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Ankur Jain, Gulab Zode, Charles Searby, Abbot F Clark, Val C Sheffield; CRISPR-Cas9 Mediated Genome Editing of Myocilin in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3187.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is a leading cause of irreversible blindness with elevated intraocular pressure (IOP) being the most important risk factor. Mutations in myocilin (MYOC) leading to MYOC misfolding and increased expression of MYOC by dexamethasone (DEX) have been associated with endoplasmic reticulum (ER) stress in the trabecular meshwork (TM), the tissue that maintains the aqueous humor outflow and regulates IOP. ER stress and/or death of the TM lead to ocular hypertension and glaucoma. We propose to relieve ER stress in human and mouse TM cells by targeting the MYOC gene using state of art CRISPR-Cas9 technology.
We generated TM cell lines overexpressing wild type and mutant human MYOC and looked for ER stress markers using quantitative PCR and western immunoblotting. MYOC was targeted by transient transfection of px330-CRISPR plasmids with guide RNAs targeting the MYOC gene (Exon1). Mouse TM cells were pretreated with Ad5px330-CRISPR virus and checked for DEX-mediated MYOC accumulation and ER stress.
TM cell lines overexpressing mutant MYOC have increased accumulation of MYOC in the ER and increased ER stress as compared to wild type. Transient transfection reduces levels of MYOC and ER stress in these cells. Mouse TM cells have reduced levels of DEX-mediated ER stress when pretreated with Ad5-CRISPR as compared to null virus controls.
This is a proof of principle study indicating that targeting the MYOC gene can relieve ER stress in TM cells, rescuing their function. We plan to translate this approach to in vivo transgenic hMYOCY437H and DEX-induced ocular hypertension mouse models.
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