| Reference | 1. J Control Release. 2015 Nov 10;217:337-44. doi: 10.1016/j.jconrel.2015.08.051. Epub 2015 Sep 3.<br />
N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice.<br />
Andries O(1), Mc Cafferty S(1), De Smedt SC(2), Weiss R(3), Sanders NN(4), Kitada T(5).<br />
Author information:<br />
(1)Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium. (2)Laboratory of General Biochemistry and Physical Pharmacy, Ghent Research Group on Nanomedicine, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium. (3)Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, 500 Technology Square, Cambridge, MA, USA. (4)Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium. Electronic address: [email protected]. (5)Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, 500 Technology Square, Cambridge, MA, USA. Electronic address: [email protected].<br />
Messenger RNA as a therapeutic modality is becoming increasingly popular in the field of gene therapy. The realization that nucleobase modifications can greatly enhance the properties of mRNA by reducing the immunogenicity and increasing the stability of the RNA molecule (the Kariko paradigm) has been pivotal for this revolution. Here we find that mRNAs containing the N(1)-methylpseudouridine (m1Ψ) modification alone and/or in combination with 5-methylcytidine (m5C) outperformed the current state-of-the-art pseudouridine (Ψ) and/or m5C/Ψ-modified mRNA platform by providing up to ~44-fold (when comparing double modified mRNAs) or ~13-fold (when comparing single modified mRNAs) higher reporter gene expression upon transfection into cell lines or mice, respectively. We show that (m5C/)m1Ψ-modified mRNA resulted in reduced intracellular innate immunogenicity and improved cellular viability compared to (m5C/)Ψ-modified mRNA upon in vitro transfection. The enhanced capability of (m5C/)m1Ψ-modified mRNA to express proteins may at least partially be due to the increased ability of the mRNA to evade activation of endosomal Toll-like receptor 3 (TLR3) and downstream innate immune signaling. We believe that the (m5C/)m1Ψ-mRNA platform presented here may serve as a new standard in the field of modified mRNA-based therapeutics.<br />
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[2]. Sultana, N., Magadum, A., Hadas, Y., Kondrat, J., Singh, N., Youssef, E., Calderon, D., Chepurko, E., Dubois, N., Hajjar, R.J. and Zangi, L., 2017.<br />
Optimizing cardiac delivery of modified mRNA.<br />
Abstract: Modified mRNA (modRNA) is a new technology in the field of somatic gene transfer that has been used for the delivery of genes into different tissues, including the heart. Our group and others have shown that modRNAs injected into the heart are robustly translated into the encoded protein and can potentially improve outcome in heart injury models. However, the optimal compositions of the modRNA and the reagents necessary to achieve optimal expression in the heart have not been characterized yet. In this study, our aim was to elucidate those parameters by testing different nucleotide modifications, modRNA doses, and transfection reagents both in vitro and in vivo in cardiac cells and tissue. Our results indicate that optimal cardiac delivery of modRNA is with N1-Methylpseudouridine-5′-Triphosphate nucleotide modification and achieved using 0.013 μg modRNA/mm2/500 cardiomyocytes (CMs) transfected with positively charged transfection reagent in vitro and 100 μg/mouse heart (1.6 μg modRNA/μL in 60 μL total) sucrose-citrate buffer in vivo. We have optimized the conditions for cardiac delivery of modRNA in vitro and in vivo. Using the described methods and conditions may allow for successful gene delivery using modRNA in various models of cardiovascular disease.<br />
Molecular Therapy, 25(6), pp.1306-1315.<br />
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[3]. Nakanishi, H. and Saito, H., 2021.<br />
Purification of Specific Cell Populations Differentiated from Stem Cells Using MicroRNA-Responsive Synthetic Messenger RNAs.<br />
Abstract: Pluripotent stem cells have the potential to differentiate into various cell types that can be used for basic biological studies, drug discovery, and regenerative medicine. To obtain reliable results using the differentiated cells, the contamination of nontarget cells should be avoided. microRNAs (miRNAs) can serve as indicators to distinguish target and nontarget cells, because the activities of miRNAs are different among cell types. In this chapter, we introduce a method to purify target cells using synthetic messenger RNAs (mRNAs) that respond to cell-specific miRNAs. The method is composed of five steps: mRNA sequence design, template DNA preparation by PCR, in vitro mRNA transcription, mRNA transfection into cells, and fluorescence-activated cell sorting. This synthetic mRNA-based cell purification method will advance various applications of pluripotent stem cells.<br />
In Mammalian Cell Engineering (pp. 73-86). Humana, New York, NY.<br />
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[4]. Ji, R.R., Qu, Y., Zhu, H., Yang, Y., Vogel, A.B., Sahin, U., Qin, C. and Hui, A., 2021.<br />
BNT162b2 Vaccine Encoding the SARS-CoV-2 P2 S Protects Transgenic hACE2 Mice against COVID-19.<br />
Abstract: BNT162b2 is a highly efficacious mRNA vaccine approved to prevent COVID-19. This brief report describes the immunogenicity and anti-viral protective effect of BNT162b2 in hACE2 transgenic mice. Prime-boost immunization with BNT162b2 elicited high titers in neutralizing antibodies against SARS-CoV-2, which correlated with viral clearance and alleviated lung lesions in these mice after viral challenge<br />
Vaccines, 9(4), p.324.
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