Topics: Humans; RNA; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Base Sequence; Proteins; DNA-Directed DNA Polymerase; RNA Stability; RNA, Viral
PubMed: 37413987
DOI: 10.1016/j.cell.2023.06.007 -
Nature Reviews. Molecular Cell Biology Oct 2023Nucleobase modifications are prevalent in eukaryotic mRNA and their discovery has resulted in the emergence of epitranscriptomics as a research field. The most abundant... (Review)
Review
Nucleobase modifications are prevalent in eukaryotic mRNA and their discovery has resulted in the emergence of epitranscriptomics as a research field. The most abundant internal (non-cap) mRNA modification is N-methyladenosine (mA), the study of which has revolutionized our understanding of post-transcriptional gene regulation. In addition, numerous other mRNA modifications are gaining great attention because of their major roles in RNA metabolism, immunity, development and disease. In this Review, we focus on the regulation and function of non-mA modifications in eukaryotic mRNA, including pseudouridine (Ψ), N,2'-O-dimethyladenosine (mAm), N-methyladenosine (mA), inosine, 5-methylcytidine (mC), N-acetylcytidine (acC), 2'-O-methylated nucleotide (Nm) and internal N-methylguanosine (mG). We highlight their regulation, distribution, stoichiometry and known roles in mRNA metabolism, such as mRNA stability, translation, splicing and export. We also discuss their biological consequences in physiological and pathological processes. In addition, we cover research techniques to further study the non-mA mRNA modifications and discuss their potential future applications.
Topics: RNA, Messenger; Gene Expression Regulation; Eukaryota; RNA Stability; RNA Splicing; RNA Processing, Post-Transcriptional
PubMed: 37369853
DOI: 10.1038/s41580-023-00622-x -
Human Vaccines & Immunotherapeutics Dec 2024The research and development of messenger RNA (mRNA) cancer vaccines have gradually overcome numerous challenges through the application of personalized cancer antigens,... (Review)
Review
The research and development of messenger RNA (mRNA) cancer vaccines have gradually overcome numerous challenges through the application of personalized cancer antigens, structural optimization of mRNA, and the development of alternative RNA-based vectors and efficient targeted delivery vectors. Clinical trials are currently underway for various cancer vaccines that encode tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), or immunomodulators. In this paper, we summarize the optimization of mRNA and the emergence of RNA-based expression vectors in cancer vaccines. We begin by reviewing the advancement and utilization of state-of-the-art targeted lipid nanoparticles (LNPs), followed by presenting the primary classifications and clinical applications of mRNA cancer vaccines. Collectively, mRNA vaccines are emerging as a central focus in cancer immunotherapy, offering the potential to address multiple challenges in cancer treatment, either as standalone therapies or in combination with current cancer treatments.
Topics: Humans; Cancer Vaccines; mRNA Vaccines; Neoplasms; Antigens, Neoplasm; RNA, Messenger; RNA; Nanoparticles
PubMed: 38282471
DOI: 10.1080/21645515.2024.2307187 -
Nature Reviews. Clinical Oncology Nov 2023Harnessing mRNA-lipid nanoparticles (LNPs) to treat patients with cancer has been an ongoing research area that started before these versatile nanoparticles were... (Review)
Review
Harnessing mRNA-lipid nanoparticles (LNPs) to treat patients with cancer has been an ongoing research area that started before these versatile nanoparticles were successfully used as COVID-19 vaccines. Currently, efforts are underway to harness this platform for oncology therapeutics, mainly focusing on cancer vaccines targeting multiple neoantigens or direct intratumoural injections of mRNA-LNPs encoding pro-inflammatory cytokines. In this Review, we describe the opportunities of using mRNA-LNPs in oncology applications and discuss the challenges for successfully translating the findings of preclinical studies of these nanoparticles into the clinic. We critically appraise the potential of various mRNA-LNP targeting and delivery strategies, considering physiological, technological and manufacturing challenges. We explore these approaches in the context of the potential clinical applications best suited to each approach and highlight the obstacles that currently need to be addressed to achieve these applications. Finally, we provide insights from preclinical and clinical studies that are leading to this powerful platform being considered the next frontier in oncology treatment.
Topics: Humans; COVID-19 Vaccines; COVID-19; Neoplasms; Nanoparticles; RNA, Messenger
PubMed: 37587254
DOI: 10.1038/s41571-023-00811-9 -
Biotechnology Advances 2023Nucleic acid-based therapies such as messenger RNA have the potential to revolutionize modern medicine and enhance the performance of existing pharmaceuticals. The key... (Review)
Review
Nucleic acid-based therapies such as messenger RNA have the potential to revolutionize modern medicine and enhance the performance of existing pharmaceuticals. The key challenges of mRNA-based therapies are delivering the mRNA safely and effectively to the target tissues and cells and controlling its release from the delivery vehicle. Lipid nanoparticles (LNPs) have been widely studied as drug carriers and are considered to be state-of-the-art technology for nucleic acid delivery. In this review, we begin by presenting the advantages and mechanisms of action of mRNA therapeutics. Then we discuss the design of LNP platforms based on ionizable lipids and the applications of mRNA-LNP vaccines for prevention of infectious diseases and for treatment of cancer and various genetic diseases. Finally, we describe the challenges and future prospects of mRNA-LNP therapeutics.
Topics: Liposomes; Drug Carriers; Excipients; RNA, Messenger; Nanoparticles; RNA, Small Interfering
PubMed: 36933868
DOI: 10.1016/j.biotechadv.2023.108130 -
Nature Reviews. Drug Discovery Jan 2024mRNA medicine is a new and rapidly developing field in which the delivery of genetic information in the form of mRNA is used to direct therapeutic protein production in... (Review)
Review
mRNA medicine is a new and rapidly developing field in which the delivery of genetic information in the form of mRNA is used to direct therapeutic protein production in humans. This approach, which allows for the quick and efficient identification and optimization of drug candidates for both large populations and individual patients, has the potential to revolutionize the way we prevent and treat disease. A key feature of mRNA medicines is their high degree of designability, although the design choices involved are complex. Maximizing the production of therapeutic proteins from mRNA medicines requires a thorough understanding of how nucleotide sequence, nucleotide modification and RNA structure interplay to affect translational efficiency and mRNA stability. In this Review, we describe the principles that underlie the physical stability and biological activity of mRNA and emphasize their relevance to the myriad considerations that factor into therapeutic mRNA design.
Topics: Humans; RNA, Messenger; Pharmaceutical Preparations
PubMed: 38030688
DOI: 10.1038/s41573-023-00827-x -
Molecular Cell Dec 2023The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined...
The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined mRNA enrichment in TIS granules (TGs) and the rough endoplasmic reticulum (ER) through particle sorting and isolated cytosolic mRNAs by digitonin extraction. When focusing on genes that encode non-membrane proteins, we observed that 52% have transcripts enriched in specific compartments. Compartment enrichment correlates with a combinatorial code based on mRNA length, exon length, and 3' UTR-bound RNA-binding proteins. Compartment-biased mRNAs differ in the functional classes of their encoded proteins: TG-enriched mRNAs encode low-abundance proteins with strong enrichment of transcription factors, whereas ER-enriched mRNAs encode large and highly expressed proteins. Compartment localization is an important determinant of mRNA and protein abundance, which is supported by reporter experiments showing that redirecting cytosolic mRNAs to the ER increases their protein expression. In summary, the cytoplasm is functionally compartmentalized by local translation environments.
Topics: Endoplasmic Reticulum; Proteins; Cytosol; RNA, Messenger; Protein Transport; Protein Biosynthesis
PubMed: 38134885
DOI: 10.1016/j.molcel.2023.11.025