Dataset Information

AAV2/4-RS1 gene therapy in the retinoschisin knockout mouse model of X-linked retinoschisis.

ABSTRACT:

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.

SUBMITTER: Scruggs BA

PROVIDER: S-EPMC9728878 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Publications

AAV2/4-RS1 gene therapy in the retinoschisin knockout mouse model of X-linked retinoschisis.

Scruggs Brittni A BA Bhattarai Sajag S Helms Megan M Cherascu Ioana I Salesevic Adisa A Stalter Elliot E Laird Joseph J Baker Sheila A SA Drack Arlene V AV

PloS one 20221207 12

<h4>Objective</h4>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.<h4>Methods</h4>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; 2μ ...[more]

PMID: 36477475

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Project description:PurposeX-linked retinoschisis (XLRS), due to loss-of-function mutations in the retinoschisin (RS1) gene, is characterized by a modest to severe decrease in visual acuity. Clinical trials for XLRS utilizing intravitreal (IVT) gene therapy showed ocular inflammation. We conducted a subretinal dose-response preclinical study using rAAV2tYF-CB-hRS1 utilizing the Rs1 knockout (Rs1-KO) mouse to investigate short- and long-term retinal rescue after subretinal gene delivery.MethodsRs1-KO mice were subretinally injected with 2 μL of rAAV2tYF-CB-hRS1 vector with 8E9 viral genomes (vg)/eye, 8E8 vg/eye, 8E7 vg/eye, or sham injection, and compared to untreated eyes. Reconstitution of human RS1 protein was detected using western blotting. Analysis of retinal function by electroretinography (ERG) and structural analysis by optical coherence tomography (OCT) were performed at 1, 2, 3, 5, 7, and 12 months post injection (MPI). Immunohistochemistry (IHC) was performed to evaluate cone rescue on the cellular level. Functional vision was evaluated using a visually guided swim assay (VGSA).ResultsWestern blotting analysis showed human RS1 protein expression in a dose-dependent manner. Quantification of western blotting showed that the RS1 protein expression in mice treated with the 8E8 vg dose was near the wild-type (WT) expression levels. ERG demonstrated dose-dependent effects: At 1 MPI the 8E8 vg dose treated eyes had higher light-adapted (LA) ERG amplitudes in 3.0 flash and 5 Hz flicker compared to untreated (p < 0.0001) and sham-treated eyes (p < 0.0001) which persisted until the 12 MPI endpoint, consistent with improved cone function. ERG b-wave amplitudes were higher in response to dark-adapted (DA) 0.01 dim flash and 3.0 standard combined response (SCR) compared to sham-treated (p < 0.01) and untreated eyes (p < 0.001) which persisted until 3 MPI, suggesting short-term improvement of the rod photoreceptors. All injections, including sham-treated, resulted in a cyst severity score of 1 (no cavities), with significant reductions compared to untreated eyes up to 3 MPI (p < 0.05). The high and low dose groups showed inconsistent ERG improvements, despite reduced cyst severity, emphasizing the dose-dependent nature of gene augmentation's efficacy and the tenuous connection between cyst reduction and ERG improvement. IHC data showed a significant cone rescue in eyes treated with the 8E8 vg dose compared to sham-treated and untreated eyes. VGSA showed better functional vision in 8E8 vg dose treated mice. Eyes treated with the highest dose showed occasional localized degeneration in the outer nuclear layer.ConclusionOur data suggest that a dose of 8E8 vg/eye subretinally improves retinal function and structure in the Rs1-KO mouse. It improves cone function, rod function, and reduces cyst severity. Sham treatment resolves schisis cysts, but 8E8 vg/eye is needed for optimal retinal electrical function rescue. These findings offer a promising path for clinical translation to human trials.

Project description:PURPOSE: To determine the clinical features and to identify mutations in the retinoschisis gene (RS1) in ten patients with X-linked retinoschisis (XLRS). METHODS: Ten male patients from nine Polish families were included in this study. Ophthalmologic examinations, including optical coherence tomography (OCT) and full-field electroretinography (ERG), were performed in all affected boys. The entire coding region encompassing six exons of the RS1 gene was amplified with PCR and directly sequenced in all the patients. RESULTS: All affected individuals showed typical retinoschisis signs and symptoms, and all appeared to have a mutation in the RS1 gene. Seven different mutations were identified, including two novel missense substitutions: c.176G>C (p.Cys59Ser), c.451T>A (p.Tyr151Asp); one novel nonsense substitution: c.218C>A (p.Ser73*); and one novel frameshift mutation: c.354_355delCA (p.Asp118Glufs*2). We also found two missense substitutions that had been previously described: c.214G>A (p.Glu72Lys) and c.626G>T (p.Arg209Leu) and one known splice site mutation in intron 5: c.522+1G>T (IVS5+1G>T). CONCLUSIONS: This study provides the first molecular genetic characteristics of patients with juvenile retinoschisis from the previously unexplored Polish population. We investigated the molecular background of XLRS in ten boys. The present study reports for the first time four novel mutations, including two missense substitutions, one nonsense substitution, and one frameshift deletion. One of these substitutions and 2-bp deletion created stop codons. Moreover, we described three substitutions that had been previously reported (one is a splicing mutation). Further genetic characterization of Polish patients with XLRS will be helpful in understanding the worldwide spectrum of RS1 mutations. Despite the mutation heterogeneity found in a small group of our patients, they presented a relatively uniform clinical picture. Identifying the causative mutation is helpful in confirming diagnosis and counseling, but cannot provide prognostic data.

Dataset Information

AAV2/4-RS1 gene therapy in the retinoschisin knockout mouse model of X-linked retinoschisis.

Objective

Methods

Results

Conclusions

Publications

AAV2/4-RS1 gene therapy in the retinoschisin knockout mouse model of X-linked retinoschisis.

Similar Datasets

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