Fig 2 Inherit trait in MCD rat It was showed a (X-linked recessive trait

1. Zuoming Zhang1, Jijing Pang2, Feng Xia1, Qun Guo1, Li Li1, Jing An1, Lei Zhang1, William W. Hauswirth2, Shaowei Yang1, Zhenfeng Li1. 1Clinical Aerospace Medicine, Fourth Military Medical University, Xi'an, China; 2Department of Ophthalmology, University of Florida, Gainesville, FL. 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 post-treatment the therapeutic effect of vector expression of the two opsin cDNAs was tested by full field photopic and flicker ERG analyses. AAV-mediated Gene Therapy Restores Cone Function In A Rat With An M-cone Opsin Deficiency, A Model For Blue Cone Monochromacy 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. 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. 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 1 Scotopic and phototic ERG in wild and MCD rat Fig 4 Retinal cone densities and thickness at different month in wild and MCD rat Bar denotes 10 μm in length. Fig 2 Inherit trait in MCD rat It was showed a (X-linked recessive trait Fig 5 Expression of Opn1mw transcripts and M-opsin protein in the retina of wild rat (SD) and MCD rats. 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 Gu YH, et al. A naturally occurring rat model of X-linked cone dysfunction. Invest Ophthalmol Vis Sci. 2003;44(12):5321-6. 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. Pang JJ,et al. Achromatopsia as a potential candidate for gene therapy.Adv Exp Med Biol, 2010;664:639-46.