AAV-mediated Gene Augmentation Therapy of CRB1 Patient-derived Retinal Organoids Restores the Histological and Transcriptional Retinal Phenotype

Overview

Journal Stem Cell Reports

Publisher Cell Press

Specialty Cell Biology

Date 2023 Apr 21

PMID 37084726

Authors

Nanda Boon

Xuefei Lu

Charlotte A Andriessen

Ioannis Moustakas

Thilo M Buck

Christian Freund

Christiaan H Arendzen

Stefan Bohringer

Hailiang Mei

Jan Wijnholds

Affiliations

Soon will be listed here.

Abstract

Retinitis pigmentosa and Leber congenital amaurosis are inherited retinal dystrophies that can be caused by mutations in the Crumbs homolog 1 (CRB1) gene. CRB1 is required for organizing apical-basal polarity and adhesion between photoreceptors and Müller glial cells. CRB1 patient-derived induced pluripotent stem cells were differentiated into CRB1 retinal organoids that showed diminished expression of variant CRB1 protein observed by immunohistochemical analysis. Single-cell RNA sequencing revealed impact on, among others, the endosomal pathway and cell adhesion and migration in CRB1 patient-derived retinal organoids compared with isogenic controls. Adeno-associated viral (AAV) vector-mediated hCRB2 or hCRB1 gene augmentation in Müller glial and photoreceptor cells partially restored the histological phenotype and transcriptomic profile of CRB1 patient-derived retinal organoids. Altogether, we show proof-of-concept that AAV.hCRB1 or AAV.hCRB2 treatment improved the phenotype of CRB1 patient-derived retinal organoids, providing essential information for future gene therapy approaches for patients with mutations in the CRB1 gene.

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References

Bulgakova N, Knust E . The Crumbs complex: from epithelial-cell polarity to retinal degeneration. J Cell Sci. 2009; 122(Pt 15):2587-96. DOI: 10.1242/jcs.023648. View

Maguire A, Russell S, Wellman J, Chung D, Yu Z, Tillman A . Efficacy, Safety, and Durability of Voretigene Neparvovec-rzyl in RPE65 Mutation-Associated Inherited Retinal Dystrophy: Results of Phase 1 and 3 Trials. Ophthalmology. 2019; 126(9):1273-1285. DOI: 10.1016/j.ophtha.2019.06.017. View

Quinn P, Buck T, Mulder A, Ohonin C, Alves C, Vos R . Human iPSC-Derived Retinas Recapitulate the Fetal CRB1 CRB2 Complex Formation and Demonstrate that Photoreceptors and Müller Glia Are Targets of AAV5. Stem Cell Reports. 2019; 12(5):906-919. PMC: 6522954. DOI: 10.1016/j.stemcr.2019.03.002. View

Kruczek K, Qu Z, Gentry J, Fadl B, Gieser L, Hiriyanna S . Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids. Stem Cell Reports. 2021; 16(2):252-263. PMC: 7878833. DOI: 10.1016/j.stemcr.2020.12.018. View

Mehalow A, Kameya S, Smith R, Hawes N, Denegre J, Young J . CRB1 is essential for external limiting membrane integrity and photoreceptor morphogenesis in the mammalian retina. Hum Mol Genet. 2003; 12(17):2179-89. DOI: 10.1093/hmg/ddg232. View

Quinn P, Alves C, Klooster J, Wijnholds J . CRB2 in immature photoreceptors determines the superior-inferior symmetry of the developing retina to maintain retinal structure and function. Hum Mol Genet. 2018; 27(18):3137-3153. DOI: 10.1093/hmg/ddy194. View

Aguilar-Aragon M, Fletcher G, Thompson B . The cytoskeletal motor proteins Dynein and MyoV direct apical transport of Crumbs. Dev Biol. 2019; 459(2):126-137. PMC: 7090908. DOI: 10.1016/j.ydbio.2019.12.009. View

van de Pavert S, Kantardzhieva A, Malysheva A, Meuleman J, Versteeg I, Levelt C . Crumbs homologue 1 is required for maintenance of photoreceptor cell polarization and adhesion during light exposure. J Cell Sci. 2004; 117(Pt 18):4169-77. DOI: 10.1242/jcs.01301. View

Pellissier L, Lundvig D, Tanimoto N, Klooster J, Vos R, Richard F . CRB2 acts as a modifying factor of CRB1-related retinal dystrophies in mice. Hum Mol Genet. 2014; 23(14):3759-71. DOI: 10.1093/hmg/ddu089. View

10.

Quinn P, Pellissier L, Wijnholds J . The CRB1 Complex: Following the Trail of Crumbs to a Feasible Gene Therapy Strategy. Front Neurosci. 2017; 11:175. PMC: 5380682. DOI: 10.3389/fnins.2017.00175. View

11.

Alves C, Pellissier L, Vos R, Garrido M, Sothilingam V, Seide C . Targeted ablation of Crb2 in photoreceptor cells induces retinitis pigmentosa. Hum Mol Genet. 2014; 23(13):3384-401. DOI: 10.1093/hmg/ddu048. View

12.

Lane A, Jovanovic K, Shortall C, Ottaviani D, Brugulat Panes A, Schwarz N . Modeling and Rescue of RP2 Retinitis Pigmentosa Using iPSC-Derived Retinal Organoids. Stem Cell Reports. 2020; 15(1):67-79. PMC: 7363745. DOI: 10.1016/j.stemcr.2020.05.007. View

13.

Warlich E, Kuehle J, Cantz T, Brugman M, Maetzig T, Galla M . Lentiviral vector design and imaging approaches to visualize the early stages of cellular reprogramming. Mol Ther. 2011; 19(4):782-9. PMC: 3070104. DOI: 10.1038/mt.2010.314. View

14.

van de Pavert S, Sanz A, Aartsen W, Vos R, Versteeg I, Beck S . Crb1 is a determinant of retinal apical Müller glia cell features. Glia. 2007; 55(14):1486-97. DOI: 10.1002/glia.20561. View

15.

den Hollander A, Ten Brink J, de Kok Y, Van Soest S, van den Born L, van Driel M . Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12). Nat Genet. 1999; 23(2):217-21. DOI: 10.1038/13848. View

16.

Pocha S, Wassmer T, Niehage C, Hoflack B, Knust E . Retromer controls epithelial cell polarity by trafficking the apical determinant Crumbs. Curr Biol. 2011; 21(13):1111-7. DOI: 10.1016/j.cub.2011.05.007. View

17.

Alves C, Bossers K, Vos R, Essing A, Swagemakers S, van der Spek P . Microarray and morphological analysis of early postnatal CRB2 mutant retinas on a pure C57BL/6J genetic background. PLoS One. 2013; 8(12):e82532. PMC: 3855766. DOI: 10.1371/journal.pone.0082532. View

18.

Tang F, Erion J, Tian Y, Liu W, Yin D, Ye J . VPS35 in Dopamine Neurons Is Required for Endosome-to-Golgi Retrieval of Lamp2a, a Receptor of Chaperone-Mediated Autophagy That Is Critical for α-Synuclein Degradation and Prevention of Pathogenesis of Parkinson's Disease. J Neurosci. 2015; 35(29):10613-28. PMC: 4510296. DOI: 10.1523/JNEUROSCI.0042-15.2015. View

19.

Ray T, Cochran K, Kozlowski C, Wang J, Alexander G, Cady M . Comprehensive identification of mRNA isoforms reveals the diversity of neural cell-surface molecules with roles in retinal development and disease. Nat Commun. 2020; 11(1):3328. PMC: 7335077. DOI: 10.1038/s41467-020-17009-7. View

20.

Roh M, Makarova O, Liu C, Shin K, Lee S, Laurinec S . The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost. J Cell Biol. 2002; 157(1):161-72. PMC: 2173254. DOI: 10.1083/jcb.200109010. View