{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T11:55:22Z","timestamp":1785412522805,"version":"3.56.0"},"reference-count":58,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T00:00:00Z","timestamp":1782172800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["82172957"],"award-info":[{"award-number":["82172957"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["81970842"],"award-info":[{"award-number":["81970842"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["82172957"],"award-info":[{"award-number":["82172957"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"award":["81970842"],"award-info":[{"award-number":["81970842"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"name":"National Key Research and Development Program of the Ministry of Science and Technology","award":["2023YFC2506100"],"award-info":[{"award-number":["2023YFC2506100"]}]},{"name":"National Key Research and Development Program of the Ministry of Science and Technology","award":["2017YFA0104101"],"award-info":[{"award-number":["2017YFA0104101"]}]},{"name":"Science & Technology Project of Guangdong Province","award":["2017B020230003"],"award-info":[{"award-number":["2017B020230003"]}]},{"name":"a Joint grant from Science & Technology Project of Guangzhou and Zhongshan Ophthalmic Center, Sun Yat-sen University","award":["202201020312"],"award-info":[{"award-number":["202201020312"]}]},{"name":"The Science & Technology Project of Guangzhou","award":["202102010288"],"award-info":[{"award-number":["202102010288"]}]},{"name":"Fundamental Research funds of the State Key Laboratory of Ophthalmology","award":["2022QN11"],"award-info":[{"award-number":["2022QN11"]}]},{"award":["2022QN11"],"award-info":[{"award-number":["2022QN11"]}],"id":[{"id":"https:\/\/ror.org\/003mfbe21","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>RPE65, an isomerohydrolase expressed in retinal pigment epithelium (RPE), is critical for the visual cycle. More than 115 missense variants of the RPE65 gene have been associated with Leber\u2019s congenital amaurosis (LCA), a severe childhood retinal dystrophy. Due to high genetic heterogeneity, the variant-specific pathogenic mechanisms remain largely uncharacterized. In this study we focus on an LCA patient carrying compound heterozygous RPE65 variants (c.200T &gt; G, c.430T &gt; C), aiming to dissect the mechanistic\/functional basis of mutated protein-driven retinal degeneration and evaluate gene therapy-mediated restoration using patient-specific hiPSCs-RPE (iRPE). Transient overexpression of wild-type\/mutant RPE65 in HEK293T cells showed both variants markedly destabilize the RPE65 protein through the autophagosome\u2013lysosome degradation pathway and its isomerohydrolase activity required for the retinoid visual cycle. We further established a patient-specific iRPE platform suitable for enzymatic activity analysis. Characterization of patient-specific iRPE cells revealed those compound heterozygous variants did not compromise iRPE morphology, most gene expression, or core canonical physiological features of iRPE. However, they significantly downregulate endogenous RPE65 protein abundance and dampen enzymatic function. Subsequently, we delivered RPE65 via adeno-associated viral (AAV) vectors driven by either the ubiquitous CMV promoter or RPE-specific VMD2 promoter into patient iRPE to validate therapeutic potency, and verified that exogenous RPE65 supplementation effectively restores deficient isomerohydrolase activity in this disease model. Collectively, this work elucidates the variant-specific pathogenesis of RPE65-associated LCA and preliminarily assesses the efficacy of gene augmentation, providing preclinical experimental evidence to support the referral of this patient for clinical RPE65 gene replacement therapy.<\/jats:p>","DOI":"10.3390\/ijms27135643","type":"journal-article","created":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T03:09:17Z","timestamp":1782270557000},"page":"5643","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Personalized Pathogenicity Assessment of RPE65 Gene Mutations Using Patient-Specific hiPSC-Derived Retinal Pigment Epithelium Model"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9593-9007","authenticated-orcid":false,"given":"Ke","family":"Ye","sequence":"first","affiliation":[{"name":"State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou 510060, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suai","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou 510060, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ping","family":"Xu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou 510060, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaojing","family":"Song","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou 510060, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuan","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou 510060, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiufeng","family":"Zhong","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou 510060, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"112","DOI":"10.12998\/wjcc.v3.i2.112","article-title":"Review and Update on the Molecular Basis of Leber Congenital Amaurosis","volume":"3","year":"2015","journal-title":"World J. 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