Bietti crystalline dystrophy like retinal degeneration (BCD) is an inherited retinal degenerative disease caused by defects in the CYP4V2 gene. Recombinant AAV-based gene replacement therapy holds promise for this disease, yet reliable enzymatic activity assays for product potency evaluation remain urgently needed. This study established and validated a method for determining the enzymatic activity of CYP4V2 based on the bioluminescent substrate Luciferin-MultiCYP.HEK293-AAVR cells were seeded at 1 × 10⁴ cells/well and transduced with rAAV-hCYP4V2 across an MOI gradient (1.17 × 10³–2.4 × 10⁶). After incubating at 37 °C for 72 h, medium was replaced with Opti-MEM containing 20 µM Luciferin-MultiCYP and incubated for 90 min ± 30 min. Next, cell supernatants were mixed with luciferase reagent, and bioluminescence was measured. Methodological validation results revealed that the method has good specificity, with an accuracy recovery rate of 100.94% ± 2.84% (RSD = 2.81%). Repeatability (GCV%) was 4.21%, and intermediate precision (RSD) was 6.16%. Linearity was good within the MOI range of 1.17 × 10³ to 2.4 × 10⁶ (R² = 0.9902 ± 0.0061, n = 9). Lastly, the method was successfully applied to activity testing the products of three AAV serotypes (AAV8, AAV2/8, and AAV2). This bioluminescent method meets regulatory potency assay requirements and serves as an effective QC tool for process development, batch release, and stability evaluation of rAAV-hCYP4V2 products.

Bietti crystalline dystrophy (BCD) is a hereditary retinal disease caused by loss-of-function mutations in the CYP4V2 gene. Gene replacement therapy using rAAV-hCYP4V2 represents a promising therapeutic strategy, requiring robust bioassays for product quality control. This study developed and validated a sensitive LC-MS/MS method for quantifying CYP4V2 enzyme activity. Lysates from HeLa-AAVR cells transduced with rAAV-hCYP4V2 (MOI = 3 × 10⁵) were used, with lauric acid as substrate supplemented with cytochrome P450 reductase, cytochrome b5, and NADPH. The ω-hydroxylated product (12-hydroxy lauric acid) was quantified using tolbutamide as an internal standard. Method validation followed ICH guidelines. Results demonstrated excellent specificity with negligible background in negative controls. Linearity was achieved over 0.5–100 ng/mL (R² > 0.99), with an average recovery of 100.6%. Intra-batch and inter-batch precision RSDs were <47.8% and <28.4%, respectively. Product stability was maintained for ≥4 weeks at −80°C. The method was successfully applied to three AAV serotypes (AAV2, AAV8, and AAV2/8), with all RSDs < 23.9%. This validated LC-MS/MS bioassay provides a crucial quality control tool for potency assessment, process development, batch release, and stability studies of rAAV-hCYP4V2 gene therapy products.

Bietti crystalline dystrophy (BCD) is a hereditary retinal disease caused by loss-of-function mutations in the CYP4V2 gene. Gene replacement therapy using rAAV-hCYP4V2 represents a promising therapeutic strategy, requiring robust bioassays for product quality control. This study developed and validated a sensitive LC-MS/MS method for quantifying CYP4V2 enzyme activity. Lysates from HeLa-AAVR cells transduced with rAAV-hCYP4V2 (MOI=3×105) were used, with lauric acid as substrate supplemented with cytochrome P450 reductase, cytochrome b5, and NADPH. The ω-hydroxylated product (12-hydroxy lauric acid) was quantified using tolbutamide as internal standard. Method validation followed ICH guidelines. Results demonstrated excellent specificity with negligible background in negative controls. Linearity was achieved over 0.5-100 ng/mL (R²>0.99), with average recovery of 100.6%. Intra-batch and inter-batch precision RSDs were <47.8% and <28.4%, respectively. Product stability was maintained ≥4 weeks at -80℃. The method was successfully applied to three AAV serotypes (AAV2, AAV8, AAV2/8), with all RSDs <23.9%. This validated LC-MS/MS bioassay provides a crucial quality control tool for potency assessment, process development, batch release, and stability studies of rAAV-hCYP4V2 gene therapy products.