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Debra A. Thompson, Lin Jia, Jingyu Yao, Kecia L. Feathers, Selina A. Azam, Alexander J. Smith, Robin R. Ali; AAV-mediated Expression Of Human Rdh12 in Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1916.
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Inherited loss-of-function mutations in RDH12 result in severe retinal degeneration often diagnosed in childhood as Leber congenital amaurosis (LCA). The purpose of our studies was to initiate feasibility testing of AAV-vector-mediated RDH12 gene-replacement therapy by evaluating outcomes in wild-type and Rdh12-knockout mice.
A human RDH12 cDNA construct previously shown to exhibit RDH activity in vitro was packaged as recombinant adeno-associated virus serotype 2/8 containing a rhodopsin-kinase promoter that drives expression in rods and cones. The resulting construct, AAV2/8-hRK-hRDH12, was administered to adult Rdh12-knockout and C57BL/6 mice by subretinal injection (1-2 υl of 1 x 108-10 viral genomes per υl). Mice were euthanized at 3 wk post injection and RDH12 expression was evaluated by western and immunohistochemical (IHC) analysis.
Using an antibody specific for mouse Rdh12, western analysis detected the endogenous protein in retinas from wild-type but not Rdh12-knockout mice, and IHC analysis showed labeling of the endogenous protein in photoreceptor inner segments and outer nuclear layer in wild-type mice. In contrast, using an antibody specific for human RDH12, the recombinant protein was detected in retinas from both Rdh12-knockout and wild-type mice injected with AAV2/8-hRK-hRDH12. In mice injected with ~109 viral genomes, the pattern of anti-human RDH12 reactivity appeared similar to the endogenous protein in both the level and pattern of expression. Similar results were obtained in mice injected at 5 and 11 wk of age. IHC analysis of rhodopsin, S-opsin, and M/L-opsin showed that expression of recombinant RDH12 did not disturb rod and cone cell integrity or diminish cell numbers.
Subretinal injection of an AAV2/8 vector in which expression is under the control of a rhodopsin-kinase promoter produces recombinant human RDH12 in the mouse retina that appears correctly localized and does not disrupt structural integrity. Future studies are needed to evaluate the ability of the recombinant protein to reconstitute functional deficits resulting from RDH12 loss-of-function. Our findings represent an important first step toward the development of a novel therapy for a subset of LCA patients.
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