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A CRISPR way to treat Usher syndrome


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Wang, Q ORCID: 0000-0003-0397-6554 2020 , 'A CRISPR way to treat Usher syndrome', PhD thesis, University of Tasmania.

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Usher syndrome (USH) is a rare and complex genetic disorder that causes both deafness and blindness. The Usher type 1F subtype (USH1F) is one of the most severe forms of this disease and is caused by mutations in the PCDH15 gene. This gene is located at the USH1F locus and encodes a protein that is expressed in the retina and cochlea; it plays an essential role in maintaining photoreceptor cells and sensory hair cells. Patients with the Arg245Ter variant in exon 8 of the gene typically have congenital deafness and develop retinal degeneration in teenage years, and there is currently no cure for this disease.
The first section of this thesis describes the use of patient-specific induced pluripotent stem cells (iPSCs) to generate a robust in vitro model of PCDH15 retinal disease and combines this technology with gene editing approaches to conduct site-specific correction of the Arg245Ter mutation. Differentiation of Usher iPSCs and mutation-corrected iPSCs to retinal organoids was undertaken to investigate disease pathogenesis. Single-cell RNA-sequencing was performed to investigate the complexity of retinal organoids and compare gene expression profiles between PCDH15 Arg245Ter and wildtype stem cell-derived retinal cells.
Additionally, an ‘exon skipping’ approach was investigated in an organoid model. The mutated exon was removed from the PCDH15 gene in stem cells using CRISPR technology, to assess the function of exon 8 in PCDH15 and to determine whether exon-skipping could be a therapy option for this disease. For all the above experiments, isogenic gene-editing H9 hESC cell lines (mutation knocked in and exon-skipped hESC) were generated and analysed in an identical manner.
To expand our genome editing applications from bench to bedside, in the last part of this thesis I tested different adenoviral vectors in mature retinal organoids to determine virus transduction efficacy. This information will be used to inform virus selection in future clinical applications.
The aims of this thesis were to:
1) establish a stem cell-based 3D organoid USH1F disease model;
2) correct the Arg245Ter mutation in human PSCs and determine if the gene-editing strategy changes the disease phenotype in retinal organoids;
3) determine which AAV can infect the retinal cells most efficiently.
Taken together, this thesis provides proof-of-principle data for a CRISPR/Cas mediated gene editing approach for USH1F, and information to guide future clinical applications.

Item Type: Thesis - PhD
Authors/Creators:Wang, Q
Keywords: Usher syndrome, CRISPR, Gene editing, Stem cells, Retinal organoid, AAV
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Copyright 2019 the author

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