Abstract
Mutations in the CRB1 gene account for around 10,000 persons with Leber congenital amaurosis (LCA) and 70,000 persons with retinitis pigmentosa (RP) worldwide. Therefore, the CRB1 gene is a key target in the fight against blindness. A proof-of-concept for an adeno-associated virus (AAV)-mediated CRB2 gene augmentation therapy for CRB1-RP was recently described. Preclinical studies using animal models such as knockout or mutant mice are crucial to obtain such proof-of-concept. In this chapter we describe a technique to deliver AAV vectors, into the murine retinas, via the subretinal route. We also present protocols to detect expression of the therapeutic protein by fluorescence immunohistochemistry and to perform histological studies using ultra-thin sections stained with toluidine blue. These techniques in combination with electroretinography and visual behavior tests are in principle sufficient to obtain proof-of-concept for new gene therapies.
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Acknowledgment
Dr. Lucie P. Pellissier and Rogier M. Vos are acknowledged for providing AAV stocks. This work was financially supported by the Foundation Fighting Blindness USA project TA-GT-0715-0665-LUMC and ZonMw project 43200004 (to JW).
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Alves, C.H., Wijnholds, J. (2018). AAV Gene Augmentation Therapy for CRB1-Associated Retinitis Pigmentosa. In: Boon, C., Wijnholds, J. (eds) Retinal Gene Therapy. Methods in Molecular Biology, vol 1715. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7522-8_10
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DOI: https://doi.org/10.1007/978-1-4939-7522-8_10
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Online ISBN: 978-1-4939-7522-8
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