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Alex Garanto, Anita Hoogendoorn, Joep J J Lurvink, Rob W J Collin; The shorter the better?: Assessing novel therapeutic strategies for CRB1-associated retinal disease. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3389. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Crumbs homolog 1 (CRB1) is a 1406-aminoacid protein with 19 EGF-like and 3 laminin G domains. It is encoded by CRB1, which is frequently mutated in several subtypes of inherited retinal disease. CRB1 is a transmembrane protein localized in the subapical region of photoreceptors. Unfortunately, conventional gene augmentation strategies using adeno-associated viruses (AAV) are challenging, due to the size of the CRB1 cDNA (4,2 kb excluding UTRs) which is at the limit of what an AAV can package. Therefore, an alternative is required. In this study, we aim to assess the residual function of shortened proteins, where some of the repetitive domains have been removed, as a potential therapeutic approach to treat CRB1-associated IRDs.
CRB1 cDNA was cloned into an expression vector and several versions producing shorter transcripts, and therefore proteins, have been generated by site-directed mutagenesis. Subsequently, these expression constructs were transfected into HEK293T or COS1 cells to study protein expression, stability and localization. In addition, using CRISPR/Cas9 technology, a knock-out zebrafish model and several lines producing shorter versions of Crb1 protein were generated to further elucidate the role of the shortened CRB1 proteins in vivo at protein, morphological and functional level. All experiments using animals were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research.
Vectors containing the different CRB1 cDNA versions were cloned and transfected into mammalian cells to study the protein expression, stability and localization. Our results showed that we were able to detect CRB1 protein by Western blot and immunocytochemistry. Currently, we are comparing the expression patterns to determine whether the localization of the shortened CRB1 proteins coincides with that of the full length protein. In addition, several zebrafish lines have been generated to study the effect of these protein in vivo. The first homozygous fish for some of the lines have been obtained. They are viable and fertile; and the retinas of the larvae are being characterized.
We have generated cellular and animal models to decipher the molecular function of shortened CRB1 proteins in order to evaluate their therapeutic potential for CRB1-associated retinal disease.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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