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Jiang-Hui Wang, Doron Hickey, Michelle E McClements, Timothy Colgan, Thomas Edwards; Validation of in vitro gene therapy for Bietti crystalline dystrophy. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3420. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Bietti crystalline dystrophy (BCD) is an inherited retinal dystrophy (IRD) caused by mutations in the CYP4V2 gene. It is a relatively common cause of IRD in east Asia. A number of features of this disease make it highly amenable to gene supplementation therapy. This study aims to validate a series of essential precursor in vitro experiments prior to developing a clinical gene therapy for BCD.
Human CYP4V2 was cloned into a mammalian expression plasmid (pCAG-CYP4V2) with a CAG (CMV-enhanced chicken beta-actin) promoter. Using standard adeno-associated virus (AAV) production methods, pCAG-CYP4V2 was packaged into AAV for viral transduction experiments. 293 cells were transfected or transduced and CYP4V2 expression was verified by qPCR, immunocytochemistry and Western blot. Functionality of CYP4V2 in 293 cells transfected with pCAG-CYP4V2 or transduced with AAV-CAG-CYP4V2 was confirmed by a nonselective cytochrome P450 enzyme assay.
As assessed by qPCR, expression of CYP4V2 in 293 cells is significantly higher (2145 ± 121.8 fold increase, p<0.001) than untreated controls. Similarly, plasmid transfection and AAV transduction of 293 cells with pCAG-CYP4V2 and AAV-CAG-CYP4V2, respectively lead to elevated protein expression levels as assessed by immunocytochemistry and Western blot, compared to untreated controls. Moreover, we observed significantly increased CYP4V2 functionality in cells transfected and transduced with pCAG-CYP4V2 (76.1% increase, p<0.001) and AAV-CAG-CYP4V2, respectively, and such increased functionality was significantly reduced (67.2% reduction, p<0.001) when transfected cells were additionally treated with a specific CYP4V2 inhibitor (10µM HET0016 for 5 minutes).
These results provide encouraging data for the preclinical assessment of a gene supplementation therapy for a currently untreatable blinding condition - BCD. The CAG promotor provided strong expression of the CYP4V2 transgene in 293 cells, both via plasmid transfection and AAV transduction. The validation of CYP4V2 antibody and functional assay in vitro allowed us to further investigate CYP4V2 expression and its function in induced pluripotent stem cell - derived retinal pigment epithelium cells from patients with BCD.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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