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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 -
International Journal of Molecular... Sep 2020Messenger ribonucleic acid (mRNA)-based drugs, notably mRNA vaccines, have been widely proven as a promising treatment strategy in immune therapeutics. The extraordinary... (Review)
Review
Messenger ribonucleic acid (mRNA)-based drugs, notably mRNA vaccines, have been widely proven as a promising treatment strategy in immune therapeutics. The extraordinary advantages associated with mRNA vaccines, including their high efficacy, a relatively low severity of side effects, and low attainment costs, have enabled them to become prevalent in pre-clinical and clinical trials against various infectious diseases and cancers. Recent technological advancements have alleviated some issues that hinder mRNA vaccine development, such as low efficiency that exist in both gene translation and in vivo deliveries. mRNA immunogenicity can also be greatly adjusted as a result of upgraded technologies. In this review, we have summarized details regarding the optimization of mRNA vaccines, and the underlying biological mechanisms of this form of vaccines. Applications of mRNA vaccines in some infectious diseases and cancers are introduced. It also includes our prospections for mRNA vaccine applications in diseases caused by bacterial pathogens, such as tuberculosis. At the same time, some suggestions for future mRNA vaccine development about storage methods, safety concerns, and personalized vaccine synthesis can be found in the context.
Topics: Drug Delivery Systems; Immunogenicity, Vaccine; RNA, Messenger; Vaccines, Synthetic; mRNA Vaccines
PubMed: 32916818
DOI: 10.3390/ijms21186582 -
Nature Biotechnology Feb 2023Circular RNAs (circRNAs) are stable and prevalent RNAs in eukaryotic cells that arise from back-splicing. Synthetic circRNAs and some endogenous circRNAs can encode...
Circular RNAs (circRNAs) are stable and prevalent RNAs in eukaryotic cells that arise from back-splicing. Synthetic circRNAs and some endogenous circRNAs can encode proteins, raising the promise of circRNA as a platform for gene expression. In this study, we developed a systematic approach for rapid assembly and testing of features that affect protein production from synthetic circRNAs. To maximize circRNA translation, we optimized five elements: vector topology, 5' and 3' untranslated regions, internal ribosome entry sites and synthetic aptamers recruiting translation initiation machinery. Together, these design principles improve circRNA protein yields by several hundred-fold, provide increased translation over messenger RNA in vitro, provide more durable translation in vivo and are generalizable across multiple transgenes.
Topics: RNA, Circular; RNA; RNA, Messenger; RNA Splicing
PubMed: 35851375
DOI: 10.1038/s41587-022-01393-0 -
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 -
Proceedings of the National Academy of... Dec 2021Lipid nanoparticles (LNPs) are a clinically mature technology for the delivery of genetic medicines but have limited therapeutic applications due to liver accumulation....
Lipid nanoparticles (LNPs) are a clinically mature technology for the delivery of genetic medicines but have limited therapeutic applications due to liver accumulation. Recently, our laboratory developed selective organ targeting (SORT) nanoparticles that expand the therapeutic applications of genetic medicines by enabling delivery of messenger RNA (mRNA) and gene editing systems to non-liver tissues. SORT nanoparticles include a supplemental SORT molecule whose chemical structure determines the LNP's tissue-specific activity. To understand how SORT nanoparticles surpass the delivery barrier of liver hepatocyte accumulation, we studied the mechanistic factors which define their organ-targeting properties. We discovered that the chemical nature of the added SORT molecule controlled biodistribution, global/apparent pK, and serum protein interactions of SORT nanoparticles. Additionally, we provide evidence for an endogenous targeting mechanism whereby organ targeting occurs via 1) desorption of poly(ethylene glycol) lipids from the LNP surface, 2) binding of distinct proteins to the nanoparticle surface because of recognition of exposed SORT molecules, and 3) subsequent interactions between surface-bound proteins and cognate receptors highly expressed in specific tissues. These findings establish a crucial link between the molecular composition of SORT nanoparticles and their unique and precise organ-targeting properties and suggest that the recruitment of specific proteins to a nanoparticle's surface can enable drug delivery beyond the liver.
Topics: Animals; Gene Editing; Humans; Liposomes; Liver; Mice; Mice, Inbred C57BL; Nanoparticle Drug Delivery System; Nanoparticles; RNA, Messenger; Tissue Distribution
PubMed: 34933999
DOI: 10.1073/pnas.2109256118 -
Cardiovascular Research Jun 2022Among several known RNA modifications, N6-methyladenosine (m6A) is the most studied RNA epitranscriptomic modification and controls multiple cellular functions during... (Review)
Review
Among several known RNA modifications, N6-methyladenosine (m6A) is the most studied RNA epitranscriptomic modification and controls multiple cellular functions during development, differentiation, and disease. Current research advancements have made it possible to examine the regulatory mechanisms associated with RNA methylation and reveal its functional consequences in the pathobiology of many diseases, including heart failure. m6A methylation has been described both on coding (mRNA) and non-coding RNA species including rRNA, tRNA, small nuclear RNA and circular RNAs. The protein components which catalyze the m6A methylation are termed methyltransferase or 'm6A writers'. The family of proteins that recognize this methylation are termed 'm6A readers' and finally the enzymes involved in the removal of a methyl group from RNA are known as demethylases or 'm6A erasers'. At the cellular level, different components of methylation machinery are tightly regulated by many factors to maintain the m6A methylation dynamics. The m6A methylation process impacts different stages of mRNA metabolism and the biogenesis of long non-coding RNA and miRNA. Although, mRNA methylation was initially described in the 1970s, its regulatory roles in various diseases, including cardiovascular diseases are broadly unexplored. Recent investigations suggest the important role of m6A mRNA methylation in both hypertrophic and ischaemic heart diseases. In the present review, we evaluate the significance of m6A methylation in the cardiovascular system, in cardiac homeostasis and disease, all of which may help to improve therapeutic intervention for the treatment of heart failure. RNA methylation in cardiovascular diseases: altered m6A RNA (coding and non-coding RNA) methylation is identified during different cardiovascular diseases. Increased cardiac hypertrophy is observed following METTL3 overexpression. In contrast, reduced FTO level was seen in mice following myocardial infarction. Increased cardiac fibroblasts activation or increased atherosclerotic plaques were also co-related with m6A RNA methylation.
Topics: Animals; Biology; Cardiovascular Diseases; Cardiovascular System; Heart Failure; Mice; RNA, Long Noncoding; RNA, Messenger
PubMed: 33956076
DOI: 10.1093/cvr/cvab160 -
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 -
Molecular Plant Jan 2020Advances in the detection and mapping of messenger RNA (mRNA) N-methyladenosine (mA) and 5-methylcytosine (mC), and DNA N-methyldeoxyadenosine (6mA) redefined our... (Review)
Review
Advances in the detection and mapping of messenger RNA (mRNA) N-methyladenosine (mA) and 5-methylcytosine (mC), and DNA N-methyldeoxyadenosine (6mA) redefined our understanding of these modifications as additional tiers of epigenetic regulation. In plants, the most prevalent internal mRNA modifications, mA and mC, play crucial and dynamic roles in many processes, including embryo development, stem cell fate determination, trichome branching, leaf morphogenesis, floral transition, stress responses, fruit ripening, and root development. The newly identified and widespread epigenetic marker 6mA DNA methylation is associated with gene expression, plant development, and stress responses. Here, we review the latest research progress on mRNA and DNA epigenetic modifications, including the detection, dynamics, distribution, functions, regulatory proteins, and evolution, with a focus on mA, mC, and 6mA. We also provide some perspectives on future research of the newly identified and unknown epigenetic modifications of mRNA and DNA in plants.
Topics: 5-Methylcytosine; Adenosine; DNA Methylation; Epigenesis, Genetic; Plants; RNA, Messenger
PubMed: 31863849
DOI: 10.1016/j.molp.2019.12.007 -
International Journal of Molecular... Feb 2023As it is well known, messenger RNA has many regulatory regions along its sequence length. One of them is the 5' untranslated region (5'UTR), which itself contains many... (Review)
Review
As it is well known, messenger RNA has many regulatory regions along its sequence length. One of them is the 5' untranslated region (5'UTR), which itself contains many regulatory elements such as upstream ORFs (uORFs), internal ribosome entry sites (IRESs), microRNA binding sites, and structural components involved in the regulation of mRNA stability, pre-mRNA splicing, and translation initiation. Activation of the alternative, more upstream transcription start site leads to an extension of 5'UTR. One of the consequences of 5'UTRs extension may be head-to-head gene overlap. This review describes elements in 5'UTR of protein-coding transcripts and the functional significance of protein-coding genes 5' overlap with implications for transcription, translation, and disease.
Topics: 5' Untranslated Regions; Protein Biosynthesis; RNA, Messenger; Gene Expression Regulation; Regulatory Sequences, Nucleic Acid
PubMed: 36769304
DOI: 10.3390/ijms24032976 -
The Keio Journal of Medicine 2022Messenger RNA was discovered in 1961 and it took 60 years until the first mRNA became FDA-approved product in the form of COVID-19 mRNA vaccine. During those years a lot...
Messenger RNA was discovered in 1961 and it took 60 years until the first mRNA became FDA-approved product in the form of COVID-19 mRNA vaccine. During those years a lot of progress has been made by hundreds of scientists. It was 1978 when the first-time isolated mRNA delivered into mammalian cells produced the encoded protein. In vitro transcription introduced in 1984 made it possible to generate any desired mRNA from the encoding plasmid using phage RNA polymerases. In the early 90s mRNA was used for therapy as well as vaccine against infectious diseases and cancer. Inflammatory nature of the mRNAs limited their in vivo use. Replacing uridine with pseudouridine made the mRNA non-immunogenic, more stable and highly translatable. Delivery of the lipid nanoparticle-formulated nucleoside-modified mRNA encoding viral antigens became a platform for effective vaccine. Labile nature of the mRNA is ideal for transient production of the viral antigen, to generate effective antibody and cellular immune response. The mRNA platform is revolutionizing the delivery of effective and safe vaccines, therapeutics and gene therapies.
Topics: Animals; COVID-19; COVID-19 Vaccines; Humans; Liposomes; Mammals; Nanoparticles; RNA, Messenger; Vaccines, Synthetic; mRNA Vaccines
PubMed: 35342149
DOI: 10.2302/kjm.71-001-ABST