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CRISPRi mediated treatment of dominant rhodopsin-associated retinitis pigmentosa.

ABSTRACT: Rhodopsin (RHO) mutations such as Pro23His, are the leading cause of dominantly inherited retinitis pigmentosa in North America. As with other dominant retinal dystrophies, these mutations lead to production of a toxic protein product, and treatment will require knockdown of the mutant allele. The purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy using catalytically inactive S. aureus Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying GFP cloned downstream of the RHO promoter fragment (nucleotides -1403 to +73), we demonstrate a ~74%-84% reduction in RHO promoter activity in RHOpCRISPRi treated vs plasmid only controls. Following subretinal transduction of human retinal explants and transgenic Pro23His mutant pigs, significant knockdown of rhodopsin protein was achieved. Suppression of mutant transgene in vivo was associated with a reduction in ER-stress and apoptosis markers and preservation of photoreceptor cell layer thickness.

ORGANISM(S): Homo sapiens

PROVIDER: GSE244786 | GEO | 2024/03/28

REPOSITORIES: GEO

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Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery

Project description:P23H is the most common mutation in the RHODOPSIN (RHO) gene leading to a dominant form of retinitis pigmentosa (RP), a rod photoreceptor degeneration that invariably causes vision loss. Specific disruption of the disease P23H RHO mutant while preserving the wild-type (WT) functional allele would be an invaluable therapy for this disease. However, various technologies tested in the past failed to achieve effective changes and consequently therapeutic benefits. We validated a CRISPR/Cas9 strategy to specifically inactivate the P23H RHO mutant, while preserving the WT allele in vitro. We, then, translated this approach in vivo by delivering the CRISPR/Cas9 components in murine Rho+/P23H mutant retinae. Targeted retinae presented a high rate of cleavage in the P23H but not WT Rho allele. This gene manipulation was sufficient to slow photoreceptor degeneration and improve retinal functions. To improve the translational potential of our approach, we tested intravitreal delivery of this system by means of adeno-associated viruses (AAVs). To this purpose, the employment of the AAV9-PHP.B resulted the most effective in disrupting the P23H Rho mutant. Finally, this approach was translated successfully in human cells engineered with the homozygous P23H RHO gene mutation. Overall, this is a significant proof-of-concept that gene allele specific targeting by CRISPR/Cas9 technology is specific and efficient and represents an unprecedented tool for treating RP and more broadly dominant genetic human disorders affecting the eye, as well as other tissues.

2019-03-20 | GSE128579 | GEO

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Project description:The goal of the study was to identify transcriptional modifications in retinal tissues from mouse model of rhodopsin mutation-associated retinitis pigmentosa (RP), Q344X compared to wild-type (WT). We implemented RNA-sequencing (RNA-seq) at poly(A) selected RNA for transcriptomic profiling. Differentially expressed genes were determined by DESeq2 using the Benjamini & Hochberg p-value adjustment and an absolute log2 fold change cutoff. The results indicate that there is specificity in transcriptional patterns in the retina from Q344x mice relative to WT, including differential expression in the potassium channel gene, Kcnv2, and differential expression in histone genes including the H1 family histone member, H1foo, the H3 histone family 3B, H3f3b, and the histone deacetylase 9, Hdac9.

2018-02-21 | GSE102247 | GEO

Modelling and rescue of RP2 Retinitis Pigmentosa using iPSC Derived Retinal Organoids

Project description:Mutations in RP2 lead to a severe form of X-linked retinitis pigmentosa (XLRP). RP2 functions as a GTPase activating protein (GAP) for the small GTPase ARL3, which is essential for cilia function and for photoreceptor development and maintenance. The mechanisms of RP2 associated retinal degeneration in humans are poorly understood, and genetically engineered animal models of RP2 XLRP present with differing retinal phenotypes and slow degeneration suggesting potential species differences. Here, we developed CRISPR gene edited isogenic RP2 knock-out (RP2 KO) induced pluripotent stem cells (iPSC) and RP2 patient derived iPSC to produce 3D retinal organoids as a human retinal disease model. The isogenic RP2 KO retinal organoids and two unrelated RP2 patient iPSC lines produced retinal organoids with a defined photoreceptor cell layer (ONL) containing rod and cone photoreceptors. Strikingly the RP2 KO and RP2 patient derived organoids showed a thinning of the ONL by 180 days (D180) of culture, which was associated with a spike in cell death in the ONL at D150 of differentiation. RNA sequencing confirmed induction of cell death pathways in the RP2 null organoids at this stage. Photoreceptor cell death and ONL thinning was attributed to cells undergoing terminal rod cell differentiation characterized by reduced RHO expression and fewer rhodopsin immunoreactive photoreceptors. Gene augmentation with a human RP2 transgene in an AAV2/5 vector efficiently transduced RP2 KO organoids and led to high levels of RP2 expression in both rods and cones. Importantly, the viral transduction significantly increased ONL thickness and restored rhodopsin expression, suggesting rescue of the RP2 degeneration phenotype. These data show that 3D retinal organoids can be used to model molecular defects associated with inherited retinal disease, photoreceptor cell death and also to test potential therapies targeted to prevent photoreceptor degeneration.

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Network biology analysis of P23H rhodopsin interactome identifies protein and mRNA quality control mechanisms

Project description:Rhodopsin is essential for phototransduction, and many rhodopsin mutations cause heritable retinal degenerations. The P23H rhodopsin variant generates a misfolded rhodopsin protein that photoreceptors quickly target for degradation by mechanisms that are incompletely understood. To gain insight into how P23H rhodopsin is removed from rods, we used mass spectrometry to identify protein interaction partners of P23H rhodopsin immunopurified from RhoP23H/P23H mice and to compare with protein interaction partners of wild-type rhodopsin from Rho+/+ mice. We identified 286 proteins associated with P23H rhodopsin and 276 proteins associated with wild-type rhodopsin. Only 113 proteins were shared between wild-type and mutant rhodopsin protein interactomes. In the P23H rhodopsin protein interactome, we saw loss of phototransduction, retinal cycle, and rhodopsin protein trafficking proteins but gain of ubiquitin-related proteins when compared with the wild-type rhodopsin protein interactome. In the P23H rhodopsin protein interactome, we saw significant enrichment of gene ontology terms related to ER-associated protein degradation, ER stress, and translation. Protein-protein interaction network analysis revealed that translational and ribosomal quality control proteins were the most significant regulators in the P23H rhodopsin protein interactome. The protein partners identified in our study may provide new insights into how photoreceptors recognize and clear mutant rhodopsin and may offer novel targets involved in retinal degeneration pathogenesis.

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Project description:A hallmark of inherited retinal degenerative diseases such as Retinitis Pigmentosa (RP) is progressive structural and functional remodeling of the remaining retinal cells as photoreceptors degenerate. Extensive remodeling of the retina stands as a barrier for the successful implementation of strategies to restore vision. To understand the molecular basis of remodeling, we performed analyses of single-cell transcriptome data from adult Zebrafish retina of wild-type and a P23H mutant rhodopsin transgenic model of RP with continuous degeneration and regeneration. We provide a benchmark atlas of retinal cell type transcriptomes in Zebrafish and find changes in all retinal cell types in the P23H model. These include widespread oxidative stress, changes in reliance on oxidative metabolism and glycolysis, widespread synaptic remodeling, and changes in circadian rhythm regulation. This comprehensive transcriptomic analysis provides a molecular road map to understand how the retina remodels in the context of chronic retinal degeneration with ongoing regeneration.

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CRISPRi mediated treatment of dominant rhodopsin-associated retinitis pigmentosa.

Project description:CRISPRi mediated treatment of dominant rhodopsin-associated retinitis pigmentosa.

| PRJNA1024894 | ENA

GENE EXPRESSION CHANGES WITHIN MÜLLER GLIAL CELLS DURING RETINITIS PIGMENTOSA

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.

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Project description:Inherited retinal degenerations (IRDs) constitute a group of clinically and genetically diverse vision-impairing disorders. Retinitis pigmentosa (RP), the most common form of IRD, is characterized by gradual dysfunction and degeneration of rod photoreceptors, followed by the loss of cone photoreceptors. Recently, we identified reserpine as a lead molecule for maintaining rod survival in mouse and human retinal organoids as well as in the rd16 mouse, which phenocopy Leber congenital amaurosis caused by mutations in the cilia-centrosomal gene CEP290 (Chen et al. eLife 2023;12:e83205. DOI: https://doi.org/10.7554/eLife.83205). Here, we show the therapeutic potential of reserpine in a rhodopsin P23H rat model of autosomal dominant RP. At postnatal day (P) 68, when males and females are analyzed together, the reserpine-treated rats exhibit higher rod-derived scotopic b-wave amplitudes compared to the controls with little or no change in scotopic a-wave or cone-derived photopic b-wave. Interestingly, the reserpine-treated female rats display enhanced scotopic a- and b-waves and photopic b-wave responses at P68, along with a better contrast threshold and increased outer nuclear layer thickness. The female rats demonstrate better preservation of both rod and cone photoreceptors following reserpine treatment. Retinal transcriptome analysis reveals sex-specific responses to reserpine, with significant upregulation of phototransduction genes and proteostasis-related pathways, and notably, genes associated with stress response. This study builds upon our previously reported results reaffirming the potential of reserpine for gene-agnostic treatment of IRDs and emphasizes the importance of biological sex in retinal disease research and therapy development.

2024-09-27 | GSE278306 | GEO

GENE EXPRESSION CHANGES WITHIN MM-CM-^\LLER GLIAL CELLS DURING RETINITIS PIGMENTOSA

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 MM-CM-<ller glia, play in the progression of the disease. Here we define gene expression changes in MM-CM-<ller 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. Retinas were dissociated and single Muller Glial Cells were picked under a dissecting microscope using a micropipette based on their distinct morphological shape. Single cell cDNAs were generated and genome wide profiles were obtained by hybridization to Affymetrix 430 2.0 microarrays. Data was normalized using MAS5.0 software. A total of 28 Muller Glial Cells were analyzed and confirmed post hoc by expression of known marker genes.

2014-10-31 | E-GEOD-35386 | biostudies-arrayexpress

Transcriptomic profiling in juvenile P23H Retinitis Pigmentosa mouse retinas

Project description:Neuronal plasticity of the inner retina has been observed in response to photoreceptor degeneration. Typically, this phenomenon has been considered maladaptive and may preclude vision restoration in the blind. However, several recent studies utilizing triggered photoreceptor ablation have shown adaptive responses in bipolar cell dendrites expected to support normal vision. Whether such homeostatic plasticity occurs during progressive photoreceptor degenerative disease to help maintain normal visual behavior is unknown. We addressed these issues in an established mouse model of Retinitis Pigmentosa caused by the P23H mutation in rhodopsin. We show robust modulation of the retinal transcriptomic network reminiscent of the neurodevelopmental state as well as potentiation of rod – rod bipolar cell signaling following rod photoreceptor degeneration. Additionally, we found highly sensitive night vision in P23H mice even when more than half of the rod photoreceptors were lost. The results implicate retinal adaptation leading to persistent visual function during photoreceptor degenerative disease.

2020-06-16 | GSE152474 | GEO

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