title: Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts.
abstract: Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5'- and 3'-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2were synthesized and cloned as five different recombinant sequence set combinations as upstream 5'-UTR and/or downstream 3'-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs.
authors.affiliation: Institute of Molecular Immunology- Experimental Oncology, Klinikum rechts der Isar, Technische Universität München, Munich, 81675, Germany.|Ethris GmbH, Planegg, 82152, Germany.
authors.forename: Mehrije
authors.initials: M
authors.lastname: Ferizi
authors.affiliation: Ethris GmbH, Planegg, 82152, Germany.
authors.forename: Manish K
authors.initials: MK
authors.lastname: Aneja
authors.affiliation: Experimental Trauma Surgery, Klinikum rechts der Isar, Technische Universität München, Munich, 81675, Germany.
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Balmayor
authors.affiliation: Institute of Molecular Immunology- Experimental Oncology, Klinikum rechts der Isar, Technische Universität München, Munich, 81675, Germany.
authors.forename: Zohreh Sadat
authors.initials: ZS
authors.lastname: Badieyan
authors.affiliation: Institute of Molecular Immunology- Experimental Oncology, Klinikum rechts der Isar, Technische Universität München, Munich, 81675, Germany.|Ethris GmbH, Planegg, 82152, Germany.
authors.forename: Olga
authors.initials: O
authors.lastname: Mykhaylyk
authors.affiliation: Ethris GmbH, Planegg, 82152, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Rudolph
authors.affiliation: Institute of Molecular Immunology- Experimental Oncology, Klinikum rechts der Isar, Technische Universität München, Munich, 81675, Germany.|Ethris GmbH, Planegg, 82152, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Plank
mesh_terms: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000072283:A549 Cells; D000273:Adipose Tissue / Q000166:cytology; D000818:Animals; D019540:Area Under Curve; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D006207:Half-Life; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D051379:Mice; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D041681:NIH 3T3 Cells; D010012:Osteogenesis; D014176:Protein Biosynthesis; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D012372:ROC Curve; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D055396:Bone Morphogenetic Protein 2; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019255:NADPH Oxidases; C086986:CYBA protein, human