Successful gene editing in a novel mouse model of aniridia towards prevention of blindness
department or school: Medicine, Faculty of
department or school: Medical Genetics, Department of
data provider: DSpace
degree campus: UBCV
creator: Mirjalili Mohanna, Seyedeh Zeinab
issued: 2023-08-15T18:37:09Z
issued: 2023
related degree: Doctor of Philosophy - PhD
degree grantor: University of British Columbia
description: Aniridia is a rare eye disorder with no cure and is caused by pathogenic variants in the paired box 6 (PAX6) gene. Patients have low vision at birth due to retinal foveal hypoplasia, which typically progresses to blindness by early adulthood due to corneal clouding and glaucoma. Since aniridia is caused by PAX6 haploinsufficiency, one potential approach to treating it is targeted CRISPR-based gene editing. Preclinical studies in an appropriate animal model are important first steps towards the translation of the research into a clinical trial. Thus, in Chapter 2, we inserted a 3xFLAG tag on the Pax6 small eye (Sey) allele, creating a novel mouse model for aniridia that is ideal for testing CRISPR strategies. Next, we developed a CRISPR strategy in vitro and subsequently evaluated its efficacy in vivo in the germline of our new mouse. We showed that our CRISPR strategy corrected the Sey variant in the mouse germline, resulting in the full rescue of the eye phenotype. Next, we worked towards developing a somatic therapy, the success of which relies on effective delivery of CRISPR components to therapeutically significant cells. In Chapter 3, we tested a novel lipid nanoparticle-based platform, and successfully edited cultured mouse cortical neurons. We then directly injected LNP-CRISPR into the mouse cornea to evaluate in vivo delivery. We demonstrated wide-spread gene editing of corneal stromal and endothelial cells but not the PAX6-expressing epithelial or limbal stem cells (LSCs). Hence, in Chapter 4, we shifted our focus to recombinant adeno-associated viruses (rAAVs), a more conventional delivery vector for CRISPR. We carried out a series of experiments to test viral capsid, dose, and route of administration. We also investigated if functional LSCs are present in the already cloudy aniridic mouse corneas and whether rAAV can transduce them. We reported unexpected ataxia and lethality in mice after intraocular rAAV-PHP.B injections. We also showed that intrastroma…
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/85471
note: Successful Gene Editing in a Novel Mouse Model of Aniridia Towards Prevention of Blindness by Seyedeh Zeinab Mirjalili Mohanna B.Sc., University of Malaya, 2014 M.Sc., The University of Sheffield, 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Medical Genetics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2023 © Seyedeh Zeinab Mirjalili Mohanna, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Successful Gene Editing in a Novel Mouse Model of Aniridia Towards Prevention of Blindness submitted by Seyedeh Zeinab Mirjalili Mohanna in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Medical Genetics Examining Committee: Dr. Elizabeth M. Simpson, Professor, Medical Genetics, UBC Supervisor Dr. Cheryl Gregory-Evans, Professor, Ophthalmology & Visual Sciences, UBC Supervisory Committee Member Dr. Perry Howard, Associate Professor, Biochemistry & Microbiology, University of Victoria External Examiner Dr. Deborah Giaschi, Professor, Ophthalmology & Visual Sciences, UBC University Examiner Dr. Robert Molday, Professor, Ophthalmology & Visual Sciences, UBC University Examiner Additional Supervisory Committee Members: Dr. Orson Moritz, Professor, Ophthalmology & Visual Sciences, UBC Supervisory Committee Member Dr. Colin Ross, Associate Professor, Medical Genetics, UBC Supervisory Committee Member iii Abstract Aniridia is a rare eye disorder with no cure and is caused by pathogenic variants in the paired box 6 (PAX6) gene. Patients have low vision at birth due to retinal foveal hypoplasia, which typically progresses to blindness by early adulthood due to corneal clouding and glaucoma. Since aniridia is caused by PAX6 haploinsufficiency, one potential approach to treating it is targeted CRISPR-based gene edi…
has type: Thesis/Dissertation
date issued: 2023-11
is shown at: 10.14288/1.0435212
language: eng
degree discipline: Medical Genetics
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivatives 4.0 International
rights URI: http://creativecommons.org/licenses/by-nc-nd/4.0/
scholar level: Graduate
contributor: Simpson, Elizabeth M.
type: Text
identifier URI: http://hdl.handle.net/2429/85471