It has been demonstrated previously that localization of cone α-transducin is perturbed in Gucy2e–/– cone photoreceptors, with α-transducin localized to the inner segments and synaptic regions of cones (Coleman and Semple-Rowland, 2005). Our results are consistent with these findings; we observed weak and mislocalized cone α-transducin staining in untreated Gucy2e–/– eyes. In the treated eyes, however, immunohistochemical staining revealed increased levels of cone α-transducin, correctly localized to the cone outer segments (Fig. 3a). It has also been reported previously that localization of cone arrestin is perturbed in Gucy2e–/– cone photoreceptors (Coleman and Semple-Rowland, 2005), and that cone arrestin remains in the outer segments and synaptic terminals regardless of light conditions. However, we did not observe a major difference in cone arrestin staining in C57BL/6J wild-type and untreated Gucy2e–/– mice (Fig. 3b). Although there was faint immunostaining throughout Gucy2e–/– cones that was not seen in wild-type cones, in light-adapted animals the bulk of the signal, as in wild-type animals, was present in cone outer segments and synaptic terminals. Furthermore, after dark adaptation, arrestin translocated normally throughout cone photoreceptor cells. The localization and translocation of cone arrestin was not altered after administration of vector (Fig. 3b).
ExperimentName: Rescue of mouse Gucy2e homozygous knockout model by AAV-borne human GUCY2D transgene
NonHumanModel: Mus musculus
GeneAlteration: The mouse Gucy2e gene was disrupted by the introduction of a neomycin-resistance gene cassette into exon 5, resulting in a truncated protein lacking its transmembrane domain (PMID: 10407028). The effects of this knockout were studied in the homozygous state.
RescueVariant: Rescue was performed with the wild-type human GUCY2D and mouse Gucy2e genes but not with a patient-derived variant.
RescuePhenotype: HP:0030465 (Undetectable light-adapted electroretinogram), HP:0030483 (Reduced amplitude of dark-adapted bright flash electroretinogram a-wave), HP:0030469 (Abnormal dark-adapted electroretinogram), HP:0000479 (Abnormal retinal morphology), HP:0008020 (Cone dystrophy)
RescueMethod: Human GUCY2D or mouse Gucy2e was cloned into an overexpression construct under the control of the human rhodopsin kinase promoter and packaged into adeno-associated (AAV2/8) viral particles for 2 subretinal injections on postnatal day 10. Efficacy was measured by ERG recordings every two weeks.
RescueEvidence: Treatment with the human GUCY2D gene restored proper localization of alpha-transducin (Figs. 3a-3b), which was first reported by PMID: 15623748 to be abnormal in this model. Treatment corrected the defect in scotopic b-wave amplitude by 65-100% (Figs. 4a-4b). Cone amplitude was corrected from 20% of wild-type to 65% of wild-type (Fig. 4c). Similar results were obtained with the mouse Gucy2e transgene (Fig. 5). Head-tracking behavior (measure of visual acuity and contrast sensitivity) was fully corrected by treatment (Fig. 6). Moderate cone preservation was also demonstrated by normalization of alpha-transducin staining, showing a 20% increase in cone numbers relative to the untreated control (Fig. 7r).
ModelVariant: The mouse model disrupts the expression of the endogenous GC1 protein product but does not feature knock-in of a variant from a human patient.