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Stefania Marsili, Shelby L. Reinstein, Sanford L. Boye, Haoyu Mao, Marina S. Gorbatyuk, William W. Hauswirth, Alfred S. Lewin, Gustavo D. Aguirre, William A. Beltran; rAAV-Mediated Gene Delivery of a Rho Knockdown and Replacement Construct in the Canine Retina. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1415.
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To assess the efficiency and safety of combined RHO suppression and hardened RHO cDNA replacement in the normal canine retina.
A construct that includes a shRNA and a hardened wild type RHO cDNA (RHO301) driven by a segment of the proximal mouse opsin promoter (mOp), was packaged into a recombinant adeno-associated viral serotype vector (rAAV2/5), and delivered via sub-retinal injection (150 µl at 6.79 x 1011 vg/ml) to the right eyes of normal adult dogs. Left eyes were sham-injected with BSS. Weekly ocular examinations were performed, and in vivo retinal imaging (Spectralis HRA/OCT) was done prior to the injections and at termination. Retinas from the dogs were collected five weeks following sub-retinal injection, and processed for morphology/IHC or RHO mRNA quantification by RT-PCR followed by restriction site analysis.
No clinical complications were seen in any of the injected eyes. Retinal lamination and ONL thickness was preserved on HRA/OCT scans as well as on histological sections. Rhodopsin IHC showed rare RHO mislocalization to the soma in rods located within the treated region of the retina. Photoreceptor morphology, including the inner and outer segments, was well preserved. RT-PCR plus restriction site analysis confirmed that the hardened RHO301 transcript was expressed in the area injected with the rAAV2/5-shRNA301-mOP-RHO301 construct.
A construct that combines RHO knockdown with replacement strategy appears to be safe and enables expression of the hardened RHO target when delivered by the means of a rAAV vector. On-going studies are evaluating its efficiency at suppressing endogenous RHO mRNA.
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