Using Crispr Interference As A Therapeutic Approach To Treat Tgf Beta 2-Induced Ocular Hypertension And Glaucoma

PURPOSE. Primary open angle glaucoma (POAG) is a leading cause of blindness world-wide with elevated intraocular pressure (IOP) as the most important risk factor. POAG IOP elevation is due to pathological changes in the trabecular meshwork (TM). Elevated TGF beta 2 contributes to these changes and increases IOP. We have shown that histone hyperacetylation is associated with TGF beta 2 elevation in the TM. In this study, we determined if clustered regularly interspaced short palindromic repeats (CRISPR) interference could specifically deacetylate histones and decrease TGF beta 2 in the TM.METHODS. We tested the efficiency of different promoters in driving KRAB-dCAS9 expression in human TM cells. We also screened and determined the optimal sgRNA sequence in the inhibition of TGF beta 2. Chromatin immunoprecipitation-qPCR was used to determine the binding of KRAB-dCAS9. An adenovirus-mediated TGF beta 2-induced ocular hypertension (OHT) mouse model was used to determine the effect of the CRISPR interference system in vivo.RESULTS. We found that the CRISPR interference system inhibited TGF beta 2 expression in human TM cells, and properly designed sgRNA targeted the promoter of the TGF beta 2 gene. Using sgRNA targeting the CMV promoter of the Ad5-CMV-TGF beta 2 viral vector, we found that lentivirus-mediated KRAB-dCAS9 and sgRNA expression was able to inhibit Ad5-CMV-TGF beta 2-induced OHT in C57BL/6J female and male mice eyes. This inhibition of OHT was associated with decreased levels of TGF beta 2 and extracellular matrix proteins in the mouse eye.CONCLUSIONS. Our results indicate that CRISPR interference is a useful tool for gene inhibition and may be a therapeutic approach to treat TGF beta 2-induced OHT.