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Ji-Jing Pang, Song Mao, Wei Shi, Wentao Deng, Shannon E. Boye, Jie Li, Xuan Liu, Sanford L. Boye, Bo Chang, William W. Hauswirth; AAV-mediated Cone Targeted Gene Therapy To Cpfl5 Mouse, A Model of Human Achromatopsia 2 with Mutations in Cnga3. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1915.
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Mutations in the gene encoding the alpha-subunit of the cone cyclic nucleotide-gated (CNGA3) channel cause cone function loss in mammals. IRBP/GNAT2 promoter is a chimeric promoter in which sequence corresponding to -1619 to -1411 of the human interphotoreceptor retinoid-binding protein (IRBP) gene was directly fused to -151 to +126 sequence of human cone transducin alpha-subunit (GNAT2) gene. mCARpro promoter is a 500 base pair mouse cone arrestin promoter, described by Li et al. 2002. We tested whether these two newly developed cone targeting promoters are cone specific and Cnga3 gene delivery can restore cone function in cpfl5 (Cone Photoreceptor Function Loss 5) mice, a natural model of human Achromatopsia 2 (ACHM2) with Cnga3 mutations.
At postnatal day 14 (P14), 1 μl of AAV5-IRBP/GNAT2-GFP or AAV5-mCARpro-mCherry vector (1013 genome containing viral particles/ml) was injected subretinally into one eye of ten P14 C57 BL/6J mice, respectively; while 1 μl of AAV5-IRBP/GNAT2-Cnga3 vector (1013 genome containing viral particles/ml) was injected subretinally into one eye of 20 cpfl5 mice. Uninjected contralateral eyes were used as controls. Dark- and light-adapted ERGs, were recorded 2 months after injection followed by harvest of injected and uninjected eyes for structural studies.
Frozen section stained with either M- or S-cone opsin showed that AAV5-IRBP/GNAT2-GFP-mediated GFP expression was observed in almost all cones, including both M- and S-cones, while AAV5-mCARpro-mCherry-mediated mCherry expression was observed not only in cones but also in RPE cells of C57 BL/6J eyes. In AAV5-IRBP/GNAT2-Cnga3 treated cpfl5 eyes, restored light-adapted ERGs were observed at 2 months after injection; the b-wave amplitudes in treated cpfl5 eyes were similar with those from the AAV5-IRBP/GNAT2-GFP injected C57 BL/6J eyes, but were about 2/3 of those from uninjected C57BL/6J eyes. In the contralateral uninjected cpfl5 eyes, the cone-driven ERGs were not recordable. In the treated cpfl5 retinas, immunohistochemical CNGA3 staining was apparent in the inner and outer segments of the cones with normal M- and S-cone opsin distribution in outer segments of cones. However in untreated partner eyes, no CNGA3 expression was observed and M- and S-cone degeneration was apparent.
The AAV5-IRBP/GNAT2 vector targets cones, including both M- and S-cones, while AAV5-mCARpro vector drives expression in both cone and RPE cells. AAV5-IRBP/GNAT2-Cnga3 mediated gene therapy corrects CNGA3 deficiency and restores cone function in a naturally occurring mouse model of human ACHM2.
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