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Mirjalili Mohanna, Seyedeh Zeinab

University of British Columbia

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 intrastromal and intravitreal delivery of rAAV9 transduced functional LSCs, as well as all PAX6-expressing retinal cell types in aniridic eye, respectively, with no observed adverse events, making rAAV9 the capsid of choice for the future gene therapy development for aniridia.

Text

2023-08-15

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/85471

Medical Genetics

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/