Characterization of human‐induced pluripotent stem cells carrying homozygous RB1 gene deletion

Deng, Xiao-Yue; Iwagawa, Toshiro; Fukushima, Masaya; Watanabe, Sumiko

doi:10.1111/gtc.12771

Cited by 7 publications

(4 citation statements)

References 17 publications

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“…Here, we designed gRNAs to target exon‐SA and ‐SD sites. We previously constructed a plasmid contains tandem‐two gRNAs, and this worked well 41 . The two gRNAs remove exons by targeting both ends of an intron.…”

Section: Discussionmentioning

confidence: 99%

“…We previously constructed a plasmid contains tandem-two gRNAs, and this worked well. 41 The two gRNAs remove exons by targeting both ends of an intron. If single-nucleotide polymorphism data are available, it is possible to target a single-nucleotide polymorphism to one allele when designing an allele-specific exon-skipping vector; this would effectively and safely "cure" mutations.…”

Section: Discussionmentioning

confidence: 99%

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Evaluation of CRISPR/Cas9 exon‐skipping vector for choroideremia using human induced pluripotent stem cell‐derived RPE

Iwagawa

Masumoto²,

Tabuchi

et al. 2022

The Journal of Gene Medicine

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Background Exon‐skipping is a powerful genetic tool, especially when delivering genes using an AAV‐mediated full‐length gene supplementation strategy is difficult owing to large length of genes. Here, we used engineered human induced pluripotent stem cells and artificial intelligence to evaluate clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9‐based exon‐skipping vectors targeting genes of the retinal pigment epithelium (RPE). The model system was choroideremia; this is an X‐linked inherited retinal disease caused by mutation of the CHM gene. Methods We explored whether artificial intelligence detected differentiation of human OTX2, PAX6 and MITF (hOPM) cells, in which OTX2, PAX6 and MITF expression was induced by doxycycline treatment, into RPE. Plasmid encoding CHM exon‐skipping modules targeting the splice donor sites of exons 6 were constructed. A clonal hOPM cell line with a frameshift mutation in exon 6 was generated and differentiated into RPE. CHM exon 6‐skipping was induced, and the effects of skipping on phagocytic activity, cell death and prenylation of Rab small GTPase (RAB) were evaluated using flow cytometry, an in vitro prenylation assay and western blotting. Results Artificial intelligence‐based evaluation of RPE differentiation was successful. Retinal pigment epithelium cells with a frameshift mutation in exon 6 showed increased cell death, reduced phagocytic activity and increased cytosolic unprenylated RABs only when oxidative stress was in play. The latter two phenotypes were partially rescued by exon 6‐skipping of CHM. Conclusions CHM exon 6‐skipping contributed to RPE phagocytosis probably by increasing RAB38 prenylation under oxidative stress.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Evaluation of CRISPR/Cas9 exon‐skipping vector for choroideremia using human induced pluripotent stem cell‐derived RPE

Iwagawa

Masumoto²,

Tabuchi

et al. 2022

The Journal of Gene Medicine

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“…Retinoblastoma is a childhood cancer of the developing retina that starts with biallelic inactivation of the retinoblastoma gene 1 (RB1) gene [ 108 , 109 ]. Children with germline mutations in RB1 have an increased risk of developing retinoblastoma and other cancers later in life.…”

Section: Construction Of Organoids Based On Ipsc Technologymentioning

confidence: 99%

Application Prospect of Induced Pluripotent Stem Cells in Organoids and Cell Therapy

Zhang,

Qian,

Song

et al. 2024

IJMS

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Since its inception, induced pluripotent stem cell (iPSC) technology has been hailed as a powerful tool for comprehending disease etiology and advancing drug screening across various domains. While earlier iPSC-based disease modeling and drug assessment primarily operated at the cellular level, recent years have witnessed a significant shift towards organoid-based investigations. Organoids derived from iPSCs offer distinct advantages, particularly in enabling the observation of disease progression and drug metabolism in an in vivo-like environment, surpassing the capabilities of iPSC-derived cells. Furthermore, iPSC-based cell therapy has emerged as a focal point of clinical interest. In this review, we provide an extensive overview of non-integrative reprogramming methods that have evolved since the inception of iPSC technology. We also deliver a comprehensive examination of iPSC-derived organoids, spanning the realms of the nervous system, cardiovascular system, and oncology, as well as systematically elucidate recent advancements in iPSC-related cell therapies.

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“…ROs have also been used to identify the disease mechanisms of Leber’s congenital amaurosis (LCA)-related RPE65 (Li et al 2019 ), CEP290 ( Shimada et al 2017 ) , AIPL1 (Lukovic et al 2020 ) and CRX (Kruczek et al 2021 ). Other ocular diseases, such as glaucoma (VanderWall et al 2020 ), macular telangiectasia type 2 (Gantner et al 2019 ), microphthalmia (Eintracht et al 2020 ), retinoblastoma (Deng et al 2020 ; Liu et al 2020 ; Saengwimol et al 2020 ), Stargardt disease (Khan et al 2020 ) and RS1- related X-linked juvenile retinoschisis as a model, moreover, a study on syndrome coronavirus 2 (SARS-CoV-2) demonstrated that SARS-CoV-2 can infect the retinal cells (Ahmad Mulyadi Lai et al 2021 ). All these studies have been conducted in the last 4 years and have increased over time, which highlights the potential of RO as a model for retinal development and diseases.…”

Section: Main Textmentioning

confidence: 99%

Retinal organoids as models for development and diseases

2021

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The evolution of pluripotent stem cell-derived retinal organoids (ROs) has brought remarkable opportunities for developmental studies while also presenting new therapeutic avenues for retinal diseases. With a clear understanding of how well these models mimic native retinas, such preclinical models may be crucial tools that are widely used for the more efficient translation of studies into novel treatment strategies for retinal diseases. Genetic modifications or patient-derived ROs can allow these models to simulate the physical microenvironments of the actual disease process. However, we are currently at the beginning of the three-dimensional (3D) RO era, and a general quantitative technology for analyzing ROs derived from numerous differentiation protocols is still missing. Continued efforts to improve the efficiency and stability of differentiation, as well as understanding the disparity between the artificial retina and the native retina and advancing the current treatment strategies, will be essential in ensuring that these scientific advances can benefit patients with retinal disease. Herein, we briefly discuss RO differentiation protocols, the current applications of RO as a disease model and the treatments for retinal diseases by using RO modeling, to have a clear view of the role of current ROs in retinal development and diseases.

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Characterization of human‐induced pluripotent stem cells carrying homozygous RB1 gene deletion

Cited by 7 publications

References 17 publications

Evaluation of CRISPR/Cas9 exon‐skipping vector for choroideremia using human induced pluripotent stem cell‐derived RPE

Evaluation of CRISPR/Cas9 exon‐skipping vector for choroideremia using human induced pluripotent stem cell‐derived RPE

Application Prospect of Induced Pluripotent Stem Cells in Organoids and Cell Therapy

Retinal organoids as models for development and diseases

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