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Ji-Jing Pang, Wentao Deng, Yuxin Zhang, Jie Li, Ping Zhu, Wolfgang Baehr, William W Hauswirth; Restoration of M-cone function in an S-cone only mouse model of human blue cone monochromacy by AAV-mediated expression of human L- or M-opsin. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4478.
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© ARVO (1962-2015); The Authors (2016-present)
Blue cone monochromacy (BCM) is an X-linked congenital disorder with severe cone dysfunction due to the absence of the long and medium wavelength-sensitive cone function. In this study, we used an M-opsin knockout (Opn1mw-/-) mice with only S-cone function remaining as a model to test gene therapy for BCM by AAV-mediated delivery of either human M- or L-opsin, homologues of the mouse M-opsin.
We generated AAV5 vectors expressing either human L-opsin or human M-opsin driven by the cone-specific PR2.1 promoter. 1 microliter (5 × 1012 vector genomes/mL) of each vector was injected subretinally into one eye of one month old Opn1mw-/- mice, while the contralateral eyes remain untreated and served as controls. M-cone mediated retinal function was analyzed by full-field photopic ERG under middle wavelength flash conditions (510nm). Human L- and M-opsin transgene expression and their cellular localization were examined by immunohistochemistry using anti-L/M-opsin, anti-S-opsin antibodies and peanut agglutinin (PNA). The levels of transgene expression were assessed by Western blot analysis.
Untreated Opn1mw-/- mice have no M-cone function and only S-cone function primarily in their ventral retina. Treatment with either human M- or L-opsin rescued middle wavelength ERG responses to about 60% of normal b-wave amplitudes. AAV delivered human M-opsin or L-opsin was detected panretinally. In dorsal retinas, M-opsin or L-opsin was localized within PNA-labeled cone sheaths in cone outer segments. In ventral retinas where S-cones are present, M-opsin or L-opsin was co-localized with endogenous mouse S-opsin in cone outer segments. Western blot analysis showed that AAV-delivered M- or L-opsin was about 65% of the wildtype level which is consistent with the level of ERG rescue.
We demonstrate that AAV-mediated expression of either human M-opsin or L-opsin can restore ERG responses to middle wavelength light in Opn1mw-/- mice. Vectored human opsins were expressed in cone outer segments in both dorsal and ventral retinas. These results also demonstrate that cones with one type of opsin, endogenous S-opsin in this case, can co-exist with a second type of vector delivered opsin, human L-opsin or M-opsin without physiological effects. These observations provide proof-of-concept for eventually treating BCM patients by gene therapy.
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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