Characterization and AAV-mediated CRB gene augmentation in human-derived CRB1KO and CRB1KOCRB2+/- retinal organoids

Nanda Boon; Xuefei Lu; Charlotte A Andriessen; Michaela Orlovà; Peter M J Quinn; Camiel J F Boon; Jan Wijnholds

doi:10.1016/j.omtm.2023.101128

Characterization and AAV-mediated CRB gene augmentation in human-derived CRB1^KO and CRB1^KOCRB2^+/- retinal organoids

Mol Ther Methods Clin Dev. 2023 Oct 10:31:101128. doi: 10.1016/j.omtm.2023.101128. eCollection 2023 Dec 14.

Authors

Nanda Boon¹, Xuefei Lu¹, Charlotte A Andriessen¹, Michaela Orlovà¹, Peter M J Quinn¹, Camiel J F Boon^{1

2}, Jan Wijnholds^{1

3}

Affiliations

¹ Department of Ophthalmology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, the Netherlands.
² Department of Ophthalmology, Amsterdam University Medical Centers, 1000 AE Amsterdam, the Netherlands.
³ Netherlands Institute for Neuroscience, an Institute of the Royal Netherlands Academy of Arts and Sciences (KNAW), Meibergdreef 47, 1105 BA Amsterdam, the Netherlands.

Abstract

The majority of patients with mutations in CRB1 develop either early-onset retinitis pigmentosa as young children or Leber congenital amaurosis as newborns. The cause for the phenotypic variability in CRB1-associated retinopathies is unknown, but might be linked to differences in CRB1 and CRB2 protein levels in Müller glial cells and photoreceptor cells. Here, CRB1^KO and CRB1^KOCRB2^+/- differentiation day 210 retinal organoids showed a significant decrease in the number of photoreceptor nuclei in a row and a significant increase in the number of photoreceptor cell nuclei above the outer limiting membrane. This phenotype with outer retinal abnormalities is similar to CRB1 patient-derived retinal organoids and Crb1 or Crb2 mutant mouse retinal disease models. The CRB1^KO and CRB1^KOCRB2^+/- retinal organoids develop an additional inner retinal phenotype due to the complete loss of CRB1 from Müller glial cells, suggesting an essential role for CRB1 in proper localization of neuronal cell types. Adeno-associated viral (AAV) transduction was explored at early and late stages of organoid development. Moreover, AAV-mediated gene augmentation therapy with AAV.hCRB2 improved the outer retinal phenotype in CRB1^KO retinal organoids. Altogether, these data provide essential information for future gene therapy approaches for patients with CRB1-associated retinal dystrophies.

Keywords: AAV; CRB1; CRB2; CRISPR-Cas9; Müller glial cells; gene therapy; hiPSC; photoreceptor; retinal degeneration; retinal organoids.