scholarly journals Allele-Specific Knockout by CRISPR/Cas to Treat Autosomal Dominant Retinitis Pigmentosa Caused by the G56R Mutation in NR2E3

2021 ◽  
Vol 22 (5) ◽  
pp. 2607
Author(s):  
Michalitsa Diakatou ◽  
Gregor Dubois ◽  
Nejla Erkilic ◽  
Carla Sanjurjo-Soriano ◽  
Isabelle Meunier ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
Autosomal Dominant ◽  
Vision Loss ◽  
Retinal Dystrophy ◽  
Common Mutation ◽  
Wild Type ◽  
Inherited Retinal Dystrophy ◽  
Allele Specific ◽  
Induced Pluripotent ◽  
Overexpression System

Retinitis pigmentosa (RP) is an inherited retinal dystrophy that causes progressive vision loss. The G56R mutation in NR2E3 is the second most common mutation causing autosomal dominant (ad) RP, a transcription factor that is essential for photoreceptor development and maintenance. The G56R variant is exclusively responsible for all cases of NR2E3-associated adRP. Currently, there is no treatment for NR2E3-related or, other, adRP, but genome editing holds promise. A pertinent approach would be to specifically knockout the dominant mutant allele, so that the wild type allele can perform unhindered. In this study, we developed a CRISPR/Cas strategy to specifically knockout the mutant G56R allele of NR2E3 and performed a proof-of-concept study in induced pluripotent stem cells (iPSCs) of an adRP patient. We demonstrate allele-specific knockout of the mutant G56R allele in the absence of off-target events. Furthermore, we validated this knockout strategy in an exogenous overexpression system. Accordingly, the mutant G56R-CRISPR protein was truncated and mis-localized to the cytosol in contrast to the (peri)nuclear localizations of wild type or G56R NR2E3 proteins. Finally, we show, for the first time, that G56R iPSCs, as well as G56R-CRISPR iPSCs, can differentiate into NR2E3-expressing retinal organoids. Overall, we demonstrate that G56R allele-specific knockout by CRISPR/Cas could be a clinically relevant approach to treat NR2E3-associated adRP.

scholarly journals Novel mutations in the 3-box motif of the BACK domain of KLHL7 associated with nonsyndromic autosomal dominant retinitis pigmentosa

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Jin Kyun Oh ◽  
Jose Ronaldo Lima de Carvalho ◽  
Young Joo Sun ◽  
Sara Ragi ◽  
Jing Yang ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
Autosomal Dominant ◽  
Fundus Autofluorescence ◽  
Retinal Dystrophy ◽  
Protein Modeling ◽  
Fundus Photography ◽  
Imaging Biomarkers ◽  
Loss Of Function ◽  
Gene Panel Testing ◽  
Sd Oct

Abstract Background Mutations in the Kelch-like protein 7 (KLHL7) represent a recently described and, to date, poorly characterized etiology of inherited retinal dystrophy. Dominant mutations in KLHL7 are a cause of isolated, non-syndromic retinitis pigmentosa (RP). In contrast, recessive loss-of-function mutations are known to cause Crisponi or Bohring-Opitz like cold induced sweating syndrome-3 (BOS-3). In this study, the phenotype and progression of five unrelated patients with KLHL7 mediated autosomal dominant RP (adRP) are characterized. Clinical evaluation of these patients involved a complete ophthalmic exam, full-field electroretinography (ffERG), and imaging, including fundus photography, spectral domain optical coherence tomography (SD-OCT), short wavelength fundus autofluorescence (SW-AF), and near-infrared fundus autofluorescence (NIR-AF). Molecular diagnoses were performed using whole-exome sequencing or gene panel testing. Disease progression was monitored in three patients with available data for a mean follow up time of 4.5 ± 2.9 years. Protein modeling was performed for all variants found in this study in addition to those documented in the literature for recessive loss-of-function alleles causing Crisponi or Bohring-Opitz like cold-induced sweating syndrome. Results Genetic testing in three patients identified two novel variants within the 3-box motif of the BACK domain: c.472 T > C:p.(Cys158Arg) and c.433A > T:p.(Asn145Tyr). Clinical imaging demonstrated hyperautofluorescent ring formation on both SW-AF and NIR-AF in three patients, with diffuse peripheral and peripapillary atrophy seen in all but one case. SD-OCT demonstrated a phenotypic spectrum, from parafoveal atrophy of the outer retina with foveal sparing to widespread retinal thinning and loss of photoreceptors. Incidence of cystoid macular edema was high with four of five patients affected. Protein modeling of dominant alleles versus recessive loss-of-function alleles showed dominant alleles localized to the BTB and BACK domains while recessive alleles were found in the Kelch domain. Conclusions We report the phenotype in five patients with KLHL7 mediated adRP, two novel coding variants, and imaging biomarkers using SW-AF and NIR-AF. These findings may influence future gene-based therapies for adRP and pave the way for mechanistic studies that elucidate the pathogenesis of KLHL7-mediated RP.

scholarly journals Targeting of the NRL Pathway as a Therapeutic Strategy to Treat Retinitis Pigmentosa

2020 ◽  
Vol 9 (7) ◽  
pp. 2224 ◽  
Author(s):  
Spencer M. Moore ◽  
Dorota Skowronska-Krawczyk ◽  
Daniel L. Chao
Keyword(s):  
Retinitis Pigmentosa ◽  
Autosomal Recessive ◽  
Therapeutic Strategy ◽  
Leucine Zipper ◽  
Retinal Dystrophy ◽  
Rod Photoreceptor ◽  
Cone Photoreceptor ◽  
Future Directions ◽  
Inherited Retinal Dystrophy ◽  
Visual Acuity Loss

Retinitis pigmentosa (RP) is an inherited retinal dystrophy (IRD) with a prevalence of 1:4000, characterized by initial rod photoreceptor loss and subsequent cone photoreceptor loss with accompanying nyctalopia, visual field deficits, and visual acuity loss. A diversity of causative mutations have been described with autosomal dominant, autosomal recessive, and X-linked inheritance and sporadic mutations. The diversity of mutations makes gene therapy challenging, highlighting the need for mutation-agnostic treatments. Neural leucine zipper (NRL) and NR2E3 are factors important for rod photoreceptor cell differentiation and homeostasis. Germline mutations in NRL or NR2E3 leads to a loss of rods and an increased number of cones with short wavelength opsin in both rodents and humans. Multiple groups have demonstrated that inhibition of NRL or NR2E3 activity in the mature retina could endow rods with certain properties of cones, which prevents cell death in multiple rodent RP models with diverse mutations. In this review, we summarize the literature on NRL and NR2E3, therapeutic strategies of NRL/NR2E3 modulation in preclinical RP models, as well as future directions of research. In summary, inhibition of the NRL/NR2E3 pathway represents an intriguing mutation agnostic and disease-modifying target for the treatment of RP.

scholarly journals Antioxidant and Biological Properties of Mesenchymal Cells Used for Therapy in Retinitis Pigmentosa

Antioxidants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 983
Author(s):  
Paolo Giuseppe Limoli ◽  
Enzo Maria Vingolo ◽  
Celeste Limoli ◽  
Marcella Nebbioso
Keyword(s):  
Retinitis Pigmentosa ◽  
Tissue Repair ◽  
Therapeutic Option ◽  
Retinal Dystrophy ◽  
Retinal Disease ◽  
Biological Properties ◽  
Mesenchymal Cells ◽  
Cell Therapies ◽  
Tissue Repair And Regeneration ◽  
Inherited Retinal Dystrophy

Both tissue repair and regeneration are a priority in regenerative medicine. Retinitis pigmentosa (RP), a complex retinal disease characterized by the progressive loss of impaired photoreceptors, is currently lacking effective therapies: this represents one of the greatest challenges in the field of ophthalmological research. Although this inherited retinal dystrophy is still an incurable genetic disease, the oxidative damage is an important pathogenetic element that may represent a viable target of therapy. In this review, we summarize the current neuroscientific evidence regarding the effectiveness of cell therapies in RP, especially those based on mesenchymal cells, and we focus on their therapeutic action: limitation of both oxidative stress and apoptotic processes triggered by the disease and promotion of cell survival. Cell therapy could therefore represent a feasible therapeutic option in RP.

A mutation in CRX causing pigmented paravenous retinochoroidal atrophy

2020 ◽  
pp. 112067212095759
Author(s):  
Jin Kyun Oh ◽  
Yan Nuzbrokh ◽  
Winston Lee ◽  
Jose Ronaldo Lima de Carvalho ◽  
Nan Kai Wong ◽  
...  
Keyword(s):  
Vision Loss ◽  
Retinal Dystrophy ◽  
Cone Dystrophy ◽  
Full Field ◽  
Inherited Retinal Dystrophy ◽  
History Of ◽  
Pigmented Paravenous Retinochoroidal Atrophy ◽  
Retinal Thinning ◽  
Bull's Eye ◽  
Sd Oct

Introduction: Mutations in the cone-rod homeobox ( CRX) gene, a known cause of inherited retinal dystrophy, are characterized by extensive phenotypic heterogeneity. We describe a novel presentation of rod-cone dystrophy (RCD) phenocopying pigmented paravenous retinochoroidal atrophy associated with a mutation in CRX. Case description: A 53-year-old man and his 48-year-old brother presented with a history of progressive vision loss and nyctalopia. Fundus examination revealed a bull’s eye lesion with chorioretinal atrophy and intraretinal pigment migration, while spectral-domain optical coherence tomography (SD-OCT) demonstrated retinal thinning with outer retinal atrophy. On short-wavelength autofluorescence (SW-AF) imaging, an atypical paravenous pattern of atrophy with a surrounding hyperautofluorescent border was observed. Full-field electroretinogram (ffERG) revealed a rod-cone pattern of dysfunction. A heterozygous pathogenic variant, c.119G>A:p.(Arg40Gln), in the CRX gene was identified in both brothers and segregated in their family. Conclusion: This case report broadens the currently known phenotypic presentations of CRX-associated retinopathy and suggests that mutations in CRX may be associated with pigmented paravenous retinochoroidal atrophy.

scholarly journals BBS Proteins Affect Ciliogenesis and Are Essential for Hedgehog Signaling, but Not for Formation of iPSC-Derived RPE-65 Expressing RPE-Like Cells

2021 ◽  
Vol 22 (3) ◽  
pp. 1345
Author(s):  
Caroline Amalie Brunbjerg Hey ◽  
Lasse Jonsgaard Larsen ◽  
Zeynep Tümer ◽  
Karen Brøndum-Nielsen ◽  
Karen Grønskov ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Dystrophy ◽  
Renal Cysts ◽  
Wild Type ◽  
Bardet Biedl Syndrome ◽  
Induced Pluripotent

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal dystrophy, renal cysts, obesity and polydactyly. BBS genes have been implicated in ciliogenesis, hedgehog signaling and retinal pigment epithelium maturation. BBS1 and BBS5 are members of the BBSome, implicated in cilia transport of proteins, and BBS10 is a member of the chaperonin-complex, mediating BBSome assembly. In this study, involvement of BBS1, BBS5 and BBS10 in ciliogenesis and hedgehog signaling were investigated in BBS-defective patient fibroblasts as well as in RPE-hTERT cells following siRNA-mediated knockdown of the BBS genes. Furthermore, the ability of BBS1-defective induced pluripotent stem-cells (iPSCs) to differentiate into RPE cells was assessed. We report that cells lacking functional BBS5 or BBS10 have a reduced number of primary cilia, whereas cells lacking functional BBS1 display shorter primary cilia compared to wild-type cells. Hedgehog signaling was substantially impaired and Smoothened, a component of hedgehog signaling, was trapped inside the cilia of the BBS-defective cells, even in the absence of Smoothened agonist. Preliminary results demonstrated the ability of BBS1-defective iPSC to differentiate into RPE-65 expressing RPE-like cells. The BBS1−/−-defective RPE-like cells were less pigmented, compared to RPE-like cells differentiated from control iPSCs, indicating an impact of BBS1 on RPE maturation.

scholarly journals The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation

2002 ◽  
Vol 115 (14) ◽  
pp. 2907-2918 ◽  
Author(s):  
Richard S. Saliba ◽  
Peter M. G. Munro ◽  
Philip J. Luthert ◽  
Michael E. Cheetham
Keyword(s):  
Quality Control ◽  
Retinitis Pigmentosa ◽  
Autosomal Dominant ◽  
Cultured Cells ◽  
State Level ◽  
Detailed Examination ◽  
Mutant Protein ◽  
Wild Type ◽  
Autosomal Dominant Retinitis Pigmentosa ◽  
Aggresome Formation

Mutations in the photopigment rhodopsin are the major cause of autosomal dominant retinitis pigmentosa. The majority of mutations in rhodopsin lead to misfolding of the protein. Through the detailed examination of P23H and K296E mutant opsin processing in COS-7 cells, we have shown that the mutant protein does not accumulate in the Golgi, as previously thought, instead it forms aggregates that have many of the characteristic features of an aggresome. The aggregates form close to the centrosome and lead to the dispersal of the Golgi apparatus. Furthermore, these aggregates are ubiquitinated, recruit cellular chaperones and disrupt the intermediate filament network. Mutant opsin expression can disrupt the processing of normal opsin, as co-transfection revealed that the wild-type protein is recruited to mutant opsin aggregates. The degradation of mutant opsin is dependent on the proteasome machinery. Unlike the situation with ΔF508-CFTR, proteasome inhibition does not lead to a marked increase in aggresome formation but increases the retention of the protein within the ER, suggesting that the proteasome is required for the efficient retrotranslocation of the mutant protein. Inhibition of N-linked glycosylation with tunicamycin leads to the selective retention of the mutant protein within the ER and increases the steady state level of mutant opsin. Glycosylation, however, has no influence on the biogenesis and targeting of wild-type opsin in cultured cells. This demonstrates that N-linked glycosylation is required for ER-associated degradation of the mutant protein but is not essential for the quality control of opsin folding. The addition of 9-cis-retinal to the media increased the amount of P23H, but not K296E, that was soluble and reached the plasma membrane. These data show that rhodopsin autosomal dominant retinitis pigmentosa is similar to many other neurodegenerative diseases in which the formation of intracellular protein aggregates is central to disease pathogenesis, and they suggest a mechanism for disease dominance.

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