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Updated: Jun 14, 2026
Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye
Published on: November 25, 2012
AAV2/4-RS1 gene therapy in the retinoschisin knockout mouse model of X-linked retinoschisis
Brittni A Scruggs1, Sajag Bhattarai1, Megan Helms1
1University of Iowa Institute for Vision Research and the Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States of America.
Objective:
To evaluate efficacy of a novel adeno-associated virus (AAV) vector, AAV2/4-RS1, for retinal rescue in the retinoschisin knockout (Rs1-KO) mouse model of X-linked retinoschisis (XLRS). Brinzolamide (Azopt®), a carbonic anhydrase inhibitor, was tested for its ability to potentiate the effects of AAV2/4-RS1.
Methods:
AAV2/4-RS1 with a cytomegalovirus (CMV) promoter (2x1012 viral genomes/mL) was delivered to Rs1-KO mice via intravitreal (N = 5; 1μL) or subretinal (N = 21; 2μL) injections at postnatal day 60-90. Eleven mice treated with subretinal therapy also received topical Azopt® twice a day. Serial full field electroretinography (ERG) was performed starting at day 50-60 post-injection. Mice were evaluated using a visually guided swim assay (VGSA) in light and dark conditions. The experimental groups were compared to untreated Rs1-KO (N = 11), wild-type (N = 12), and Rs1-KO mice receiving only Azopt® (N = 5). Immunofluorescence staining was performed to assess RS1 protein expression following treatment.
Results:
The ERG b/a ratio was significantly higher in the subretinal plus Azopt® (p<0.0001), subretinal without Azopt® (p = 0.0002), and intravitreal (p = 0.01) treated eyes compared to untreated eyes. There was a highly significant subretinal treatment effect on ERG amplitudes collectively at 7-9 months post-injection (p = 0.0003). Cones showed more effect than rods. The subretinal group showed improved time to platform in the dark VGSA compared to untreated mice (p<0.0001). RS1 protein expression was detected in the outer retina in subretinal treated mice and in the inner retina in intravitreal treated mice.
Conclusions:
AAV2/4-RS1 shows promise for improving retinal phenotype in the Rs1-KO mouse model. Subretinal delivery was superior to intravitreal. Topical brinzolamide did not improve efficacy. AAV2/4-RS1 may be considered as a potential treatment for XLRS patients.
Insights
Novel adeno-associated virus (AAV) vector AAV2/4-RS1 shows promise for treating X-linked retinoschisis (XLRS) in mice. Subretinal delivery improved retinal function, though brinzolamide did not enhance efficacy.
Area of Science:
- Ophthalmology
- Gene Therapy
- Retinal Diseases
Background:
- X-linked retinoschisis (XLRS) is a genetic disorder causing vision loss.
- The retinoschisin knockout (Rs1-KO) mouse model mimics human XLRS.
- Adeno-associated virus (AAV) vectors are being explored for gene therapy in retinal diseases.
Purpose of the Study:
- To evaluate the efficacy of AAV2/4-RS1 for retinal rescue in Rs1-KO mice.
- To assess if brinzolamide potentiates the therapeutic effects of AAV2/4-RS1.
- To compare intravitreal versus subretinal delivery of AAV2/4-RS1.
Main Methods:
- Rs1-KO mice received intravitreal or subretinal AAV2/4-RS1 injections.
- Some mice received topical brinzolamide concurrently with subretinal therapy.
- Electroretinography (ERG) and visually guided swim assays (VGSA) were used for functional assessment.
- RS1 protein expression was analyzed via immunofluorescence staining.
Main Results:
- Subretinal AAV2/4-RS1 delivery significantly improved ERG b/a ratios and amplitudes compared to controls.
- Subretinal treatment enhanced performance in dark VGSA, indicating improved visual function.
- RS1 protein was detected in the outer retina after subretinal delivery.
- Brinzolamide did not show a significant additive effect on efficacy.
Conclusions:
- AAV2/4-RS1 gene therapy demonstrates potential for treating XLRS in a mouse model.
- Subretinal delivery is a more effective route than intravitreal for this vector.
- Further investigation into AAV2/4-RS1 as a therapeutic strategy for XLRS is warranted.
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