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Marie O. Pequignot, Aude Conscience, Lorenne Robert, Nicolas Cereso, Christian P. Hamel, Vasiliki Kalatzis; Development of human in vitro and murine in vivo tools for gene therapy studies of Retinitis Punctata Albescens. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1891.
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© ARVO (1962-2015); The Authors (2016-present)
We aim to perform pre-clinical gene therapy studies for Retinitis Punctata Albescens (RPA). RPA is caused by mutations in the RLBP1 gene, which codes for the visual cycle protein CRALBP. A CRALBP-deficiency results in a malfunction of the retinal pigment epithelium (RPE) and leads to RPE and photoreceptor (PR) death. The early apparition of characteristic clinical signs (e.g. night blindness in childhood and small white dots observed by fundoscopy) allows an early diagnosis thus providing a large therapeutic window. An Rlbp1-deficient mouse model has been previously generated but it does not fully reproduce the disease course. As a consequence, in parallel to this model, we are generating an in vitro human retinal model of the RPA RPE for gene transfer studies.
We transferred the white Rlbp1-deficient mouse colony onto a pigmented background. In parallel, three RPA patients volunteered for skin biopsies from which we generated a stock of CRALBP1-deficient fibroblasts. These cells were reprogrammed into induced pluripotent stem (iPS) cells.
We are currently analyzing the phenotype of our pigmented Rlbp1-/- mice to determine the functional tests that can be used as readouts for gene therapy studies. In parallel, we obtained iPS-like clones for 2 of the 3 patient fibroblasts and are currently characterizing their pluripotency status. The reprogrammation of the 3rd patient’s fibroblasts is in process. We have set up the in vitro differentiation of control iPS cells to RPE and are developing the tools necessary to characterize its functions (e.g. phagocytosis, visual cycle analysis). The control and CRALBP-deficient RPE will constitute the human cellular model for gene transfer studies.
The human RPE model is potentially a unique tool as i) it allows a better understanding of the disease by providing the possibility to perform tests that cannot be done in patients, and ii) it represents a more biologically relevant model than the mouse eye for gene therapy studies. Consequently, the validation of this model will allow a considerable reduction in the future number of disease-specific animals necessary for testing novel therapies, as well as providing a model for testing novel therapies for diseases for which an animal model does not exist.
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