New publication: AAV Gene therapy for Bietti crystalline dystrophy
The team has tested the use of Adeno-associated viruses (AAV) as a gene therapy for Bietti crystalline dystrophy in cell models in the lab.
AAV2-mediated gene therapy for Bietti crystalline dystrophy provides functional CYP4V2 in multiple relevant cell models
Jiang-Hui Wang, Grace Lidgerwood, Maciej Daniszewski, Monica Hu, Georgina Roberts, Raymond Ching-Bong Wong, Sandy Hung, Michelle McClements, Alex Hewitt, Alice Pebay, Doron Hickey, Tom Edwards
Scientific Reports, 2022, in press
Purpose: Bietti crystalline dystrophy (BCD) is an inherited retinal disease (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.
Methods: Human wild-type or codon-optimized CYP4V2 was cloned into a mammalian expression plasmid with a CAG (CMV-enhanced chicken beta-actin) promoter (p.wtCYP4V2 or p.coCYP4V2). and packaged into AAV2 (AAV2.wtCYP4V2 or AAV2.coCYP4V2) for viral transduction experiments. HEK293 or ARPE19 cells or BCD patient specific induced pluripotent stem cell-derived RPE cells (iPS-RPE) were transduced, and CYP4V2 expression was verified by immunocytochemistry and western blot. Functionality of CYP4V2 in transfected or transduced cells was confirmed by a cytochrome P450 enzyme assay. Expression of CYP4V2 was assessed in ex vivo AAV2-transduced human retina explant from a healthy donor.
Results: HEK293, ARPE19, and patient induced pluripotent stem cell (iPSC)- derived RPE cells transduced with AAV2.coCYP4V2 resulted in elevated protein expression levels of CYP4V2 compared to those transduced with AAV2.wtCYP4V2, as assessed by immunocytochemistry and western blot. Similarly, we observed significantly increased CYP4V2 enzyme activity in cells transduced with AAV2.coCYP4V2 compared to those transduced with AAV2.wtCYP4V2. We also showed CYP4V2 expression in human RPE/choroid explants transduced with AAV2.coCYP4V2 compared to those transduced with AAV2.wtCYP4V2.
Conclusions: These preclinical data support the further development of a gene supplementation therapy for a currently untreatable blinding condition – BCD. Codon-optimized CYP4V2 transgene was superior to wild type in terms of protein expression and enzyme activity. Ex vivo culture of human RPE cellsprovided an effective approach to test AAV-mediated transgene delivery.
Read the publication here.