Sustained Vision Recovery by OSK Gene Therapy in a Mouse Model of Glaucoma

Abstract

Glaucoma, a chronic neurodegenerative disease, is a leading cause of age-related blindness worldwide and characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons. Previously, we developed a novel epigenetic rejuvenation therapy, based on the expression of the three transcription factors Oct4, Sox2, and Klf4 (OSK), which safely rejuvenates RGCs without altering cell identity in glaucomatous and old mice after 1 month of treatment. In the current year-long study, mice with continuous or cyclic OSK expression induced after glaucoma-induced vision damage had occurred were tracked for efficacy, duration, and safety. Surprisingly, only 2 months of OSK fully restored impaired vision, with a restoration of vision for 11 months with prolonged expression. In RGCs, transcription from the doxycycline (DOX)-inducible Tet-On AAV system, returned to baseline 4 weeks after DOX withdrawal. Significant vision improvements remained for 1 month post switching off OSK, after which the vision benefit gradually diminished but remained better than baseline. Notably, no adverse effects on retinal structure or body weight were observed in glaucomatous mice with OSK continuously expressed for 21 months providing compelling evidence of efficacy and safety. This work highlights the tremendous therapeutic potential of rejuvenating gene therapies using OSK, not only for glaucoma but also for other ocular and systemic injuries and age-related diseases.

Keywords: aging; gene therapy; glaucoma; neuron; rejuvenation; retina.

FIG. 1.

Full recovery of visual function through inducible OSK AAVs in microbead-induced glaucomatous animals. (a) Schematic illustration of the experimental timeline. Visual acuity measurements, by OMR, were made at baseline (day −7), after induction of glaucoma (day 28), and after AAV-OSK treatment (week 4 and 8). (b) IOP measurements for the first 4 weeks after microbeads or saline. (c) Schematic illustration of the OMR setup. Reflexive head movements of each mouse were tracked in response to the rotation of a moving striped pattern that increased in spatial frequency (cyc/deg). (d) Optomotor acuity was measured at D-7 (baseline) and 4 weeks postbeads' injection, and 4 and 8 weeks post-AAV treatment in four groups of mice: Saline (nonglaucomatous control); Beads (Tet-ON OSK, no DOX); Beads (Tet-On OSK, plus DOX); Beads (Tet-Off OSK, continuous OSK expression) (n = 15, 7, 8, 10 mice per group). Two experiments each with saline control are combined, see individual batch results in Supplementary Figure S1. Data are presented as mean ± SEM *p < 0.05, ****p < 0.0001. DOX, doxycycline; IOP, intraocular pressure; OMR, optomotor response; OSK, Oct4, Sox2, and Klf4; SEM, standard error of mean.

FIG. 2.

Inducibility and stringency of Tet-On AAV system in RGCs. (a) Schematic illustration of the experimental timeline; (b–d) expression of Oct4, Sox2, and Klf4 from the whole retina measured by qPCR (relative to AAV2-TRE-OSK injected eye without DOX treatment) 2 weeks after IVT injection of AAV2-TRE-OSK or AAV2-TRE-OSK; CMV-rtTA and treated with or without DOX; (e, i) representative confocal images from retinal flat-mounts stained with anti-Brn3a (red), an RGC-specific marker, and Klf4 (green). Images from retinal center, midperiphery, and periphery, 2 weeks after IVT injection of AAV2-TRE-OSK; CMV-rtTA and treated +DOX (e) and after additional 3–4 weeks DOX withdrawal (i). Scale bars are 50 μm. (f–h) Expression of Oct4, Sox2, and Klf4 from the whole retina (of which 0.1% cells are RGCs) measured by qPCR (relative to AAV2-TRE-OSK-injected eye without DOX) 2 weeks after IVT injection of TRE-OSK or TRE-OSK-rtTA and treated with or without DOX, followed by 3–4 weeks DOX withdrawal. IVT, intravitreal; qPCR, quantitative PCR; RGC, retinal ganglion cell. *p < 0.05; **p < 0.01.

FIG. 3.

Long-term, safe restoration of visual function in mice. (a) Schematic illustration of the experimental timeline for the 1-year vision follow-up. (b) IOP measurements taken by rebound tonometry in mice injected with microbeads or saline. Data are presented as mean IOP ± SD, saline group: n = 10, microbeads for cyclic OSK: n = 8; microbeads for continuous OSK: n = 10. (c) The cumulative IOP was significantly elevated in mice that received microbeads as compared with control mice receiving saline. No difference of cumulative IOP elevation between the bead-treated groups (cyclic vs. continuous OSK). (d) A schematic illustration of the continuous (tTA; TRE-OSK—green) and the dox-inducible cyclic (rtTA; TRE-OSK—orange) AAV vectors. (e) Optomotor acuity follow-up with the nonglaucomatous (saline) and glaucomatous (beads) eyes, either treated with cyclic OSK or continues OSK AAVs. (f, g) Longitudinal statistical analysis of visual acuity in the continuous (f) or cyclic (g) OSK-treated groups comparing the visual acuity at the damage baseline (glaucoma) up to 12 months of treatment. SD, standard deviation. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

FIG. 4.

Retina health and animal body weight at 21 months post-AAV treatment. (a) Schematic illustration of the experimental timeline and endpoint measurements. (b) Body weight (g) measured at 21 months post-AAV treatment revealed no significant differences between the saline and beads-injected AAV-injected groups. (c) Representative retinal cross-section B-scan images (retinal layers: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer and choroid). (d) Quantification of total retinal thickness shows no changes in retinal structure and no tumor development in any group (n = 4 uninjected eye, n = 4 saline injected, n = 6 continuous OSK, n = 6 cyclic OSK). (e) Representative H&E-stained retinal cross-sections (retinal layers: GCL; INL; ONL; PR, photoreceptor; RPE, retinal pigment epithelium and choroid). Scale bar = 20 μm. H&E, Hematoxylin and Eosin.