Fuchs endothelial corneal dystrophy (FECD) is characterized by endothelial cell death, corneal edema, and vision loss. FECD affects approximately 4% of the US population. The only definitive treatment is endothelial keratoplasty, and FECD was the leading indication for corneal transplant in the US in 2013. FECD is a corneal disease with major public health impact in the US and thus is relevant to the mission of the National Institutes of Health. A major unmet need for this important corneal disorder is better understanding of pathophysiology and improved, non-surgical treatments. Approximately 70% of FECD cases are caused by a trinucleotide repeat (TNR) expansion in the TCF4 gene. Major pathogenic mechanisms of TNR expansions involve 1) RNA toxicity from antisense transcription and 2) protein toxicity from repeat associated non-ATG (RAN) translation. Both of these mechanisms remain uncharacterized for the TCF4 mutation, and successful completion of this project would substantially expand our understanding of TNR associated pathogenesis of FECD. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) 9 nuclease are an extremely versatile and accurate approach to cut genomic DNA in numerous experimental systems. CRISPR/Cas9 have been used to delete and replace genomic sequence using non-homologous end joining (NHEJ) or homology directed repair (HDR). Use of this system for correction of the TCF4 TNR repeat in FECD has not been investigated and could lead to new treatment approaches. Overall goals of this project are to assess 1) the roles of antisense RNAs and RAN translation as pathogenic mechanisms and 2) the potential for CRISPR/Cas9 to correct the TCF4 TNR expansion.
Aim I will test the hypothesis that the TCF4 TNR expansion: 1) produces toxic antisense RNAs which bind the RNA processing protein, muscleblind-like 1 (MBNL1) and 2) produces aberrant proteins resulting from repeat associated non-ATG (RAN) translation. Toxic antisense RNAs and MBNL1 binding will be identified by assessing for colocalization of RNA foci using labeled oligonucleotides and MBNL1 using fluorescence in situ hybridization. RAN translation products will be assessed by Western blotting of transformed human corneal endothelial cells transfected with plasmids encoding predicted RAN proteins and immunohistochemistry of FECD patient endothelial tissue using polyclonal antibodies raised against predicted RAN proteins.
Aim II will test the hypothesis that CRISPR/Cas9 can be used to correct the TCF4 TNR expansion. Guide RNAs (gRNAs) will be designed on either side of the TCF4 TNR and will be assessed singly for cutting activity in 293 cells. The best gRNAs will be singly or doubly transfected with flanking oligonucleotides to assess for HDR, and doubly transfected without an oligonucleotide to assess for NHEJ in 293 cells and subsequently in transformed FECD corneal endothelial cells with the TCF4 TNR expansion.
Fuchs endothelial corneal dystrophy (FECD) is a common disease in which loss of cells lining the inner surface of the cornea causes clouding of the cornea and decreased vision. FECD affects approximately 4% of the US population and was the leading cause of corneal transplant surgery in 2013. The purpose of this study is to investigate the causes of cell death in FECD and possible gene therapy approaches which may lead to improved treatments for FECD.