{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T15:41:35Z","timestamp":1785426095960,"version":"3.56.0"},"reference-count":27,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,4,24]],"date-time":"2026-04-24T00:00:00Z","timestamp":1776988800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100009592","name":"Beijing Municipal Science and Technology Commission","doi-asserted-by":"publisher","award":["Z251100004625002"],"award-info":[{"award-number":["Z251100004625002"]}],"id":[{"id":"10.13039\/501100009592","id-type":"DOI","asserted-by":"publisher"}]},{"award":["Z251100004625002"],"award-info":[{"award-number":["Z251100004625002"]}],"id":[{"id":"https:\/\/ror.org\/034k14f91","id-type":"ROR","asserted-by":"publisher"}]},{"name":"the National Key R&amp;D Program of China","award":["2023YFC3403305"],"award-info":[{"award-number":["2023YFC3403305"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>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 \u00d7 105) were used, with lauric acid as substrate supplemented with cytochrome P450 reductase, cytochrome b5, and NADPH. The \u03c9-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\u2013100 ng\/mL (R2 &gt; 0.99), with an average recovery of 100.6%. Intra-batch and inter-batch precision RSDs were &lt;47.8% and &lt;28.4%, respectively. Product stability was maintained for \u22654 weeks at \u221280\u00b0C. The method was successfully applied to three AAV serotypes (AAV2, AAV8, and AAV2\/8), with all RSDs &lt; 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.<\/jats:p>","DOI":"10.3390\/molecules31091417","type":"journal-article","created":{"date-parts":[[2026,4,24]],"date-time":"2026-04-24T14:28:23Z","timestamp":1777040903000},"page":"1417","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development and Validation of a Quantitative LC-MS\/MS Method for Measuring CYP4V2 Enzyme Activity via 12-Hydroxylauric Acid in rAAV-hCYP4V2 Gene Therapy Products"],"prefix":"10.3390","volume":"31","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-4482-8811","authenticated-orcid":false,"given":"Ge","family":"Ren","sequence":"first","affiliation":[{"name":"National Institutes for Food and Drug Control, Beijing 100050, China"},{"name":"State Key Laboratory of Drug Regulatory Science, Beijing 100050, China"},{"name":"School of Life and Health Sciences, Hubei University of Technology, Wuhan 430068, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xi","family":"Qin","sequence":"additional","affiliation":[{"name":"National Institutes for Food and 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\/0009-0009-1884-4440","authenticated-orcid":false,"given":"Yiran","family":"Li","sequence":"additional","affiliation":[{"name":"National Institutes for Food and 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 Food and Drug Control, Beijing 100050, China"},{"name":"State Key Laboratory of Drug Regulatory Science, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenjing","family":"Luo","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":"Yang","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Life and Health Sciences, Hubei University of Technology, Wuhan 430068, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yong","family":"Zhou","sequence":"additional","affiliation":[{"name":"National Institutes for Food and 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 Food and 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":[[2026,4,24]]},"reference":[{"key":"ref_1","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. 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