Fuchs? endothelial corneal dystrophy is a degenerative disorder of the eye?s corneal endothelium and the most common indication for corneal transplantation. The majority of patients carry a trinucleotide repeat expansion in the TCF4 gene, causing sequestration of the MBNL1 splicing factor and widespread mis-splicing of MBNL1- regulated transcripts. Here we propose to use patient-derived cells to provide a characterization of the molecular pathology of this disease and evaluate the potential of our newly developed RNA-targeting CRISPR/Cas9 system as a therapeutic modality to eliminate toxic repeat expansions in TCF4 RNA transcripts.
Several human diseases are linked to expansions in short DNA sequences whose transcripts produce toxic RNA products. One of these repeat expansion diseases, Fuchs? endothelial corneal dystrophy or FECD affects approximately 4% of adults over age 40 and ultimately causes vision loss if untreated. Recent studies have linked the presence of CUG repeat RNA foci originating from the TCF4 locus and associated RNA splicing defects indicating that the molecular pathology associated with FECD largely manifests on the level of RNA, likely due to sequestration of the MBNL1 splicing factor from its natural RNA targets. In this study, we generate a molecular description of FECD as pre-requisites for the identification of disease biomarkers and the evaluation of the efficacy of potential therapeutics. We employ RNA sequencing of single corneal epithelial cells from FECD patient corneas and corneal endothelial cells from FECD patient-derived induced pluripotent stem cells to achieve sensitive detection of RNA splicing dysfunction linked to FECD. This effort will generate a comprehensive picture of the MBNL-associated molecular pathology of FECD and may reveal MBNL- independent splicing changes associated with FECD that distinguish this disease other CTG repeat expansion diseases, providing a detailed molecular description of the role of MBNL1 in FECD and will constitute initial efforts towards identifying molecular features associated with FECD that are unique among CTG repeat expansion conditions. In addition, we recently established the ability of CRISPR/Cas9 to bind RNA (RNA- targeting Cas9 or RCas9) in living human cells in a programmable fashion determined by nucleic acid specificity alone and demonstrated the ability of RCas9 to eliminate RNAs produced from microsatellite repeat expansions and have also generated minimized versions of the RCas9 system that are compatible with the limited packaging capacity of adeno-associated viral vectors (AAV), which provide an established transduction mechanism in the human eye. We will apply this methodology to test if targeting TCF4 repeat expansions for degradation can revert or attenuate the molecular (mis-splicing) and cellular (RNA foci) consequences of FECD. If successful, long-lived expression of the RCas9 system supported by an adeno-associated viral (AAV) vector has the potential to provide a permanent, single dose cure for Fuchs? and, by extension, other conditions caused by RNA repeats.
|Gottesfeld, Joel M; Carey, Michael F (2018) Introduction to the Thematic Minireview Series: Chromatin and transcription. J Biol Chem 293:13775-13777|