The product that will result from this proposal is a nucleoside-modified mRNA encoding erythropoietin for treatment of red blood cell deficiency (anemia). In vitro transcribed mRNAs encoding physiologically important proteins have considerable potential for therapeutic applications. However, mRNA is naturally labile, inefficiently translated and immunogenic and has therefore been traditionally unsuited for therapy. RNARx is developing a technology that modifies mRNA by incorporating non-classical nucleosides, such as pseudouridine. Our preliminary data suggest that this improves the translational efficiency and overall stability of mRNA, as well as diminishing its immunogenicity in vivo. These favorable new properties provide an opportunity to develop deliverable pseudouridine-modified mRNAs as vectors for expressing clinically beneficial proteins safely and effectively in vivo. RNARx will collaborate with the University of Pennsylvania in this Fast-track proposal to develop the first of these therapeutic vectors for delivery of human erythropoietin (EPO). In Phase 1, nucleoside-modified mRNA encoding EPO will be developed, characterized and delivered to mice for verification of biological EPO activity in the absence of immune activation. In Phase 2, a suitable (preferably non-injection) delivery system will be developed and modified mRNA will be tested in small and large animal systems. In addition, based on new models of autoimmunity immunopathogenesis, we will investigate the potential of mRNA to exacerbate a model of autoimmunity (SLE) and whether non-immunogenic nucleoside-modified mRNA avoids this potential. At the completion of these studies, we plan to file an IND to initiate clinical trials of EPO-encoding modified mRNA. Future objectives will include the use of the mRNA platform for other biologics and for intracellular protein delivery (gene therapy), an important therapeutic need for which there are currently no products beyond human clinical trials. Deliverable proteins such as erythropoietin, insulin, and clotting factors are an enormously important arsenal of medical therapies that nevertheless carry a risk of dangerous allergic reactions. In addition, many proteins in the human body, including cell structural proteins, cannot be replaced by conventional protein administration and alternative therapies, such as gene therapy, have not performed to expectation. The product we are developing, a structurally-modified messenger RNA (the intermediary between DNA and protein), is an alternative to protein delivery and gene therapy that will enable the safe (non-allergic) and efficient replacement or enhancement of proteins (in this proposal erythropoietin) including cell structural proteins. ? ? ?

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Small Business Technology Transfer (STTR) Grants - Phase II (R42)
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Special Emphasis Panel (ZRG1-HEME-D (10))
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Qasba, Pankaj
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Boros, Gábor; Karikó, Katalin; Muramatsu, Hiromi et al. (2016) Transfection of Human Keratinocytes with Nucleoside-Modified mRNA Encoding CPD-Photolyase to Repair DNA Damage. Methods Mol Biol 1428:219-28
Boros, Gábor; Miko, Edit; Muramatsu, Hiromi et al. (2015) Identification of Cyclobutane Pyrimidine Dimer-Responsive Genes Using UVB-Irradiated Human Keratinocytes Transfected with In Vitro-Synthesized Photolyase mRNA. PLoS One 10:e0131141
Pardi, Norbert; Muramatsu, Hiromi; Weissman, Drew et al. (2013) In vitro transcription of long RNA containing modified nucleosides. Methods Mol Biol 969:29-42
Weissman, Drew; Pardi, Norbert; Muramatsu, Hiro et al. (2013) HPLC purification of in vitro transcribed long RNA. Methods Mol Biol 969:43-54
Anderson, B R; Karikó, K; Weissman, D (2013) Nucleofection induces transient eIF2? phosphorylation by GCN2 and PERK. Gene Ther 20:136-42
Boros, Gábor; Miko, Edit; Muramatsu, Hiromi et al. (2013) Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: a new approach with future therapeutic potential. J Photochem Photobiol B 129:93-9
Karikó, Katalin; Muramatsu, Hiromi; Keller, Jason M et al. (2012) Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin. Mol Ther 20:948-53
Karikó, Katalin; Muramatsu, Hiromi; Ludwig, János et al. (2011) Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res 39:e142
Anderson, Bart R; Muramatsu, Hiromi; Jha, Babal K et al. (2011) Nucleoside modifications in RNA limit activation of 2'-5'-oligoadenylate synthetase and increase resistance to cleavage by RNase L. Nucleic Acids Res 39:9329-38
Anderson, Bart R; Muramatsu, Hiromi; Nallagatla, Subba R et al. (2010) Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res 38:5884-92

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