Project description:Retinitis pigmentosa (RP) and Leber congenital amaurosis are inherited retinal dystrophies caused by mutations in, among others, the Crumbs homologue 1 (CRB1) gene. CRB1 is required for organizing apical-basal polarity and adhesion between photoreceptors and Müller glial cells. Using human CRB1 patient induced pluripotent stem cells from RP patients we derived CRB1 retinal organoids, with diminished expression of variant CRB1 protein with 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 to isogenic controls. Adeno-associated viral (AAV) vector-mediated hCRB2 or hCRB1 gene augmentation in Müller glial and photoreceptor cells partially restored the histological and differentially expressed genes phenotype. 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.
Project description:Retinitis Pigmentosa (RP) is a progressive retinal degeneration in which the retina loses nearly all of its photoreceptor cells and undergoes major structural changes. Little is known regarding the role the resident glia, the Müller glia, play in the progression of the disease. Here we define gene expression changes in Müller glial cells (MGCs) from two different mouse models of RP, the retinal degeneration 1 (rd1) and rhodopsin knock-out (Rhod-ko) models. The RNA repertoire of 28 single MGCs was comprehensively profiled, and a comparison was made between MGC from wild type (WT) and mutant retinas. Two time points were chosen for analysis, one at the peak of rod photoreceptor death and one during the period of cone photoreceptor death. MGCs have been shown to respond to retinal degeneration by undergoing gliosis, a process marked by the upregulation of GFAP. In this data, many additional transcripts were found to change. These can be placed into functional clusters, such as retinal remodeling, stress response, and immune related response. It is noteworthy that a high degree of heterogeneity among the individual cells was observed, possibly due to their different spatial proximities to dying cells, and/or inherent heterogeneity among MGCs.
Project description:In mammals, retinal damage is followed by Müller glia cell activation and proliferation. While retinal gliosis persists in adult mammals after an insult or disease, some vertebrates, including zebrafish, have the capacity to regenerate. We believe we are the first group to show that gliosis is a fibrotic-like process in mammals’ eyes caused by differential activation of canonical and non-canonical TGFβ signaling pathways.
Project description:Transcriptome perturbations within NIH3T3 cell lines expressing rhodopsin and its retinitis pigmentosa mutant and implication for drug screening
Project description:Recessive retinitis pigmentosa (RP) is often caused by nonsense mutations that lead to low mRNA levels as a result of nonsense-mediated decay. Some RP genes are expressed at detectable levels in leukocytes as well as in the retina. We designed a microarray-based method to find recessive RP genes based on low lymphoblast mRNA expression levels Keywords: Recessive mutations; mRNA expression; nonsense mediated-decay; retinitis pigmentosa; lymphocyte; Affymetrix genechip Human Genome U133Plus2.0.
