AAV-mediated Gene Therapy Restores M-Cone Function in S-opsin only Opn1mw KO Mice

Abstract

Purpose: Color vision is facilitated by cones expressing different opsin photopigments. Cones containing M- or S-opsin are sensitive to middle or short wavelength visible light, respectively. Opn1mw knock-out (M-opsin KO) mice lack of M-opsin expression while maintaining a normal number of cones and S-opsin expression. Similarly, Blue Cone Monochromacy (BCM) patients lack functioning middle and long wavelength opsins and only express S-opsin. Therefore, the Opn1mw KO mouse is a good model to establish pre-clinical efficacy and safety data in future BCM gene therapy clinical trial. We therefore tested whether AAV-mediated M-opsin expression in cones can restore M-cone function/structure in this model.

Methods: Opn1mw KO mice were generated by inserting a gene trap into intron 2 of the mouse Opn1mw gene. At postnatal day 14 (P14), one μl of AAV5-PR2.1-mouse-M-opsin vector (10¹³ vector genome particles/ml) was injected subretinally into one eye of each Opn1mw KO mice. The other eye was uninjected and served as a control. M-cone mediated ERGs were recorded at two months after injection. Treated and untreated eyes were harvested immediately after ERG recording for immunohistochemical studies.

Results: Two months after treatment, nearly normal dark-adapted rod ERG waveforms were recorded in either treated or untreated Opn1mw KO eyes with a mild reduction of ERG amplitudes in treated eyes, likely due to slight subretinal injection-related damage. Cone mediated light-adapted ERGs or S-cone mediated ERGs were also recorded from treated and untreated eyes; M-cone mediated ERGs were restored only in treated but not in untreated Opn1mw KO eyes. Cone-opsin staining showed that S-opsin was present in both treated and untreated eyes, mainly in central and inferior retina; no M-opsin was evident in untreated eyes. In contrast, abundant M-opsin positive cones were observed in treated eyes throughout the retina. AAV-mediated M-opsin expression not only co-localized with S-opsin expression in central and inferior cone outer segments, but was also found in the superior retina which has little or no S-opsin.

Conclusions: Since Opn1mw KO mice express only S-opsin, they serve as a model for human BCM. AAV5-PR2.1-mouse-M-opsin mediated gene therapy restores M-opsin expression and M-opsin function in Opn1mw KO mice. These results serve as a baseline for studying long-term M-cone rescue in Opn1mw KO mice and for developing a BCM gene therapy.