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Non-Viral Delivery of Genome-Editing Nucleases for Gene Therapy
Published in Yashwant Pathak, Gene Delivery, 2022
Studies reported that the modification of mRNA with a combination of 2-thiouridine and 5-methylcytidine effectively reduced the immune stimulation through pattern recognition receptors, such as TLR3, TLR7, TLR8, and retinoic acid receptor responder protein 3 also known as RIG-I [2, 23]. In addition, the inclusion of pseudouridine in the mRNA (Ψ-mRNA) blocked activation of pattern recognition receptors18 and 2ʹ-5ʹ-oligoadenylate synthetase19. These modifications can also stabilize the mRNA against cleavage and ultimately increase expression rates [24]. A direct intramyocardial injection of vascular endothelial growth factor A (VEGF-A)-encoding modified mRNA (modRNA) complexed with RNAiMAX in myocardial infarction mice decreased the infract size and apoptotic cell frequency, as compared to the control group [3, 23, 24]. In another study, pulmonary surfactant associated protein deficient mice showed 0% survival by five days, whereas mice with administration of modified PSPB-encoding mRNA showed >80% survival by day 30 [3]. Luciferase mRNA successfully delivered into mice after intranasal administration in which mRNA made complex transfection reagent Stemfect or to a hydrophobic poly(β-amino ester) that had been coated with a positively charged lipid layer.[25, 26]. This complex also could express luciferase in the spleen after IV administration [3, 26].
Human Noroviruses
Published in Dongyou Liu, Handbook of Foodborne Diseases, 2018
G. Sanchez, W. Randazzo, D.H. D'Souza
HNoV RdRp is essential for viral replication and in the synthesis of both, the negative-sense RNA and also the newly synthesized positive-sense genomic RNA.221 Suramin, a nonnucleoside inhibitor drug and its analog that consists of naphthalene-trisulfonic acid moiety, is reported to bind to HNoV RdRp and inhibit its activity.221,241 Furthermore, nucleoside-analogs such as 2′-C-methylcytidine (2CMC) and ribavirin are reported to inhibit MNV and HNoV replication in vitro.242,243 As technologies and approaches for high-throughput screening and structure-function analysis advance, noncytotoxic yet potent and broad-spectrum antiviral or inhibitory drugs can be engineered to provide health-care providers with treatment options against chronic and persistent or recurring HNoV infections.221
Hepatitis C Virus and Its Inhibitors
Published in Satya Prakash Gupta, Cancer-Causing Viruses and Their Inhibitors, 2014
Selecting replicons resistant to 2′-deoxy-2′-fluoro-2′-C-methylcytidine (PSI- 6130, 11) proved very difficult; on the other hand, clones resistant to a nonnucleoside RdRp inhibitor (HCV-796) or an HCV protease inhibitor (VX-950) readily emerged (McCown et al. 2008). It took prolonged culturing (more than 20 passages) of HCV replicon harboring cells in the presence of increasing concentrations of 11 to finally isolate resistant clones. Sequencing revealed multiple mutations in the polymerase; among them, S282T was the only substitution recurring in all selected and analyzed replicons. In contrast to other 2′-C-methyl modified nucleosides, the potency of 2′-fluoro-2′-C-methylcytidine and its prodrug mericitabine (13) against the mutant NS5B was only moderately affected with a less than 3.5-fold increase in EC50 in the replicon assay and a twofold increase in Ki in an enzyme assay compared to wild type. Interestingly, 2′-deoxy-2′-fluoro-2′-C-methyluridine (12, 16, sofosbuvir 17), a metabolite of PSI-6130, was more susceptible to the S282T mutation in a replicon assay with a 19-fold higher EC90 compared to wild-type replicons—a value similar to that observed for 2′-C-methylcytidine (Table 3.6). The S96T mutation, which reduced the efficacy of R1479, hardly affected the potency of PSI-6130, meaning that no cross-resistance existed between these two compounds. In fact, PSI-6130 and its uridine analog sofosbuvir were twice as potent against an S96T, or an S96T/N142T mutant, respectively, as against the wild type (Ali et al. 2008; Sofia et al. 2010).
Quantifying RNA modifications by mass spectrometry: a novel source of biomarkers in oncology
Published in Critical Reviews in Clinical Laboratory Sciences, 2022
Amandine Amalric, Amandine Bastide, Aurore Attina, Armelle Choquet, Jerome Vialaret, Sylvain Lehmann, Alexandre David, Christophe Hirtz
In addition to m6A, the urine concentration of other modified nucleosides varied in cancer patients. For example, 5-methylcytidine (m5C) and 5-hydroxymethylcytidine (hm5C) levels were decreased in the urine of colorectal cancer patients [48]. In this study [48], the urine concentration of hm5C was evaluated using LC-MS/MS and found to be four-fold lower than m5C. By contrast, another LC-MS/MS study reported that hm5C and 2′-O-methyl-5-hydroxymethylcytidine (hm5Cm) urine levels were increased in lymphoma and gastric and esophageal cancers [47]. Another nucleoside oxidation product, ho8G, was higher in colorectal cancer patient urine than in healthy controls; this was consistent with the fact that oxidative RNA damage is associated with cancer development [53]. The urine content of N4-acetylcytidine (ac4C) was also altered under pathological conditions; the ac4C level decreased in ovarian cancer [49] while it increased in colorectal [38], urogenital [42] and bladder [45] cancers. Furthermore, the methyl group(s) at the N2 position of guanosine is associated with numerous cancers. m2,2G was abundant in colorectal cancer patient urine [38], while urogenital [43,44] and breast [39,52] cancers had elevated singly (m2G) and m2,2G .
An overview of rational design of mRNA-based therapeutics and vaccines
Published in Expert Opinion on Drug Discovery, 2021
Kenneth K.W. To, William C.S. Cho
The use of conventional mRNA is associated with low stability and strong immunogenicity, therefore hindering their clinical applications. There has been extensive research in the last decade to understand and improve the production, stability, translation, and pharmacokinetics of mRNA therapeutics. Kariko and colleagues pioneered to modify specific nucleosides (cytidine and uridine) to 5-methylcytidine and pseudouridine, respectively, which rendered the resulting mRNA molecules stable inside the cells and dramatically less immunogenic [34–36]. Interestingly, while pseudouridine is primarily found in tRNA, rRNA and small nuclear RNAs, pseudouridine-containing mRNAs are readily translated and produced even more protein compared to unmodified mRNA [34,36]. Thus, modification of nucleosides has been suggested the method of choice for preparing IVT mRNAs. The use of nucleoside modifications was further confirmed by Rossi et al. to potently enhance protein expression and suppress cytokine secretion using enhanced green fluorescent protein mRNA [37]. Other modified nucleosides/nucleotides, such as n6-methyladenosine, 5-methylcytidine triphosphate, n6-methyladenosine-5ʹ-triphosphate and 2-thiouridine triphosphate have also been successfully applied to promote protein production and suppress immunostimulation by mRNA therapeutics [38,39]. Recently, Wadhwa et al. have provided an excellent review about the various strategies to enhance the stability and translation efficiency but to reduce the immunogenicity of mRNA-based therapeutics [40].
Environmental exposures and RNA N6-Methyladenosine modified long Non-Coding RNAs
Published in Critical Reviews in Toxicology, 2020
The term “epitranscriptome” collectively refers to modifications in coding and non-coding RNAs (Saletore et al. 2012). RNA modifications are observed in tRNAs, rRNAs, mRNAs, and long-noncoding RNAs (lncRNA) in various cellular localizations including the nucleus, cytoplasm, and mitochondrion. The first modification in RNAs was discovered in 1957 (Davis and Allen 1957). Today, more than 100 different RNA modifications in different bases and with different chemical groups have been reported in a range of organisms (Gilbert et al. 2016). Several modifications including 6-methyladenosine (m6A), 5-methylcytidine (m5C), N1-methyladenosine (m1A), 5-hydroxylmethylcytidine (hm5C), inosine, and pseudouridine have commonly been reported in eukaryotic organisms (Dominissini et al. 2012; Meyer et al. 2012; Squires et al. 2012; Li et al. 2016).