{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T15:44:30Z","timestamp":1786549470060,"version":"build-2736575974"},"reference-count":23,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T00:00:00Z","timestamp":1786492800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Institutes for Drug Control","award":["Z251100004625002"],"award-info":[{"award-number":["Z251100004625002"]}]},{"name":"National Institutes for Drug Control","award":["2025SKLDRS0354"],"award-info":[{"award-number":["2025SKLDRS0354"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>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 \u00d7 104 cells\/well and transduced with rAAV-hCYP4V2 across an MOI gradient (1.17 \u00d7 103\u20132.4 \u00d7 106). After incubating at 37 \u00b0C for 72 h, medium was replaced with Opti-MEM containing 20 \u00b5M Luciferin-MultiCYP and incubated for 90 min \u00b1 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% \u00b1 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 \u00d7 103 to 2.4 \u00d7 106 (R2 = 0.9902 \u00b1 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.<\/jats:p>","DOI":"10.3390\/molecules31162811","type":"journal-article","created":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T14:45:06Z","timestamp":1786545906000},"page":"2811","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development and Validation of a Method to Determine CYP4V2 Enzyme Activity in rAAV-hCYP4V2 Gene Therapy Products Using a Bioluminescent Substrate Assay"],"prefix":"10.3390","volume":"31","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-1884-4440","authenticated-orcid":false,"given":"Yiran","family":"Li","sequence":"first","affiliation":[{"name":"National Institutes for Drug Control, Beijing 100050, China"},{"name":"State Key Laboratory of Drug Regulatory Science, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenhong","family":"Fan","sequence":"additional","affiliation":[{"name":"National Institutes for Drug Control, Beijing 100050, China"},{"name":"State Key Laboratory of Drug Regulatory Science, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuting","family":"Hou","sequence":"additional","affiliation":[{"name":"National Institutes for Drug Control, Beijing 100050, China"},{"name":"State Key Laboratory of Drug Regulatory Science, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yue","family":"Ding","sequence":"additional","affiliation":[{"name":"National Institutes for Drug Control, Beijing 100050, China"},{"name":"State Key Laboratory of Drug Regulatory Science, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiuqing","family":"Jia","sequence":"additional","affiliation":[{"name":"National Institutes for Drug Control, Beijing 100050, China"},{"name":"State Key Laboratory of Drug Regulatory Science, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xi","family":"Zhu","sequence":"additional","affiliation":[{"name":"Shanghai Vitalgen BioPharma Co., Ltd., Shanghai 201210, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuemeng","family":"Yuan","sequence":"additional","affiliation":[{"name":"Shanghai Vitalgen BioPharma Co., Ltd., Shanghai 201210, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yufei","family":"Zhang","sequence":"additional","affiliation":[{"name":"Shanghai Vitalgen BioPharma Co., Ltd., Shanghai 201210, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanrong","family":"Cao","sequence":"additional","affiliation":[{"name":"Shanghai Vitalgen BioPharma Co., Ltd., Shanghai 201210, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xi","family":"Qin","sequence":"additional","affiliation":[{"name":"National Institutes for Drug Control, Beijing 100050, China"},{"name":"State Key Laboratory of Drug Regulatory Science, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8916-6872","authenticated-orcid":false,"given":"Chenggang","family":"Liang","sequence":"additional","affiliation":[{"name":"National Institutes for Drug Control, Beijing 100050, China"},{"name":"State Key Laboratory of Drug Regulatory Science, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lan","family":"Wang","sequence":"additional","affiliation":[{"name":"National Institutes for Drug Control, Beijing 100050, China"},{"name":"State Key Laboratory of Drug Regulatory Science, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"101377","DOI":"10.1016\/j.preteyeres.2025.101377","article-title":"Unravelling CYP4V2: Clinical features, genetic insights, pathogenic mechanisms and therapeutic strategies in Bietti crystalline corneoretinal dystrophy","volume":"107","author":"Jia","year":"2025","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"953","DOI":"10.2147\/OPTH.S388292","article-title":"Diagnostic and Management Strategies of Bietti Crystalline Dystrophy: Current Perspectives","volume":"17","author":"Saatci","year":"2023","journal-title":"Clin. Ophthalmol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"3773","DOI":"10.1038\/s41467-024-48092-9","article-title":"In vivo genome editing via CRISPR\/Cas9-mediated homology-independent targeted integration for Bietti crystalline corneoretinal dystrophy treatment","volume":"15","author":"Meng","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1186\/s12964-024-01710-x","article-title":"Uncovering the role of ferroptosis in Bietti crystalline dystrophy and potential therapeutic strategies","volume":"22","author":"Shen","year":"2024","journal-title":"Cell Commun. Signal."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"9525","DOI":"10.1038\/s41598-022-12210-8","article-title":"AAV2-mediated gene therapy for Bietti crystalline dystrophy provides functional CYP4V2 in multiple relevant cell models","volume":"12","author":"Wang","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1042\/BCJ20230041","article-title":"The biochemistry and physiology of long-chain dicarboxylic acid metabolism","volume":"480","author":"Houten","year":"2023","journal-title":"Biochem. J."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"114841","DOI":"10.1016\/j.bcp.2021.114841","article-title":"CYP4V2 fatty acid omega hydroxylase, a druggable target for the treatment of metabolic associated fatty liver disease (MAFLD)","volume":"195","author":"Osborne","year":"2022","journal-title":"Biochem. Pharmacol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"116241","DOI":"10.1016\/j.bcp.2024.116241","article-title":"The fatty acid omega hydroxylase genes (CYP4 family) in the progression of metabolic dysfunction-associated steatotic liver disease (MASLD): An RNA sequence database analysis and review","volume":"228","author":"Leahy","year":"2024","journal-title":"Biochem. Pharmacol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"100875","DOI":"10.1016\/j.bj.2025.100875","article-title":"IRE1\u03b1-mediated endoplasmic reticulum stress response regulates oxidative damage in CYP4V2 deficient human retinal pigment epithelial cells","volume":"48","author":"Hsiao","year":"2025","journal-title":"Biomed. J."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"256","DOI":"10.63500\/mv_v31_256","article-title":"Bietti crystalline corneoretinal dystrophy: Advances in understanding and gene therapeutic approaches","volume":"31","author":"Zhao","year":"2025","journal-title":"Mol. Vis."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1001\/jamaophthalmol.2024.5619","article-title":"Safety and Vision Outcomes Following Gene Therapy for Bietti Crystalline Dystrophy: A Nonrandomized Clinical Trial","volume":"143","author":"Chen","year":"2025","journal-title":"JAMA Ophthalmol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1007\/s13318-024-00912-5","article-title":"Personalized Medicine Approach to Proteomics and Metabolomics of Cytochrome P450 Enzymes: A Narrative Review","volume":"49","author":"Fetse","year":"2024","journal-title":"Eur. J. Drug Metab. Pharmacokinet."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1038\/s41392-024-01806-3","article-title":"Gene replacement therapy in Bietti crystalline corneoretinal dystrophy: An open-label, single-arm, exploratory trial","volume":"9","author":"Wang","year":"2024","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"101460","DOI":"10.1016\/j.omtm.2025.101460","article-title":"Preclinical studies of an AAV8-CYP4V2 gene therapy VGR-R01 for the treatment of Bietti crystalline dystrophy","volume":"33","author":"Luo","year":"2025","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_15","unstructured":"U.S. Food and Drug Administration (2023). Potency Assurance for Cellular and Gene Therapy Products, Draft Guidance for Industry. Docket No. FDA-2023-D-4299."},{"key":"ref_16","unstructured":"European Medicines Agency (2025). Guideline on Quality, Non-clinical and Clinical Requirements for Investigational Advanced Therapy Medicinal Products in Clinical Trials, EMA\/CAT\/22473\/2025."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1371","DOI":"10.1089\/hum.2024.249","article-title":"Advancing Potency Assay Development for Advanced Therapy Medicinal Products: A Comprehensive Approach and Regulatory Insights","volume":"36","author":"Abdellatif","year":"2025","journal-title":"Hum. Gene Ther."},{"key":"ref_18","first-page":"601","article-title":"convenient test system for the identification of CYP4V2 inhibitors","volume":"27","author":"Sharma","year":"2021","journal-title":"Mol. Vis."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"108913","DOI":"10.1016\/j.bioelechem.2025.108913","article-title":"Cytochrome P450 electrochemical biosensors transforming in vitro metabolism testing\u2014Opportunities and challenges","volume":"163","author":"Vieira","year":"2025","journal-title":"Bioelectrochemistry"},{"key":"ref_20","unstructured":"Chinese Pharmacopoeia Commission (2020). Pharmacopoeia of the People\u2019s Republic of China 2020 Edition, Volume III, General Chapter 9401: Guidelines for Validation of Bioactivity\/Potency Assays for Biological Products, China Medical Science and Technology Press."},{"key":"ref_21","unstructured":"International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) (2023). Validation of Analytical Procedures: Q2(R2), ICH."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1210","DOI":"10.1016\/S0022-2275(20)32545-1","article-title":"Involvement of cytochrome P450 2E1 in the (omega-1)-hydroxylation of oleic acid in human and rat liver microsomes","volume":"39","author":"Adas","year":"1998","journal-title":"J. Lipid Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1042","DOI":"10.1007\/s12010-022-04184-0","article-title":"Exploring the Chemical Space of Proluciferins as Probe Substrates for Human Cytochrome P450 Enzymes","volume":"195","author":"Zhao","year":"2023","journal-title":"Appl. Biochem. Biotechnol."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/16\/2811\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T14:45:11Z","timestamp":1786545911000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/16\/2811"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,12]]},"references-count":23,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["molecules31162811"],"URL":"https:\/\/doi.org\/10.3390\/molecules31162811","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,12]]}}}