Human L- and M-opsins restore M-cone function in a mouse model for human blue cone monochromacy.

Purpose: Blue cone monochromacy (BCM) is an X-linked congenital vision disorder characterized by complete loss or severely reduced L- and M-cone function. Patients with BCM display poor visual acuity, severely impaired color discrimination, myopia, nystagmus, and minimally detectable cone-mediated electroretinogram. Recent studies of patients with BCM with adaptive optics scanning laser ophthalmoscopy (AOSLO) showed that they have a disrupted cone mosaic with reduced numbers of cones in the fovea that is normally dominated by L- and M-cones. The remaining cones in the fovea have significantly shortened outer segments but retain sufficient structural integrity to serve as potential gene therapy targets. In this study, we tested whether exogenously expressed human L- and M-opsins can rescue M-cone function in an M-opsin knockout (Opn1mw-/- ) mouse model for BCM. Methods: Adeno-associated virus type 5 (AAV5) vectors expressing OPN1LW, OPN1MW, or C-terminal tagged OPN1LW-Myc, or OPN1MW-HA driven by a cone-specific promoter were injected subretinally into one eye of Opn1mw-/- mice, while the contralateral eye served as the uninjected control. Expression of cone pigments was determined with western blotting and their cellular localization identified with immunohistochemistry. M-cone function was analyzed with electroretinogram (ERG). Antibodies against cone phototransduction proteins were used to study cone outer segment (OS) morphology in untreated and treated Opn1mw-/- eyes. Results: We showed that cones in the dorsal retina of the Opn1mw-/- mouse do not form outer segments, resembling cones that lack outer segments in the human BCM fovea. We further showed that AAV5-mediated expression of either human M- or L-opsin individually or combined promotes regrowth of cone outer segments and rescues M-cone function in the treated Opn1mw-/- dorsal retina. Conclusions: Exogenously expressed human opsins can regenerate cone outer segments and rescue M-cone function in Opn1mw-/- mice, thus providing a proof-of-concept gene therapy in an animal model of BCM.

Rescue of M-cone Function in Aged Opn1mw-/- Mice, a Model for Late-Stage Blue Cone Monochromacy.

Purpose: Previously we showed that AAV5-mediated expression of either human M- or L-opsin promoted regrowth of cone outer segments and rescued M-cone function in the treated M-opsin knockout (Opn1mw-/-) dorsal retina. In this study, we determined cone viability and window of treatability in aged Opn1mw-/- mice. Methods: Cone viability was assessed with antibody against cone arrestin and peanut agglutinin (PNA) staining. The rate of cone degeneration in Opn1mw-/- mice was quantified by PNA staining. AAV5 vector expressing human L-opsin was injected subretinally into one eye of Opn1mw-/- mice at 1, 7, and 15 months old, while the contralateral eyes served as controls. M-cone-mediated retinal function was analyzed 2 and 13 months postinjection by full-field ERG. L-opsin transgene expression and cone outer segment structure were examined by immunohistochemistry. Results: We showed that dorsal M-opsin dominant cones exhibit outer segment degeneration at an early age in Opn1mw-/- mice, whereas ventral S-opsin dominant cones were normal. The remaining M-opsin dominant cones remained viable for at least 15 months, albeit having shortened or no outer segments. We also showed that AAV5-mediated expression of human L-opsin was still able to rescue function and outer segment structure in the remaining M-opsin dominant cones when treatment was initiated at 15 months of age. Conclusions: Our results showing that the remaining M-opsin dominant cones in aged Opn1mw-/- mice can still be rescued by gene therapy is helpful for establishing the window of treatability in future blue cone monochromacy clinical trials.