10.3390/polym8030084

Vollständiger Abstract

Worum geht es in dieser Arbeit?

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 × 10 4 cells/well and transduced with rAAV-hCYP4V2 across an MOI gradient (1.17 × 10 3 -2.4 × 10 6 ). 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 × 10 3 to 2.4 × 10 6 (R 2 = 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.

Abstract: PubMed · Datensatz