Preservation of retinal structure and function in two mouse models of inherited retinal degeneration by ONL1204, an inhibitor of the Fas receptor

Abstract

Due to the large number of genes and mutations that result in inherited retinal degenerations (IRD), there has been a paucity of therapeutic options for these patients. There is a large unmet need for therapeutic approaches targeting shared pathophysiologic pathways in a mutation-independent manner. The Fas receptor is a major activator and regulator of retinal cell death and inflammation in a variety of ocular diseases. We previously reported the activation of Fas-mediated photoreceptor (PR) cell death in two different IRD mouse models, rd10 and P23H, and demonstrated the protective effect of genetic Fas inhibition. The purpose of this study was to examine the effects of pharmacologic inhibition of Fas in these two models by intravitreal injection with a small peptide inhibitor of the Fas receptor, ONL1204. A single intravitreal injection of ONL1204 was given to one eye of rd10 mice at P14. Two intravitreal injections of ONL1204 were given to the P23H mice, once at P14 and again at 2-months of age. The fellow eyes were injected with vehicle alone. Fas activation, rate of PR cell death, retinal function, and the activation of immune cells in the retina were evaluated. In both rd10 and P23H mice, ONL1204 treatment resulted in decreased number of TUNEL (+) PRs, decreased caspase 8 activity, enhanced photoreceptor cell counts, and improved visual function compared with vehicle treated fellow eyes. Treatment with ONL1204 also reduced immune cell activation in the retinas of both rd10 and P23H mice. The protective effect of pharmacologic inhibition of Fas by ONL1204 in two distinct mouse models of retinal degeneration suggests that targeting this common pathophysiologic mechanism of cell death and inflammation represents a potential therapeutic approach to preserve the retina in patients with IRD, regardless of the genetic underpinning.

Fig. 1. ONL1204 injection reduced Fas-mediated photoreceptor cell death and immune cell activation in the P23H retina.

A Quantification for caspase-8 activity in the retina of ONL1204 and vehicle injected (CTL) P23H mice at 1 month, normalized to age matched C57 mice. (Pooled 2–3 retinas for each sample; 7 mice for P23H, 4 mice for C57). B Representative TUNEL staining images and (C) quantification of TUNEL-positive cells in the ONL of vehicle and ONL1204 injected P23H at 1 month of age (n = 13 mice). D Paired line graph represents the value of ONL1204 and control vehicle injected eye from each mouse. E Representative immunostaining images of retinal sections from inferior retinas of vehicle and ONL1204 injected P23H mice at 1 month of age stained with Iba-1 and DAPI. F Representative images of the outer nuclear layer from inferior area of the retinal whole mount of ONL1204 and vehicle injected P23H mice stained with Iba1 at 1 month of age. G Quantification of Iba1-positive cells in the ONL and subretinal space of the inferior retina of vehicle-injected P23H and ONL1204-injected P23H mice (n = 7 mice). H paired line graph shows the value of ONL1204, and control vehicle injected eye from each individual mouse. one-tailed Wilcoxon test. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer, IS inner segment,OS outer segment.

Fig. 2. Increased photoreceptor survival in ONL1204 injected P23H retina compared with vehicle injected control eyes.

A Representative H&E staining images show preserved photoreceptor layer (yellow bars indicate the ONL) in the retina of ONL1204 injected eyes compared with control vehicle injected eyes at 4 months of age. B Fundus image demonstrates the representative OCT images in (C) were extracted from the comparable area from both superior and inferior of the retina. C Representative OCT images of superior (sup) and inferior (inf) retina of 4-month-old ONL1204-injected P23H and vehicle-injected P23H control. D Quantification of the thickness of the ONL (indicated by yellow bars in the OCT images) of the superior and inferior retina measured at both 250 and 500 µm from the optic nerve head by OCT in ONL1204 and control vehicle injected P23H mice at the age of 4 months (n = 16 mice). **p < 0.01; ***p < 0.001; ****p < 0.0001. one-tailed Wilcoxon test. GCL ganglion cell layer, H&E hematoxylin-eosin, INL inner nuclear layer, ONL outer nuclear layer, IS inner segment, OS outer segment, Sup superior, Inf inferior.

Fig. 3. Preserved retinal function in the eyes of ONL1204 injected P23H mice.

A Representative scotopic (at 0.01, 1, 10, 32 and 64 cd s/m²) ERG at 3 months of age in ONL1204-injected P23H, vehicle-injected P23H, and C57 mice. B Quantification of amplitudes of scotopic a-wave and scotopic b-wave (32 cd s/m²) at 3 months and (C) 4 months of age confirms the preservation in retinal function. Line graph shows the paired value of both eyes from each individual mouse. (n = 20 mice for 3 months of age, n = 13 mice for 4 months of age); D Representative immunostaining images of inferior retina of four-month-old C57 and injected P23H mice, stained with rhodopsin (RHO in red), m-opsin (green), and DAPI (blue). The white rectangular inserts are zoomed-in areas highlighting the staining in the inner and outer segments of the retinas. one-tailed Wilcoxon test. INL inner nuclear layer, ONL outer nuclear layer, IS inner segment, OS outer segment. Scale bar: 25 μm.

Fig. 4. Intravitreal injection of ONL1204 reduced photoreceptor cell death in the rd10 retina.

A Quantification of caspase 8 activity in the pooled retinas of vehicle and ONL1204 injected eyes of rd10 mice at P21. (Pooled 2 retinas for each sample; 10 mice). B Representative TUNEL staining images and (C) quantification of TUNEL-positive cells in the ONL for control vehicle and ONL1204 injected rd10 mice at P21. D Line graph shows paired value of both eyes from each individual mouse. (n = 13 mice), one-tailed Wilcoxon test. INL inner nuclear layer, ONL outer nuclear layer.

Fig. 5. Increased photoreceptor survival in ONL1204 injected eyes of rd10 mice compared with vehicle injected eyes.

A Representative H&E staining images show preserved photoreceptors (yellow bars indicated ONL) in the retina of ONL1204-injected rd10 at P42. B Red “+” on the fundus image demonstrate the measure points of ONL thickness at 500 µm from the optic nerve at superior, inferior, temporal, and nasal areas of the retina. C Representative retinal OCT images crossing optic nerve (yellow line in B) of control vehicle and ONL1204 injected rd10 at P28. D Quantification of the thickness of the ONL measured at 500 µm from the optic nerve head by OCT in control vehicle and ONL1204 injected rd10 mice at P28 (n = 21 mice), P35 (n = 16 mice), and P42 (n = 12 mice). Line graphs show paired value of both eyes from each individual mouse. One-tailed Wilcoxon test. INL inner nuclear layer, ONL outer nuclear layer.

Fig. 6. Improved retinal function in ONL1204 injected eyes in rd10 mice.

A Representative immunostaining retinal images of C57, control vehicle and ONL1204 injected rd10 eyes at P42, stained with rhodopsin (RHO in red), m-opsin (green), and DAPI (blue). The white rectangular inserts are zoomed-in areas highlighting the staining in the inner and outer segments of the retinas. B Representative scotopic ERG traces of C57, and injected rd10 mice. C Quantification of amplitudes of scotopic a-wave, and (D) b-wave of ONL1204 and vehicle injected rd10 mice at P42 (36 cd s/m²). E Representative photopic ERG traces of C57, and injected rd10 mice. F Quantification of amplitudes of scotopic b-wave of ONL1204 and vehicle injected rd10 mice at P42 (100 cd s/m²). Line graphs show paired value of both eyes from each individual mouse. n = 10 mice; one-tailed Wilcoxon test; INL inner nuclear layer, ONL outer nuclear layer, IS inner segment, OS outer segment.

Fig. 7. Injection of ONL1204 decreased the activation of immune cells in retina of rd10 mice.

A Representative immunostaining images of retinal sections and (B) ONL from retinal whole mount of ONL1204 and control vehicle injected rd10 eyes at P21 stained with Iba-1 and DAPI. C Quantification of Iba1-positive cells in the ONL and subretinal space of control vehicle and ONL1204 injected rd10 eyes (n = 8 mice). Line graph shows paired value of both eyes from each individual mouse. One-tailed Wilcoxon test. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer.