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Gene therapy of dominant CRX-Leber congenital amaurosis using patient retinal organoids II

ABSTRACT: Mutations in the cone-rod homeobox (CRX) transcription factor lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs mutation, we established an in vitro model of CRX-LCA in retinal organoids that exhibit defective photoreceptor maturation by histology and gene profiling including diminished expression of visual opsins. Gene therapy by delivery of an additional correct CRX allele using an AAV vector partially restored photoreceptor phenotype and expression of phototransduction-related genes as revealed by single cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying a K88N mutation revealed loss of opsin expression as a common phenotype, which could also be alleviated by AAV-mediated overexpression of CRX. Our studies provide the proof-of-concept for development of gene therapy for dominant CRX-LCA and other CRX-retinopathies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE153099 | GEO | 2021/02/17

REPOSITORIES: GEO

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Gene therapy of dominant CRX-Leber congenital amaurosis using patient retinal organoids I

Project description:Mutations in the cone-rod homeobox (CRX) transcription factor lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs mutation, we established an in vitro model of CRX-LCA in retinal organoids that exhibit defective photoreceptor maturation by histology and gene profiling including diminished expression of visual opsins. Gene therapy by delivery of an additional correct CRX allele using an AAV vector partially restored photoreceptor phenotype and expression of phototransduction-related genes as revealed by single cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying a K88N mutation revealed loss of opsin expression as a common phenotype, which could also be alleviated by AAV-mediated overexpression of CRX. Our studies provide the proof-of-concept for development of gene therapy for dominant CRX-LCA and other CRX-retinopathies.

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Gene therapy of dominant CRX-Leber congenital amaurosis using patient retinal organoids

Project description:Gene therapy of dominant CRX-Leber congenital amaurosis using patient retinal organoids

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Gene therapy of dominant CRX-Leber congenital amaurosis using patient retinal organoids I

Project description:Gene therapy of dominant CRX-Leber congenital amaurosis using patient retinal organoids I

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Gene therapy of dominant CRX-Leber congenital amaurosis using patient retinal organoids II

Project description:Gene therapy of dominant CRX-Leber congenital amaurosis using patient retinal organoids II

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Project description:Leber congenital amaurosis (LCA) includes congenital or early-onset blinding diseases, characterized by vision loss together with nystagmus and nonrecordable electroretinogram (ERG). At least 19 genes are associated with LCA. While most LCA is recessive, mutations in the homeodomain transcription factor gene CRX lead to autosomal dominant LCA. The mechanism of CRX-LCA is not understood. Here, we report a new spontaneous mouse mutant carrying a frameshift mutation in Crx (CrxRip). We show that, unlike Crx-/- mouse retina, the dominant Crx c.763del1 mutation in CrxRip results in congenital blindness with complete loss of ERG, yet the photoreceptors do not degenerate. Dominant CRX frameshift mutations associated with LCA mimic the CrxRip phenotype that can be rescued by Crx. RNA-Seq profiling reveals progressive and complete loss of rod differentiation factor Nrl in CrxRip, while residual Nrl remains in Crx-/- retina. Moreover, Nrl partially restores the rod phenotype in CrxRip/+ mice. We show that the binding of Otx2 to Nrl promoter is obliterated in CrxRip mutant, and ectopic Otx2 can rescue the rod phenotype. Therefore, Otx2 is required to maintain Nrl expression in developing rods to consolidate rod fate. Our studies provide the mechanism of congenital blindness caused by dominant CRX mutations and should assist in therapeutic design. Retinal samples were harvested from WT, CrxRip/+, CrxRip/Rip, Crx-/- and Nrl-/- retina at postnatal days 2 and 21 for whole transcriptome sequencing (RNAseq). Each sample included 2 independent frozen retina and experiments were performed in duplicates. RNA-seq transcriptome libraries were constructed from 1 ?g of total RNA.

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