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PubMed · 42263801

Optimized AAV5-RPGR ORF15 Gene Therapy Rescues Photoreceptor Structure and Function in X-Linked Retinitis Pigmentosa Mouse Model.

Xiang Chen·Xintong Xu·Shanshan Cao·Chunyan Pan·Shihui Wei·Baoke Hou·Huanfen Zhou
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Abstract

PURPOSE: To develop and evaluate an rAAV5-based gene therapy vector expressing an optimized human RPGR ORF15 transgene (rAAV5-RPGR) for the treatment of X-linked retinitis pigmentosa caused by RPGR mutations, addressing the challenges of cloning the unstable wild-type ORF15 sequence. DESIGN: This was a prospective experimental study. SUBJECTS: This was an animal study. METHODS: An optimized RPGR ORF15 sequence was designed to eliminate problematic secondary structures and cryptic splice sites. In vitro expression was validated in HEK 293T and photoreceptor-like 661 W cells. A complete Rpgr knockout mouse model (Rpgr-knockout [KO]) was generated and characterized phenotypically. Therapeutic efficacy was assessed in Rpgr-KO mice via subretinal injection of rAAV5-RPGR at low (1 &#xd7; 10&#x2079; vg/eye), medium (3 &#xd7; 10&#x2079; vg/eye), or high (1 &#xd7; 10&#xb9;&#x2070; vg/eye) doses. Structural and functional outcomes were evaluated at 12- and 14-month postinjection. Short-term safety was assessed in rabbits 1 month after subretinal injection. MAIN OUTCOME MEASURES: Level of RPGR protein expression and Protein isoform profile (elimination of truncated isoforms), Cellular localization of transgene expression and Dose-dependence of expression, outer nuclear layer thickness, and electroretinography parameters. RESULTS: (1) The optimized vector increased RPGR protein expression 3.3-fold in vitro compared to wild-type and eliminated truncated isoforms. (2) Subretinal delivery of rAAV5-RPGR in mice demonstrated dose-dependent transgene expression localized correctly to photoreceptor inner segments. (3) In Rpgr-KO mice, high-dose treatment significantly preserved outer nuclear layer thickness at the injection site (42% greater than controls at 14 months, P < .01) and central retina (P < .05), reduced aberrant rhodopsin mislocalization (P < .01), and partially restored retinal function. ERG showed significantly improved scotopic a-wave (&#x2265;100 vs <90 &#xb5;V in controls at 10 cd&#xb7;s/m&#xb2;) and photopic b-wave amplitudes (49-66 vs 31-46 &#xb5;V at 30 cd&#xb7;s/m&#xb2;) in treated mice. (4) No vector-related toxicity was observed in rabbits. CONCLUSIONS: rAAV5-RPGR mediated efficiently, targeted expression of optimized RPGR-ORF15, significantly preserved photoreceptor structure and function in a severe X-linked retinitis pigmentosa mouse model, and demonstrated a favorable safety profile. This study provides preclinical proof-of-concept for RPGR-targeted gene replacement therapy.

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AnimalsRetinitis PigmentosaGenetic TherapyMiceDisease Models, AnimalDependovirusGenetic VectorsElectroretinographyRabbitsMice, KnockoutEye ProteinsHumansPhotoreceptor Cells, VertebrateGene Therapy AgentsProspective StudiesMice, Inbred C57BLHEK293 CellsGenetic Diseases, X-LinkedFemaleCarrier ProteinsJournal Article

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Xiang Chen, Xintong Xu, Shanshan Cao, Chunyan Pan, Shihui Wei, Baoke Hou, Huanfen Zhou. 2026-06-09. Optimized AAV5-RPGR ORF15 Gene Therapy Rescues Photoreceptor Structure and Function in X-Linked Retinitis Pigmentosa Mouse Model.. https://doi.org/10.1016/j.ajo.2026.06.005

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Journal Article resourceAnimals subjectDisease Models, Animal subjectFemale subjectHumans subjectJournal Article subjectMice subject

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