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.

Nonsense suppression therapies in ocular genetic diseases

“…More recently, a study has demonstrated that treatment of R120X fibroblasts with G418 caused an increase in 40 % of RP2 mRNA and 20 % of protein levels, while PTC124 can restore up to 13 % of RP2 protein levels, but did not significantly increase RP2 mRNA levels. The stored RP2 protein was functional in reversing the cellular phenotypes in cells lacking RP2 proteins [50].…”

Das Überlesen von Nonsense-Mutationen

“…Mutationen im Retinitis pigmentosa-2-Gen (RP2) verursacheneine X-chromosomal gekoppelte RP. RP2-Patienten zeigen häufig NonsenseMutationen, wobei die p.(Arg120*) die häufigste Mutation darstellt [22]. In Fibroblasten von Patienten und in retinalen Pigmentepithelzellen (RPE), die aus induzierten pluripotenten Stammzellen ("induced pluripotent stem cells"; iPSC) generiert wurden, konnte die wiederhergestellte Expression von RP2 nach der Behandlung mit Ataluren und G418 gezeigt werden.…”

“…In Fibroblasten von Patienten und in retinalen Pigmentepithelzellen (RPE), die aus induzierten pluripotenten Stammzellen ("induced pluripotent stem cells"; iPSC) generiert wurden, konnte die wiederhergestellte Expression von RP2 nach der Behandlung mit Ataluren und G418 gezeigt werden. Darüber hinaus wurde phä-notypisch die korrekte Lokalisation des Zilienproteins IFT20 und der Golgi-Architektur durch Gentamicin-und Ataluren-Behandlung wiederhergestellt [22]. Eine Nonsense-Mutation in MERTK (MER Rezeptortyrosinkinase, RP38), konnte durch die Applikation von G418 und Ataluren überlesen werden.…”

Induced Pluripotent Stem Cell Therapies for Degenerative Disease of the Outer Retina: Disease Modeling and Cell Replacement

“…54 iPSC-derived RPE from patients with mutations in the MERTK gene, which causes RP, exhibits defective phagocytosis 55 and from patients with mutations in RP2 shows defects in IFT20 localization, Golgi cohesion, and protein trafficking, which could be corrected either by protein overexpression or by translational read through inducing drugs. 56 iPSC lines from AMD-affected individuals have also been used to derive RPE cells, to model the disease in vitro or for drug screening. 57,58 Although AMD-iPSC are able to undergo normal RPE differentiation, they express lower levels of ZO-1 and RPE65, suggesting defective tight junction formation.…”