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Timothy P Day, Trevor Lee, Jan Wijnholds, David Schaffer, John Gerard Flannery; Characterization of CRB1 gene therapy in a retinal degeneration mouse model. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3633.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the crumbs homologue 1 gene (crb1) are associated with recessive retinitis pigmentosa, Leber congenital amaurosis, and other clinically relevant retinal degenerations. For potential crb1 gene therapy treatment, adeno-associated virus (AAV) is the vector of choice for delivery but the necessary transgene using traditional promoters is too large for the packaging capacity of AAV. Therefore, smaller synthetic CMV promoters were evaluated for the ability to express crb1 in the retina. AAV vectors driven by a small synthetic promoter were then tested in the rd8 mouse for characterization.
GFP and a myc-tagged crb1 transgene, both driven by a 323 base-pair (bp) synthetic CMV promoter, were packaged in the Müller glia-specific AAV variant, ShH10. These vectors were intravitreally injected into wild-type mice to determine expression and localization using fundus imaging and immunohistochemistry. A crb1 transgene packaged in ShH10 was intravitreally injected into rd8 mice, and optical coherence tomography (OCT) and electroretinograms (ERGs) were performed to determine the effect of a gene therapy intervention.
Fundus imaging demonstrated that the 323 bp synthetic CMV promoter was capable of driving expression in the retina. Utilizing ShH10 for delivery, immunohistochemistry showed expression and localization of myc-tagged CRB1 to Müller glia where it is natively expressed in mice. ERG and OCT allowed for further characterization of the rd8 mouse as model for CRB1 gene therapy.
In humans crb1 is expressed in both photoreceptors and Müller glia cells, while in mice it is only expressed in Müller glia. This discrepancy necessitates a careful evaluation of mouse models of the disease. Our study further confirms the ability of small synthetic promoters to drive expression in the retina and proper localization of CRB1. Additionally, we have further characterized the rd8 mouse as a model for the disease. This helps to pave the way for future gene therapy studies in larger animals.
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