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 pheno-typic... Show moreThe 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 pheno-typic variability in CRB1-associated retinopathies is unknown, but might be linked to differences in CRB1 and CRB2 protein levels in Muller glial cells and photoreceptor cells. Here, CRB1KO and CRB1KOCRB2+/-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 CRB1KO and CRB1KOCRB2+/-retinal organoids develop an additional inner retinal phenotype due to the complete loss of CRB1 from Muller glial cells, suggesting an essential role for CRB1 in proper local-ization 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 pheno-type in CRB1KO retinal organoids. Altogether, these data pro -vide essential information for future gene therapy approaches for patients with CRB1-associated retinal dystrophies. Show less
Mutations in the CRB1 gene can cause retinal dystrophies such as Retinitis Pigmentosa or Leber Congenital Amaurosis . These patients experience progressive vision loss which ultimately leads to... Show moreMutations in the CRB1 gene can cause retinal dystrophies such as Retinitis Pigmentosa or Leber Congenital Amaurosis . These patients experience progressive vision loss which ultimately leads to blindness. Currently, there are no treatment options available for these patients.This thesis provides novel information on AAV.hCRB gene augmentation therapy in CRB1 mutant animal and human-derived models. We show the phenotype of (1) a novel mouse model with CRB2 ablation specifically in rod photoreceptor cells with loss of retinal function (Chapter 2), (2) a Crb1 mutant brown Norway rat with severe and early onset progressive vision loss (Chapter 3), (3) CRB1 patient-derived retinal organoids (Chapter 4), and (4) CRB1KO and CRB1KOCRB2+/- LCA-like retinal organoids (Chapter 5). Next, AAV-mediated gene augmentation was explored in Crb1 mutant rats (Chapter 3) and CRB1 patient-derived and CRB1KO LCA retinal organoids (Chapter 4 and 5). Finally, single-cell RNA sequencing was performed on AAV.hCRB treated CRB1 patient-derived retinal organoids (Chapter 4). To our knowledge this is the first time that an improved phenotype after AAV.hCRB gene augmentation in CRB1 RP patient-derived and CRB1KO LCA retinal organoids is observed, providing essential information for future gene therapy possibilities in patients with a mutation in the CRB1 gene. Show less
CRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging,... Show moreCRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging, necessitating the development of alternatives. We generated human induced pluripotent stem cell (iPSC)-derived retinal organoids of patients with retinitis pigmentosa caused by biallelic CRB1 mutations and evaluated them against autologous gene-corrected hiPSCs and hiPSCs from healthy individuals. Patient organoids show decreased levels of CRB1 and NOTCH1 expression at the retinal outer limiting membrane. Proximity ligation assays show that human CRB1 and NOTCH1 can interact via their extracellular domains. CRB1 patient organoids feature increased levels of WDFY1+ vesicles, fewer RAB11A+ recycling endosomes, decreased VPS35 retromer complex components, and more degradative endolysosomal compartments relative to isogenic control organoids. Taken together, our data demonstrate that patient-derived retinal organoids enable modeling of retinal degeneration and highlight the importance of CRB1 in early endosome maturation receptor recycling in the retina. Show less
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... Show moreRetinitis 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 Muller 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 Muller 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. Show less
Background Crumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identified CRB2 mutations as a novel cause of steroid-resistant... Show moreBackground Crumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identified CRB2 mutations as a novel cause of steroid-resistant nephrotic syndrome (SRNS).Methods To study the function of Crb2 at the renal filtration barrier, mice lacking Crb2 exclusively in podocytes were generated. Gene expression and histologic studies as well as transmission and scanning electron microscopy were used to analyze these Crb2(podKO) knockout mice and their littermate controls. Furthermore, high-resolution expansion microscopy was used to investigate Crb2 distribution in murine glomeruli. For pull-down experiments, live cell imaging, and transcriptome analyses, cell lines were applied that inducibly express fluorescent protein-tagged CRB2 wild type and mutants.Results Crb2(podKO) mice developed proteinuria directly after birth that preceded a prominent development of disordered and effaced foot processes, upregulation of renal injury and inflammatory markers, and glomerulosclerosis. Pull-down assays revealed an interaction of CRB2 with Nephrin, mediated by their extracellular domains. Expansion microscopy showed that in mice glomeruli, Crb2 and Nephrin are organized in adjacent clusters. SRNS-associated CRB2 protein variants and a mutant that lacks a putative conserved O-glycosylation site were not transported to the cell surface. Instead, mutants accumulated in the ER, showed altered glycosylation pattern, and triggered an ER stress response.Conclusions Crb2 is an essential component of the podocyte's slit diaphragm, interacting with Nephrin. Loss of slit diaphragm targeting and increasing ER stress are pivotal factors for onset and progression of CRB2-related SRNS. Show less
Mutations in the Crumbs homologue 1 (CRB1) gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported... Show moreMutations in the Crumbs homologue 1 (CRB1) gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported with a spontaneous insertion-deletion (indel) mutation in exon 6 of Crb1. It has been reported that these Crb1 mutant rats show vascular abnormalities associated with retinal telangiectasia and possess an early-onset retinal degenerative phenotype with outer limiting membrane breaks and focal loss of retinal lamination at 2 months of age. Here, we further characterized the morphological phenotype of new-born and adult Crb1 mutant rats in comparison with age-matched Brown Norway rats without a mutation in Crb1. A significantly decreased retinal function and visual acuity was observed in Crb1 mutant rats at 1 and 3 months of age, respectively. Moreover, in control rats, the subcellular localization of canonical CRB1 was observed at the subapical region in Muller glial cells while CRB2 was observed at the subapical region in both photoreceptors and Muller glial cells by immuno-electron microscopy. CRB1 localization was lost in the Crb1 mutant rats, whereas CRB2 was still observed. In addition, we determined the tropism of subretinal or intravitreally administered AAV5-, AAV9- or AAV6-variant ShH10(Y445F) vectors in new-born control and Crb1 mutant rat retinas. We showed that subretinal injection of AAV5 and AAV9 at postnatal days 5 (P5) or 8 (P8) predominantly infected the retinal pigment epithelium (RPE) and photoreceptor cells; while intravitreal injection of ShH10(Y445F) at P5 or P8 resulted in efficient infection of mainly Muller glial cells. Using knowledge of the subcellular localization of CRB1 and the ability of ShH10(Y445F) to infect Muller glial cells, canonical hCRB1 and hCRB2 AAV-mediated gene therapy were explored in new-born Crb1 mutant rats. Enhanced retinal function after gene therapy delivery in the Crb1 rat was not observed. No timely rescue of the retinal phenotype was observed using retinal function and visual acuity, suggesting the need for earlier onset of expression of recombinant hCRB proteins in Muller glial cells to rescue the severe retinal phenotype in Crb1 mutant rats. Show less
Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are inherited degenerative retinal dystrophies with vision loss that ultimately lead to blindness. Several genes have been shown to be... Show moreRetinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are inherited degenerative retinal dystrophies with vision loss that ultimately lead to blindness. Several genes have been shown to be involved in early onset retinal dystrophies, includingCRB1andRPE65. Gene therapy recently became available for young RP patients with variations in theRPE65gene. Current research programs test adeno-associated viral gene augmentation or editing therapy vectors on various disease models mimicking the disease in patients. These include several animal and emerging human-derived models, such as human-induced pluripotent stem cell (hiPSC)-derived retinal organoids or hiPSC-derived retinal pigment epithelium (RPE), and human donor retinal explants. Variations in theCRB1gene are a major cause for early onset autosomal recessive RP with patients suffering from visual impairment before their adolescence and for LCA with newborns experiencing severe visual impairment within the first months of life. These patients cannot benefit yet from an available gene therapy treatment. In this review, we will discuss the recent advances, advantages and disadvantages of differentCRB1human and animal retinal degeneration models. In addition, we will describe novel therapeutic tools that have been developed, which could potentially be used for retinal gene augmentation therapy for RP patients with variations in theCRB1gene. Show less
Variations in the Crumbs homolog-1 (CRB1) gene are associated with a wide variety of autosomal recessive retinal dystrophies, including early onset retinitis pigmentosa (RP) and Leber congenital... Show moreVariations in the Crumbs homolog-1 (CRB1) gene are associated with a wide variety of autosomal recessive retinal dystrophies, including early onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). CRB1 belongs to the Crumbs family, which in mammals includes CRB2 and CRB3. Here, we studied the specific roles of CRB2 in rod photoreceptor cells and whether ablation of CRB2 in rods exacerbates the Crb1-disease. Therefore, we assessed the morphological, retinal, and visual functional consequences of specific ablation of CRB2 from rods with or without concomitant loss of CRB1. Our data demonstrated that loss of CRB2 in mature rods resulted in RP. The retina showed gliosis and disruption of the subapical region and adherens junctions at the outer limiting membrane. Rods were lost at the peripheral and central superior retina, while gross retinal lamination was preserved. Rod function as measured by electroretinography was impaired in adult mice. Additional loss of CRB1 exacerbated the retinal phenotype leading to an early reduction of the dark-adapted rod photoreceptor a-wave and reduced contrast sensitivity from 3-months-of-age, as measured by optokinetic tracking reflex (OKT) behavior testing. The data suggest that CRB2 present in rods is required to prevent photoreceptor degeneration and vision loss. Show less
Meurs, L.; Mbow, M.; Boon, N.; Vereecken, K.; Amoah, A.S.; Labuda, L.A.; ... ; Polman, K. 2014
