Abstract
Purpose :
Fuchs’ endothelial corneal dystrophy (FECD) is a genetic disease which leads to eye pain, significant loss of vision and corneal lesions called guttae. Late-onset FECD is characterized by the expanded repeat trinucleotide sequence (CTG)n (n>30-40) in the TCF4 gene. The accumulation of CUG RNA in the nucleus forms cytotoxic RNA foci. Recently, we developed a recombinant variant of DICER, OptiDicer, which can degrade double-strand RNA through RNaseIII activity. In this study, we examined whether OptiDicer can decrease CUG RNA accumulation in corneal endothelial cells from patients with Fuchs' dystrophy.
Methods :
F35T cells, Human corneal endothelial cells from an FECD patient with (CTG)n n>1000, were used in this study. pCMV-NLS-OptiDicer (Nuclear localization signal conjugated OptiDicer) was transfected by Lipofectamine-3000 in an Fibronectin coated 8-well chamber slide. As a control, we used D2A-OptiDicer (which has no RNaseIII activity by introducing two alanine scanning mutations in OptiDicer). Two days post transfection, the cells were fixed by 4% PFA, and then subjected to in situ hybridization using Alexa647 conjugated (CAG)8 2O-methyl RNA probes in order to detect CUG RNA accumulation. The images were obtained with an EVOS fluorescence microscope, and the number of CUG RNA accumulation was counted.
Results :
The transfection efficiency was estimated as 50%. We did not find significant cell loss by pCMV-NLS-OptiDicer or pCMV-D2A-OptiDicer transfection. Accumulation of CUG RNA was counted in 276 OptiDicer transfected and 222 D2A-OptiDcier transfected F35T cells. The average number of CUG RNA accumulation was 1.9+/-1.4 in OptiDicer-F35T and 2.9+/-1.7 in D2A-OptiDicer control F35T (p<0.001), respectively.
Conclusions :
We found that OptiDicer significantly decreased CUG-RNA accumulation in late-onset FECD patient derived corneal endothelial cells, although the low transfection efficiency may underestimate OptiDicer effect. Our result suggests OptiDicer can be a potential treatment for long CUG RNA repeat derived FECD. Future studies will explore OptiDicer in other cell lines from FECD patients with other transfection methods.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.