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AAV-mediated Gene Therapy Restores Cone Function In A Rat With An M-cone Opsin Deficiency, A Model For Blue Cone Monochromacy Zuoming 1Clinical 1 Zhang , Jijing 2 Pang , Feng 1 Xia , Qun 1 Guo , Li 1 Li , Jing 1 An , Lei 1 Zhang , William W. 2 Hauswirth , Shaowei 1 Yang , Zhenfeng 1 Li . Aerospace Medicine, Fourth Military Medical University, Xi'an, China; 2Department of Ophthalmology, University of Florida, Gainesville, FL. Purpose: Using an AAV vector targeting human L-cone opsin or rat M-cone opsin expression to cones, we aimed to test gene therapy in a naturally occurring m-cone opsin mutant middle-wavelength opsin cone dysfunction, MCD) rat model. Methods: Abnormal cone response phenotype male Sprague-Dawley (SD) rats (outbreed strain) were analyzed to identify the inherited trait and the causative mutation. To develop a gene therapy, two AAV vectors were constructed. The first was a serotype 5 AAV with the human red opsin promoter (PR2.1) driving expression of a human L-opsin cDNA (hROps). The second was a serotype 8 AAV containing a Y-F mutation at capsid position 733 (AAV8-733) with the same promoter driving a rat M-opsin cDNA. One microliter of each vector containing 1010 vector genomes was subretinally injected into one eye of a cohort of 30 mutant rats, respectively at postnatal day 14 (P14). At 2 months posttreatment the therapeutic effect of vector expression of the two opsin cDNAs was tested by full field photopic and flicker ERG analyses. Results: The rat with abnormal cone function has an X-linked recessive trait and the causative mutation is in the Opn1mw gene. There was no cone response or flicker ERG under standard intensity flashes in untreated rats, a phenotype that was stably maintained for 16 generations over 7 years. Histologically, there was no obvious change in retinal thickness, structure or the number of M-cones. After subretinal injection of the AAV5 vector expressing the human L-opsin in rat cones, there was no obviously change in cone single flash or flicker ERG at 2 months post-treatment. In contrast, the AAV8 (733) vector encoding the rat M-opsin yielded robust cone function rescue. Fig 2 Inherit trait in MCD rat It was showed a (X-linked recessive trait Fig 4 Retinal cone densities and thickness at different month in wild and MCD rat Bar denotes 10 μm in length. Fig 3 Identification of the rat mcd mutation locus In the rats, the nucleotide conversion G-to-T indicated by the box occurred at the invariant splicing acceptor site AG of intron 4. Fig 5 Expression of Opn1mw transcripts and M-opsin protein in the retina of wild rat (SD) and MCD rats. Fig 1 Scotopic and phototic ERG in wild and MCD rat Fig 6 ERGs after the AAV8 (733) vector encoding treatment in MCD rat Uper trace was right eye (treated eye) down trace was left eye (un treated eye) Conclusions: Since there is no L-cone opsin in the rat, this rat strain lacking M-cone function can be considered a model for combined L and M cone function loss in humans, a condition termed Blue Cone Monochromacy. Replacement gene therapy using a subretinally delivered AAV vector restores cone function in this model when using the homologous rat M-opsin cDNA but not when using the human L-opsin cDNA perhaps due to incompatibility of the human opsin with one or more components of the rat phototransduction complex. Reference 1. Gu YH, et al. A naturally occurring rat model of X-linked cone dysfunction. Invest Ophthalmol Vis Sci. 2003;44(12):5321-6. 2. Xie B, et al. A novel middle-wavelength opsin (M-opsin) null-mutation in the retinal cone dysfunction rat. Exp Eye Res. 2010;91(1):26-33. 3. Pang JJ,et al. Achromatopsia as a potential candidate for gene therapy.Adv Exp Med Biol, 2010;664:639-46.