AAV Gene Augmentation Therapy for CRB1-Associated Retinitis Pigmentosa

TY - JOUR

T1 - AAV Gene Augmentation Therapy for CRB1-Associated Retinitis Pigmentosa

AU - Alves, C Henrique

AU - Wijnholds, J.

PY - 2018

Y1 - 2018

N2 - 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.

AB - 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.

KW - Journal Article

U2 - 10.1007/978-1-4939-7522-8_10

DO - 10.1007/978-1-4939-7522-8_10

M3 - Article

C2 - 29188511

SN - 1064-3745

VL - 1715

SP - 135

EP - 151

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

ER -