Molecular Mechanism And Gene Therapy Research Of Bietti Crystalline Corneoretinal Dystrophy

Purposes:Bietti crystalline corneoretinal dystrophy(BCD)is an autosomal recessive retinal degeneration,resulting in blindness in most patients.The pathogenic gene is CYP4V2,which is highly expressed in retinal pigment epithelium(RPE)cells and plays an important role in the metabolism of steroids and fatty acids.The disease has a great impact on the visual function of patients,but there are no effective treatments currently available and the underlying mechanisms of diseses are not clear.Therefore,this study intends to explore the molecular mechanism of BCD by using induced pluripotent stem cells(iPSCs),and to design and optimize the gene therapy vector of BCD for clinical application.Methods:1.Skin fibroblasts and blood lymphocytes were prepared from BCD patients and normal controls,which were induced into iPSCs using Sendai virus,and the characteristics of stem cells were also identified.The iPSCs were then differentiated into RPE cells,which can be used as a cell model for disease.Annexin V-FITC and Tunel were used to detect apoptosis;mitochondrial reactive oxygen species and DHE were used to detect oxidative stress;western blot(WB)and Fluo-4 AM were used to detect the endoplasmic reticulum stress levels.2.Lipidomics was used for serum and iPSC-RPE cells from patients and normal controls.Statistical analysis of lipidomics results were performed using R software.The orthogonal partial least squares discriminant analysis(OPLS-DA)was utilized to establish a predictive model.A volcano plot was constructed based on the P-value and fold change.RNA was extracted from iPSC-RPE cells of patients and normal controls for transcriptome,statistical analysis was performed including difference significance analysis and functional enrichment analysis.3.The iPSCs from one BCD patient were further differentiated into retinal organoids,and immunofluorescence(IF)was used to detect the presence of various types of retinal cells and mature photoreceptor cells in the constructed retinal organoids.4.Optimizing the capsid,promoter,and transgene sequences of adeno-associated virus(AAV)for BCD gene therapy.The promoter and transgene verification experiments were carried out by constructing the corresponding plasmid and to transfect ARPE-19 cells,and WB and quantitative PCR(qPCR)were used to detect which optimization could improve more expression of CYP4V2 in cells.The capsid was injected with GFP virus into the vitreous cavity of mice and simultaneously infected iPSC-RPE cells and retinal organoids.The efficiency of capsids was evaluated by the immunofluorescence of retinal section,retinal flatmount,and GFP expression of cells and retinal organoids.Results:1.The iPSCs from 5 BCD patients and 4 normal subjects were successfully generated,and all of them had the characteristics of stem cells and then differentiated into RPE cells.It was found that iPSC-RPE cells from BCD patients were more likely to form vacuolar structures and cell death than cells from normal subjects.Moreover,apoptosis increased,oxidative stress and endoplasmic reticulum stress increased in BCD.2.The serum and iPSC-RPE cells of BCD patients and normal subjects were analyzed by lipidomics and transcriptome.It was found that there were significant abnormalities in lipid metabolism in BCD.Plasmenyl-phosphatidylethanolamines(PEp)was abnormal in both serum and RPE cells.It is suggested that peroxisome may play an important role in the occurrence and development of BCD,and endoplasmic reticulum dysfunction may also be an important fantor affecting lipid metabolism.3.A BCD patient-specific retinal organoid was successfully generated with various types of retinal cells and mature photoreceptor cells.4.The optimal promoter,trangene sequence and capsid structure of AAV for BCD gene therapy were preliminarily determined,which could provide the expression of CYP4V2 protein.Conclusions:Abnormal lipid metabolism plays an important role in the development of BCD disease,and abnormal endoplasmic reticulum function may be associated with abnormal lipid and promote the degeneration and apoptosis of RPE cells in BCD patients.It is also found that peroxisome may affect lipid metabolism of BCD.Retinal organoid with mature photoreceptor cells lay a foundation for further research.The improvement and optimization of AAV vector provide basis for further clinical trials of gene therapy for BCD patients.