title: Efficient delivery and functional expression of transfected modified mRNA in human embryonic stem cell-derived retinal pigmented epithelial cells.
abstract: Gene- and cell-based therapies are promising strategies for the treatment of degenerative retinal diseases such as age-related macular degeneration, Stargardt disease, and retinitis pigmentosa. Cellular engineering before transplantation may allow the delivery of cellular factors that can promote functional improvements, such as increased engraftment or survival of transplanted cells. A current challenge in traditional DNA-based vector transfection is to find a delivery system that is both safe and efficient, but using mRNA as an alternative to DNA can circumvent these major roadblocks. In this study, we show that both unmodified and modified mRNA can be delivered to retinal pigmented epithelial (RPE) cells with a high efficiency compared with conventional plasmid delivery systems. On the other hand, administration of unmodified mRNA induced a strong innate immune response that was almost absent when using modified mRNA. Importantly, transfection of mRNA encoding a key regulator of RPE gene expression, microphthalmia-associated transcription factor (MITF), confirmed the functionality of the delivered mRNA. Immunostaining showed that transfection with either type of mRNA led to the expression of roughly equal levels of MITF, primarily localized in the nucleus. Despite these findings, quantitative RT-PCR analyses showed that the activation of the expression of MITF target genes was higher following transfection with modified mRNA compared with unmodified mRNA. Our findings, therefore, show that modified mRNA transfection can be applied to human embryonic stem cell-derived RPE cells and that the method is safe, efficient, and functional.
authors.affiliation: From the Center of Regenerative Medicine in Barcelona, 08003 Barcelona, Spain, magnus.l.hansson@ki.se.
authors.forename: Magnus L
authors.initials: ML
authors.lastname: Hansson
authors.affiliation: From the Center of Regenerative Medicine in Barcelona, 08003 Barcelona, Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: Albert
authors.affiliation: From the Center of Regenerative Medicine in Barcelona, 08003 Barcelona, Spain, the Universitat de Barcelona, 08007 Barcelona, Spain, and.
authors.forename: Louisa
authors.initials: L
authors.lastname: González Somermeyer
authors.affiliation: From the Center of Regenerative Medicine in Barcelona, 08003 Barcelona, Spain.
authors.forename: Rubén
authors.initials: R
authors.lastname: Peco
authors.affiliation: From the Center of Regenerative Medicine in Barcelona, 08003 Barcelona, Spain.
authors.forename: Eva
authors.initials: E
authors.lastname: Mejía-Ramírez
authors.affiliation: From the Center of Regenerative Medicine in Barcelona, 08003 Barcelona, Spain.
authors.forename: Núria
authors.initials: N
authors.lastname: Montserrat
authors.affiliation: the Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, 92037 California belmonte@salk.edu.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Izpisua Belmonte
mesh_terms: D021581:Active Transport, Cell Nucleus; D015153:Blotting, Western; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000235:genetics / Q000378:metabolism; D051739:Microphthalmia-Associated Transcription Factor / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D051857:Otx Transcription Factors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D019745:cis-trans-Isomerases / Q000235:genetics / Q000378:metabolism; D000099178:Retinoid Isomerohydrolase
chemical_list: D050838:Interferon Regulatory Factor-3; D051739:Microphthalmia-Associated Transcription Factor; D051857:Otx Transcription Factors; D012333:RNA, Messenger; D051996:Transcription Factor RelA; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C494232:IRF3 protein, human; C497234:MITF protein, human; C497659:OTX2 protein, human; C498143:RELA protein, human
keywords: Differentiation; Embryonic Stem Cell; Induced Pluripotent Stem Cell (iPS Cell, iPSC); Modified mRNA; Retinal Pigmented Epithelial Cell; Transfection; mRNA