title: Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: a new approach with future therapeutic potential.
abstract: UVB irradiation induces harmful photochemical reactions, including formation of Cyclobutane Pyrimidine Dimers (CPDs) in DNA. Accumulation of unrepaired CPD lesions causes inflammation, premature ageing and skin cancer. Photolyases are DNA repair enzymes that can rapidly restore DNA integrity in a light-dependent process called photoreactivation, but these enzymes are absent in humans. Here, we present a novel mRNA-based gene therapy method that directs synthesis of a marsupial, Potorous tridactylus, CPD-photolyase in cultured human keratinocytes. Pseudouridine was incorporated during in vitro transcription to make the mRNA non-immunogenic and highly translatable. Keratinocytes transfected with lipofectamine-complexed mRNA expressed photolyase in the nuclei for at least 2days. Exposing photolyase mRNA-transfected cells to UVB irradiation resulted in significantly less CPD in those cells that were also treated with photoreactivating light, which is required for photolyase activity. The functional photolyase also diminished other UVB-mediated effects, including induction of IL-6 and inhibition of cell proliferation. These results demonstrate that pseudouridine-containing photolyase mRNA is a powerful tool to repair UVB-induced DNA lesions. The pseudouridine-modified mRNA approach has a strong potential to discern cellular effects of CPD in UV-related cell biological studies. The mRNA-based transient expression of proteins offers a number of opportunities for future application in medicine.
authors.affiliation: Department of Dermatology, Medical and Health Science Center, University of Debrecen, Nagyerdei korut 98, 4032 Debrecen, Hungary. Electronic address: gaba@med.unideb.hu.
authors.forename: Gábor
authors.initials: G
authors.lastname: Boros
authors.forename: Edit
authors.initials: E
authors.lastname: Miko
authors.forename: Hiromi
authors.initials: H
authors.lastname: Muramatsu
authors.forename: Drew
authors.initials: D
authors.lastname: Weissman
authors.forename: Eszter
authors.initials: E
authors.lastname: Emri
authors.forename: Dávid
authors.initials: D
authors.lastname: Rózsa
authors.forename: Georgina
authors.initials: G
authors.lastname: Nagy
authors.forename: Attila
authors.initials: A
authors.lastname: Juhász
authors.forename: István
authors.initials: I
authors.lastname: Juhász
authors.forename: Gijsbertus
authors.initials: G
authors.lastname: van der Horst
authors.forename: Irén
authors.initials: I
authors.lastname: Horkay
authors.forename: Éva
authors.initials: É
authors.lastname: Remenyik
authors.forename: Katalin
authors.initials: K
authors.lastname: Karikó
authors.forename: Gabriella
authors.initials: G
authors.lastname: Emri
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects / Q000528:radiation effects; D004260:DNA Repair; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008027:Light; D008055:Lipids / Q000737:chemistry; D048328:Potoroidae / Q000378:metabolism; D011560:Pseudouridine / Q000737:chemistry* / Q000378:metabolism; D011740:Pyrimidine Dimers / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D014466:Ultraviolet Rays
chemical_list: D015850:Interleukin-6; D008055:Lipids; C086724:Lipofectamine; D011740:Pyrimidine Dimers; D012333:RNA, Messenger; D011560:Pseudouridine; D004255:Deoxyribodipyrimidine Photo-Lyase
keywords: Cyclobutane pyrimidine dimers; Human keratinocyte; Photolyase; Pseudouridine-modified mRNA; UVB; mRNA therapy