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Wentao Deng, Ji-Jing Pang, Jie Li, Ping Zhu, Wolfgang Baehr, W. Clay Smith, William W Hauswirth; Co-expression of human L- or M-opsin restores M-Cone Function in the Opn1mw Knock-out mouse, a model for blue cone monochromacy (BCM). Invest. Ophthalmol. Vis. Sci. 2017;58(8):4477.
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BCM is an X-linked congenital disorder characterized by loss of both L- and M- cone function. Here we tested gene therapy for BCM using an M-cone knock out mouse model by AAV-mediated expression of either or both human M- and L-opsin.
Since human L- and M-opsin are highly homologous there is no antibody that can distinguish between them. We generated AAV5 vectors expressing either C-terminal myc tagged L-opsin-myc cDNA or C-terminal HA tagged M-opsin-HA cDNA, both driven by the cone-specific PR2.1 promoter. Vectors were delivered either individually or as a mixture containing equal numbers of vector genomes of each. 1 μL of vector(s) was injected subretinally into one eye of one month old M-opsin Knockout (Opn1mw-/-) mice, while the contralateral eyes served as controls. M-cone function was assessed by photopic ERG under middle wavelength (510nm) flash conditions. In retinal sections human L- and M-opsin expression were analyzed by myc and HA antibodies respectively, and their cellular localizations analyzed by peanut agglutinin (PNA) and anti-human L/M-opsin and anti-mouse S-opsin.
Retinal functional analysis of Opn1mw-/- mice treated with either vector individually or as an equal mixture showed significant ERG rescue compared to untreated controls. In eyes treated with individual vectors, expression of myc, HA and M/L-opsin were detected and co-localized correspondingly. In the dorsal retina where no mouse M-opsin is expressed, human M- or/and L-opsin expression co-localized with PNA labeled cone sheaths. In the ventral retina, where mouse S-opsin is present in cone outer segments, they co-localized with endogenous S-opsin. In retinas treated with the vector mixture, a majority of the cones co-express both M- and L-opsin as seen by HA and Myc co-localization. We also note that individual cones can contain different ratios of human M- and L-opsin.
We demonstrate that C-terminal tagging human opsins does not interfere either function or cellular localization. Therefore, these tags can be employed to distinguish L- from M-opsin in the same retina. Functional and structural analyses in Opn1mw-/- retinas confirm that either human opsin or their mixture can restore both significant M-cone function and cone outer segments. These observations provide preclinical proof-of-principle justifying gene therapy treatment of human BCM.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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