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Updated: Apr 14, 2026
Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium
Published on: August 7, 2015
A long-term efficacy study of gene replacement therapy for RPGR-associated retinal degeneration
Zhijian Wu1, Suja Hiriyanna2, Haohua Qian2
1National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA wuzh@mail.nih.gov swaroopa@nei.nih.gov.
Abstract:
Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for >70% of X-linked retinitis pigmentosa (XLRP) and 15-20% of all inherited retinal degeneration. Gene replacement therapy for RPGR-XLRP was hampered by the relatively slow disease progression in mouse models and by difficulties in cloning the full-length RPGR-ORF15 cDNA that includes a purine-rich 3'-coding region; however, its effectiveness has recently been demonstrated in four dogs with RPGR mutations. To advance the therapy to clinical stage, we generated new stable vectors in AAV8 or AAV9 carrying mouse and human full-length RPGR-ORF15-coding sequence and conducted a comprehensive long-term dose-efficacy study in Rpgr-knockout mice. After validating their ability to produce full-length proteins that localize to photoreceptor connecting cilia, we evaluated various vector doses in mice during a 2-year study. We demonstrate that eyes treated with a single injection of mouse or human RPGR-ORF15 vector at an optimal dose maintained the expression of RPGR-ORF15 throughout the study duration and exhibited higher electroretinogram amplitude, thicker photoreceptor layer and better targeting of opsins to outer segments compared with sham-treated eyes. Furthermore, mice that received treatment at an advanced age also showed remarkable preservation of retinal structure and function. Retinal toxicity was observed at high vector doses, highlighting the importance of careful dose optimization in future clinical experiments. Our long-term dose-efficacy study should facilitate the design of human trials with human RPGR-ORF15 vector as a clinical candidate.
Insights
Gene therapy using RPGR-ORF15 vectors shows promise for X-linked retinitis pigmentosa (XLRP). Optimal doses preserved retinal structure and function in mice, paving the way for human clinical trials.
Area of Science:
- Ophthalmology
- Genetics
- Gene Therapy
Background:
- Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are a primary cause of X-linked retinitis pigmentosa (XLRP) and other inherited retinal degenerations.
- Previous gene replacement therapy efforts for RPGR-XLRP faced challenges due to slow disease progression in animal models and difficulties in cloning the full-length RPGR-ORF15 cDNA.
Purpose of the Study:
- To develop and evaluate AAV vectors carrying mouse and human full-length RPGR-ORF15 for gene replacement therapy.
- To conduct a long-term dose-efficacy study of RPGR-ORF15 gene therapy in Rpgr-knockout mice.
Main Methods:
- Generation of stable AAV8 and AAV9 vectors encoding mouse and human RPGR-ORF15.
- Long-term (2-year) dose-efficacy study in Rpgr-knockout mice, including electroretinography, histological analysis, and protein localization.
- Evaluation of treatment in mice at different ages, including those with advanced disease.
Main Results:
- Single injection of optimal dose RPGR-ORF15 vectors (mouse or human) led to sustained protein expression and preserved retinal structure and function in treated mice.
- Treated eyes showed improved electroretinogram amplitudes, thicker photoreceptor layers, and better opsin targeting compared to controls.
- Therapy was effective even when administered to older mice with advanced disease, preserving retinal integrity.
- High vector doses resulted in observed retinal toxicity, underscoring the need for precise dose optimization.
Conclusions:
- AAV-mediated gene replacement therapy with RPGR-ORF15 is effective in preserving retinal structure and function in a mouse model of RPGR-XLRP.
- The study provides crucial long-term dose-efficacy data to support the clinical development of human RPGR-ORF15 vector as a therapeutic candidate for XLRP.
- Careful dose optimization is critical to maximize therapeutic benefit while minimizing potential retinal toxicity in future clinical applications.
