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Naga Pradeep Rayana, Chenna Kesavulu Sugali, Jiannong Dai, Michael Peng, Shaohui Liu, Yucheng Zhang, Jun Wan, Weiming Mao; Using CRISPR Interference as a Therapeutic Approach to Treat TGFβ2-Induced Ocular Hypertension and Glaucoma. Invest. Ophthalmol. Vis. Sci. 2021;62(12):7. doi: https://doi.org/10.1167/iovs.62.12.7.
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Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide 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 TGFβ2 contributes to these changes and increases IOP. We have shown that histone hyperacetylation is associated with TGFβ2 elevation in the TM. In this study, we determined if clustered regularly interspaced short palindromic repeats (CRISPR) interference could specifically deacetylate histones and decrease TGFβ2 in the TM.
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 TGFβ2. Chromatin immunoprecipitation-qPCR was used to determine the binding of KRAB-dCAS9. An adenovirus-mediated TGFβ2-induced ocular hypertension (OHT) mouse model was used to determine the effect of the CRISPR interference system in vivo.
We found that the CRISPR interference system inhibited TGFβ2 expression in human TM cells, and properly designed sgRNA targeted the promoter of the TGFβ2 gene. Using sgRNA targeting the CMV promoter of the Ad5-CMV-TGFβ2 viral vector, we found that lentivirus-mediated KRAB-dCAS9 and sgRNA expression was able to inhibit Ad5-CMV-TGFβ2-induced OHT in C57BL/6J female and male mice eyes. This inhibition of OHT was associated with decreased levels of TGFβ2 and extracellular matrix proteins in the mouse eye.
Our results indicate that CRISPR interference is a useful tool for gene inhibition and may be a therapeutic approach to treat TGFβ2-induced OHT.
GAPDH24 – Forward: 5’-GGTGAAGGTCGGAGTCA-AC-3′ Reverse: 5′-CCATGGGTGGAATCATATTG-3′ TGFβ2 – Forward: 5′-AGAGTGCCTGAACAACGGATT-3′ Reverse: 5′-CCATTCGCCTTCTGCTCTT-3′
GAPDH24 – Forward: 5’-GGTGAAGGTCGGAGTCA-AC-3′
TGFβ2 – Forward: 5′-AGAGTGCCTGAACAACGGATT-3′
TGFβ2 1 – Forward - 5’-TTTCCACACTCCCTCAA-CGG-3’ Reverse – 5’-TCTCTGAACCACGTGTCTGC-3’ TGFβ2 5 – Forward - 5’-CCCACAGCGGTCCTCAT-TTC-3’ Reverse – 5’-TGCCAGCAGATAACATCACGA-3’
TGFβ2 1 – Forward - 5’-TTTCCACACTCCCTCAA-CGG-3’
Reverse – 5’-TCTCTGAACCACGTGTCTGC-3’
TGFβ2 5 – Forward - 5’-CCCACAGCGGTCCTCAT-TTC-3’
Reverse – 5’-TGCCAGCAGATAACATCACGA-3’
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