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Alun R. Barnard, Peter Charbel Issa, Mandeep Singh, Haidong Shan, Robert E. MacLaren; Restoration Of Cone Function By rAAV2/5.CNGA3 Gene Therapy In A Mouse Model Of Achromatopsia Developed To Assess Visual Behaviour. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5476.
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Achromatopsia is a severe visual impairment caused by recessive mutations in genes encoding channel proteins or enzymes of the cone visual transduction cascade. CNGA3 is a cone-specific channel protein causing achromatopsia and cone dystrophy in humans. Although non-functional, cones are relatively well preserved into adulthood, which in theory makes gene therapy an attractive approach for restoring vision. Although mouse models of achromatopsia do exist, they remain overwhelmingly dependent on rod-mediated vision. Hence assessing successful cone gene therapy for behavioural responses such as light avoidance can be problematic. We therefore utilised a triple knockout (TKO) mouse, which is homozygous for 3 targeted mutations: Gnat1 (rod specific α-transducin); Cnga3 (cone specific cyclic nucleotide channel subunit) and Opn4 (ganglion cell specific melanopsin). We assessed the effects of CNGA3 gene replacement in this model.
5-6 week old TKO mice received 1µl subretinal injections containing 1 x 109 genome particles of rAAV2/5.GNGA3, with rAAV2/5.GFP used as a control vector. 2-3 months after injection, electroretinograms (ERGs) were recorded to assess restoration of visual function and confocal scanning laser ophthalmoscopy was used to examine transduced retinae in vivo. Eyes were removed post-mortem, fixed in 4% paraformaldehyde, embedded and sectioned (18µm) for immunohistochemistry to detect and localise CNGA3 protein.
Immunohistochemistry in the retinas of TKO mice showed strong staining for CNGA3 in the outer segments of cones located in the area of injection of rAAV2/5.CNGA3, but not in un-injected areas of retina, un-injected control eyes or in rAAV2/5.GFP injected controls. Restoration of cone function was apparent from recordable ERG responses to bright photopic flashes in rAAV2/5.CNGA3 injected TKO eyes but not un-injected or rAAV2/5.GFP injected controls. The major component (b-wave) of the ERG was ~20% of the equivalent wild-type value and oscillatory potentials could also be observed.
CNGA3 gene therapy can restore cone function in a retina which is minimally light responsive. This model will be useful to assess whether a largely dormant retinofugal pathway can be activated de novo, following successful gene therapy applied after the period of visual development.
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