title: Translational read-through of the RP2 Arg120stop mutation in patient iPSC-derived retinal pigment epithelium cells.
abstract: Mutations in the RP2 gene lead to a severe form of X-linked retinitis pigmentosa. RP2 patients frequently present with nonsense mutations and no treatments are currently available to restore RP2 function. In this study, we reprogrammed fibroblasts from an RP2 patient carrying the nonsense mutation c.519C>T (p.R120X) into induced pluripotent stem cells (iPSC), and differentiated these cells into retinal pigment epithelial cells (RPE) to study the mechanisms of disease and test potential therapies. RP2 protein was undetectable in the RP2 R120X patient cells, suggesting a disease mechanism caused by complete lack of RP2 protein. The RP2 patient fibroblasts and iPSC-derived RPE cells showed phenotypic defects in IFT20 localization, Golgi cohesion and Gβ1 trafficking. These phenotypes were corrected by over-expressing GFP-tagged RP2. Using the translational read-through inducing drugs (TRIDs) G418 and PTC124 (Ataluren), we were able to restore up to 20% of endogenous, full-length RP2 protein in R120X cells. This level of restored RP2 was sufficient to reverse the cellular phenotypic defects observed in both the R120X patient fibroblasts and iPSC-RPE cells. This is the first proof-of-concept study to demonstrate successful read-through and restoration of RP2 function for the R120X nonsense mutation. The ability of the restored RP2 protein level to reverse the observed cellular phenotypes in cells lacking RP2 indicates that translational read-through could be clinically beneficial for patients.
authors.affiliation: UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK.
authors.forename: Nele
authors.initials: N
authors.lastname: Schwarz
authors.affiliation: UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK.
authors.forename: Amanda-Jayne
authors.initials: AJ
authors.lastname: Carr
authors.affiliation: UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK.
authors.forename: Amelia
authors.initials: A
authors.lastname: Lane
authors.affiliation: Johannes Gutenberg-University Muellerweg 6, 55099 Mainz, Germany and.
authors.forename: Fabian
authors.initials: F
authors.lastname: Moeller
authors.affiliation: UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK.
authors.forename: Li Li
authors.initials: LL
authors.lastname: Chen
authors.affiliation: UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK.
authors.forename: Mònica
authors.initials: M
authors.lastname: Aguilà
authors.affiliation: UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK.
authors.forename: Britta
authors.initials: B
authors.lastname: Nommiste
authors.affiliation: UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK, Moorfields Eye Hospital, 162 City Road, London EC1V 2PD, UK.
authors.forename: Manickam N
authors.initials: MN
authors.lastname: Muthiah
authors.affiliation: UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK.
authors.forename: Naheed
authors.initials: N
authors.lastname: Kanuga
authors.affiliation: Johannes Gutenberg-University Muellerweg 6, 55099 Mainz, Germany and.
authors.forename: Uwe
authors.initials: U
authors.lastname: Wolfrum
authors.affiliation: Johannes Gutenberg-University Muellerweg 6, 55099 Mainz, Germany and.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Nagel-Wolfrum
authors.affiliation: UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK, Moorfields Eye Hospital, 162 City Road, London EC1V 2PD, UK.
authors.forename: Lyndon
authors.initials: L
authors.lastname: da Cruz
authors.affiliation: UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Coffey
authors.affiliation: UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK, michael.cheetham@ucl.ac.uk.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Cheetham
authors.affiliation: UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK.
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Hardcastle
mesh_terms: D002454:Cell Differentiation; D065150:Cellular Reprogramming; D002923:Cilia / Q000378:metabolism / Q000473:pathology; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism*; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D019204:GTP-Binding Proteins; D015870:Gene Expression; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010069:Oxadiazoles / Q000494:pharmacology; D010641:Phenotype; D014176:Protein Biosynthesis / Q000187:drug effects; D021381:Protein Transport; D055213:Retinal Pigment Epithelium / Q000166:cytology*; D055815:Young Adult
chemical_list: D005136:Eye Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010069:Oxadiazoles; C072178:RP2 protein, human; D019204:GTP-Binding Proteins; C515878:ataluren