Allele-specific gene editing to rescue dominant CRX-associated LCA7 phenotypes in a retinal organoid model

Chirco, Kathleen R.; Chew, Shereen; Moore, Anthony T.; Duncan, Jacque L.; Lamba, Deepak A.

doi:10.1016/j.stemcr.2021.09.007

Cited by 36 publications

(24 citation statements)

References 24 publications

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“…3D retinal organoid cultures recapitulate the developmental timeline of human retinogenesis by encompassing all the various retinal cell types present in vivo . Retinal organoids were differentiated using our previously published protocols [42] ( Figure 1A) . Retinal organoids were easily identified by phase contrast live imaging ( Figure 1B ) displaying typical features of neuroepithelium formation (1B1-B2) and visible laminated pattern (1B3).…”

Section: Resultsmentioning

confidence: 99%

Biophysical, Molecular and Proteomic profiling of Human Retinal Organoids derived Exosomes

Arthur

Kandoi

Lee

et al. 2022

Preprint

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Extracellular vesicles (EVs) are phospholipid bilayer-bound particles released by cells that play a role in cell-cell communication, signal transduction, and extracellular matrix remodeling. There is a growing interest in EVs for ocular applications as therapeutics, biomarkers, and drug delivery vehicles. EVs secreted from mesenchymal stem cells (MSCs) have shown to provide therapeutic benefits in ocular conditions. However, very little is known about the properties of bioreactors cultured-3D human retinal organoids secreted EVs. This study provides a comprehensive morphological, nanomechanical, molecular, and proteomic characterization of retinal organoid EVs and compares it with human umbilical cord (hUC) MSCs. Nanoparticle tracking analysis indicated the average size of EV as 100–250 nm. Atomic force microscopy showed that retinal organoid EVs are softer and rougher than the hUCMSC EVs. Gene expression analysis by qPCR showed a high expression of exosome biogenesis genes in late retinal organoids derived EVs (>120 days). Immunoblot analysis showed highly expressed exosomal markers Alix, CD63, Flotillin-2, HRS and Hsp70 in late retinal organoids compared to early retinal organoids EVs (<120 days). Protein profiling of retinal organoid EVs displayed a higher differential expression of retinal function-related proteins and EV biogenesis/marker proteins than hUCMSC EVs, implicating that the use of retinal organoid EVs may have a superior therapeutic effect on retinal disorders. This study adds supplementary knowledge on the properties of EVs secreted by retinal organoids and suggests their potential use in the diagnostic and therapeutic treatments for ocular diseases.

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Section: Resultsmentioning

confidence: 99%

Biophysical, Molecular and Proteomic profiling of Human Retinal Organoids derived Exosomes

Arthur

Kandoi

Lee

et al. 2022

Preprint

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“…Retinal organoid differentiation. Retinal organoids were differentiated via the embryoid body (EB) approach as described previously (Chirco et al, 2021). Notch inhibition.…”

Section: Methodsmentioning

confidence: 99%

“…Using previously published 3D retinal organoid differentiation protocols, 3,4 we generated organoids from a hiPSC line 17 and a modified hESC line (CRXp-GFP H9 18 ). Both lines consistently made retinal organoids, signified by a bright ring of retinal tissue.…”

Section: Small Molecule γ-Secretase Inhibitor Blocks Notch Pathway Ac...mentioning

confidence: 99%

“…Human pluripotent stem cells (hPSCs) have been utilized to generate retinal photoreceptors in a monolayer or organoid setting to study development and disease. [1][2][3][4] Alternatively, lab-generated photoreceptors could be used as a pool of photoreceptors for transplantation. [5][6][7][8] Over the course of development, retinal progenitor cells (RPCs) differentiate into their final cell fate in the neural retina, including cone and rod photoreceptors (PRs), retinal ganglion cells (RGCs), amacrine cells (ACs), horizontal cells (HCs), bipolar cells (BCs), and Müller glia (MG).…”

Section: Introductionmentioning

confidence: 99%

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Timed Notch Inhibition drives Photoreceptor fate specification in Human Retinal Organoids

Chew

Martinez

Chirco

et al. 2022

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PurposeTransplanting photoreceptors from human pluripotent stem cell derived retinal organoids have the potential to reverse vision loss in affected individuals. However, transplantable photoreceptors are only a subset of all cells in the organoids. Hence the goal of our current study was to accelerate and synchronize photoreceptor differentiation in retinal organoids by inhibiting the Notch signaling pathway at different developmental time-points using a small molecule, PF-03084014 (PF).MethodsHuman induced pluripotent stem cell (hiPSC)- and embryonic stem cells (hESC)-derived retinal organoids were treated with 10μM PF for three days at day 45 (D45), D60, D90 and D120 of differentiation. Organoids collected at 14-, 28-, and 42-days post-PF treatment were analyzed for progenitor and photoreceptor markers and Notch pathway inhibition by immunohistochemistry (IHC), quantitative PCR (qPCR) and bulk RNA-seq (n=3-5 organoids from 3 independent experiments).ResultsRetinal organoids collected at 14-days post-PF treatment showed a decrease in progenitor markers (KI67, VSX2, PAX6, and LHX2) and an increase in differentiated pan-photoreceptor markers (OTX2, CRX, and RCVRN) at all organoid stages except D120. PF-treated organoids at D45 and D60 exhibited an increase in cone photoreceptor markers (RXRG and ARR3). PF-treatment at D90 revealed an increase in cone and rod photoreceptors markers (ARR3, NRL, and NR2E3). Bulk RNA-seq analysis mirrored the IHC data and qPCR confirmed Notch effector inhibition.ConclusionsTiming the Notch pathway inhibition in human retinal organoids to align with progenitor competency stages can yield an enriched population of early cone or rod photoreceptors.

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“…In the past few years, CRISPR-Cas has become a particularly useful tool for editing mutations causing different hereditary blindness conditions, such as retinitis pigmentosa, 23 , 39 , 40 enhanced S-cone syndrome, 41 Leber congenital amaurosis, 42 , 43 and Usher syndrome type 2, 44 in patient-derived iPSCs or in mouse models of retinal degeneration. Thus, gene editing holds a promising potential for translational progress in the vision field.…”

Section: Introductionmentioning

confidence: 99%

CRISPR-AsCas12a Efficiently Corrects a GPR143 Intronic Mutation in Induced Pluripotent Stem Cells from an Ocular Albinism Patient

et al. 2022

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Mutations in the GPR143 gene cause X-linked ocular albinism type 1 (OA1), a disease that severely impairs vision. We recently generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of an OA1 patient carrying a point mutation in intron 7 of GPR143 . This mutation activates a new splice site causing the incorporation of a pseudoexon. In this study, we present a high-performance CRISPR-Cas ribonucleoprotein strategy to permanently correct the GPR143 mutation in these patient-derived iPSCs. Interestingly, the two single-guide RNAs available for SpCas9 did not allow the cleavage of the target region. In contrast, the cleavage achieved with the CRISPR-AsCas12a system promoted homology-directed repair at a high rate. The CRISPR-AsCas12a-mediated correction did not alter iPSC pluripotency or genetic stability, nor did it result in off-target events. Moreover, we highlight that the disruption of the pathological splice site caused by CRISPR-AsCas12a-mediated insertions/deletions also rescued the normal splicing of GPR143 and its expression level.

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Allele-specific gene editing to rescue dominant CRX-associated LCA7 phenotypes in a retinal organoid model

Cited by 36 publications

References 24 publications

Biophysical, Molecular and Proteomic profiling of Human Retinal Organoids derived Exosomes

Biophysical, Molecular and Proteomic profiling of Human Retinal Organoids derived Exosomes

Timed Notch Inhibition drives Photoreceptor fate specification in Human Retinal Organoids

CRISPR-AsCas12a Efficiently Corrects a GPR143 Intronic Mutation in Induced Pluripotent Stem Cells from an Ocular Albinism Patient

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