UBC Theses and Dissertations

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UBC Theses and Dissertations

Development of a CRISPR gene correction strategy for aniridia employing a minimally humanized mouse embryonic stem cell-based disease model Adair, Bethany


Aniridia is a rare congenital blindness caused by heterozygous variants in the PAX6 gene. There is no vision-saving therapy, but one exciting approach is the gene editing capabilities of CRISPR systems to permanently correct the genomic variants. Pre-clinical studies to develop such a therapy in animal models face the challenge of showing efficacy when binding human DNA. To answer this challenge, while developing a CRISPR therapy for aniridia, we proposed the “CRISPR Humanized Minimally Mouse Models” (CHuMMMs) strategy. I hypothesize that a CRISPR gene strategy can be developed and optimized in humanized mouse embryonic stem cells that will be able to distinguish between the patient variant and non-variant chromosomes, laying the foundation for correcting aniridic congenital blindness in humans. Thus, we minimally humanized Pax6 exon 9, the location of the most common aniridia variant c.718C>T, creating a 312 bp “landing pad” of human DNA. Initially, we generated a non-variant mouse to show humanization did not disrupt Pax6 function. Then, we generated a CHuMMMs cell-based disease model, in which we tested five CRISPR enzymes for efficacy. In this system, base editor ABE8e had the highest correction of the variant at 76.8%. Finally, we used LNPs to deliver ABE8e to primary neurons ex vivo, which altered a second patient variant and rescued 24.8% Pax6 expression. Thus, we demonstrated the CHuMMMs approach, and showed the first genomic editing by ABE8e encapsulated as an LNP-RNP. Furthermore, we laid the foundation for translation of the proposed CRISPR therapy to human cells and eventually aniridia patients.

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