The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-h RS1 in a mouse model of X-linked retinoschisis

Salma Hassan; Ying Hsu; Jacob M Thompson; Emily Kalmanek; Joel A VandeLune; Sarah Stanley; Arlene V Drack

doi:10.3389/fmed.2024.1304819

The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-h RS1 in a mouse model of X-linked retinoschisis

Front Med (Lausanne). 2024 Feb 13:11:1304819. doi: 10.3389/fmed.2024.1304819. eCollection 2024.

Authors

Salma Hassan^{1

2}, Ying Hsu¹, Jacob M Thompson^{1

3}, Emily Kalmanek¹, Joel A VandeLune¹, Sarah Stanley¹, Arlene V Drack^{1

2

4}

Affiliations

¹ Department of Ophthalmology and Visual Sciences, Institute for Vision Research, and Carver College of Medicine, University of Iowa, Iowa City, IA, United States.
² Biomedical Science-Cell and Developmental Biology Graduate Program, Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA, United States.
³ Department of Epidemiology, College of Public Health, University of Iowa, Iowa City, IA, United States.
⁴ Department of Pediatrics, University of Iowa, Iowa City, IA, United States.

Abstract

Purpose: X-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.

Methods: Rs1-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).

Results: Western 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.

Conclusion: Our 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.

Keywords: Rs1 knockout mouse; X-linked retinoschisis; dose-response; electroretinogram; functional vision; subretinal gene therapy; visually guided swim assay.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. Our funding is from the Chakraborty Family Foundation, and the Ronald Keech Professorship. The research was also supported in part by a National Institutes of Health Center Support grant (P30 EY025580) to the University of Iowa and a grant from Research to Prevent Blindness. Dr. Hsu is supported by a career starter grant from the Knights Templar Eye Foundation.