An osmolarity dependent mechanism partially ameliorates retinal cysts and rescues cone function in a mouse model of X-linked Retinoschisis

Abstract

Introduction X-linked retinoschisis (XLRS) is a vitreoretinal dystrophy caused by RS1 gene mutations which disrupt retinoschisin protein function. A vital protein for maintaining retinal architecture, the absence of functional retinoschisin leads to the development of intraretinal cysts. The preliminary goal of this study was to investigate a low dose gene therapy in Rs1 knockout (Rs1-KO) mice; however, our experiments revealed an unexpected therapeutic effect of a hypertonic buffer, which led to further exploration of this effect. Methods 10 Rs1-KO mice were subretinally injected with an AAV2/4 vector containing the RS1 gene driven by an Ef1a promoter. 16 Rs1-KO mice were subretinally injected with a hypertonic buffer (180 mM NaCl 0.001% F68/PBS (pH 7.4)) or an isotonic buffer (155.2 mM NaCl 0.001% F68/PBS, pH 7.0) as a sham control. Endpoints included electroretinogram (ERG), optical coherence tomography (OCT), and a visually guided swim assay (VGSA). An immunohistochemistry assay was used to quantify cone density in buffer injected and treatment-naive eyes. Results Unexpectedly, hypertonic buffer-injected eyes had significantly reduced cyst severity at 1 month post-injection (MPI) (p=<0.0001), significantly higher amplitudes in cone-dominant ERGs persisting to 5 months post-injection (5 Hz flicker; p=0.0018; 3.0 Flash; p=0.0060) and demonstrated improved navigational vision in the light compared to untreated Rs1-KO eyes (p<0.0001). To investigate the role of tonicity on this effect, an isotonic buffer-injected cohort was created (155.2 mM NaCl 0.001% F68/PBS, pH 7.0) (n=6). Surprisingly, hypertonic buffer-injected eyes exhibited a greater reduction in cyst severity and demonstrated improved cone-dominant ERG metrics over isotonic buffer-injected eyes. Using an immunohistochemistry assay, we demonstrated greater cone density in hypertonic buffer-injected eyes than untreated controls (p=0.0147), suggesting a possible cone preservation mechanism. Moreover, our findings reveal a negative correlation between the peak severity of cysts and long-term cone-dominant ERG metrics, implying that effectively managing cysts could yield enduring benefits for cone function. Discussion/Conclusion This study presents evidence that cyst resolution can be triggered through an osmosis-dependent pathway, and cyst resolution can have long term effects on cone signaling and survival, offering potential insights for the development of novel treatments for patients with XLRS.

The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-hRS1 in a mouse model of X-linked retinoschisis

“…It is worth mentioning that eyes treated with the 8E8 vg dose showed better amplitudes in response to the 5 Hz flicker test compared to eyes treated with 8E9 vg and 8E7 vg doses at 1 and 3 MPI, p < 0.05 ( Figure 4C ). Importantly, the group treated with isotonic sham injections demonstrated slightly better amplitudes in response to the 5 Hz flicker test compared to untreated eyes, aligning with findings from a prior study by our laboratory ( 31 ). However, while the sham-treated eyes performed better than untreated eyes, the observed improvements were of a much lower magnitude than those observed with gene therapy.…”

“…At 2 MPI (3 months of age), cysts in uninjected eyes had worsened; treatment naïve eyes possessed a significantly higher cyst severity score compared to eyes that received the sham treatment or the rAAV2-tYF-CB-h RS1 viral vector (8E8 and 8E7 vg: p < 0.0001; 8E9: p = 0.004; sham treatment: p = 0.008) ( Figure 7B ). It is worth noting that our group has previously reported the beneficial effects of injecting buffer diluent alone on cyst severity in Rs1 -KO mice ( 31 ). At 3 MPI, eyes that received different doses of the rAAV2-tYF-CB-h RS1 viral vector had less severe cysts compared to eyes that received the sham treatment ( Figure 7A ).…”

“…Eyes that received 8E9, 8E8, 8E7 vg/eye of rAAV2-tYF-CB-h RS1 viral vector were compared to eyes that received buffer diluent only (sham-treated), as well as completely uninjected eyes. A study from this laboratory has previously demonstrated the beneficial effects of buffer injection alone on RS1 phenotypes ( 31 ), necessitating the inclusion of a control group consisting of eyes subretinally injected with buffer/diluent, in addition to uninjected eyes. To demonstrate the expression levels of RS1 protein after administration of different doses of the gene therapy vector, retinas were lysed, and western blotting was performed at 1 MPI ( Figure 3A ).…”

“…Eyes that received the 8E9 vg dose had higher amplitudes compared to uninjected eyes, but not compared to sham-injected eyes. Comparison with sham-injected eyes is important for determining the efficacy of gene therapy, since unexpected beneficial effects were observed after injection of buffer/diluent alone in Rs1- KO mice ( 31 ). The improvement in cone function by the 5 Hz flicker test was not limited to the 1 MPI time point but was consistently observed at 2, 3, 5 and 7 MPI ( Figure 4C ).…”

“…Immunostaining and imaging techniques, as previously detailed ( 31 ), were employed for eye sections. Initially, sections underwent permeabilization with 0.3% Triton X-100 in PBS for 10 min at room temperature.…”