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Carlaw, Tiffany Marie

University of British Columbia

There are currently no effective treatments for 95% of the over 7,000 known genetic diseases [1, 2]. The use of CRISPR/Cas9 has recently emerged as a powerful approach to directly repair disease-causing mutations [3-8]. The goal of this project was to optimize a novel CRISPR/Cas9 ‘base editing’ approach to treat genetic diseases by specifically repairing the disease-causing mutation in situ in the patients’ DNA. This technology uses a fusion of a partially deactivated Cas9 (nCas9) protein coupled with a nucleotide modifying enzyme to achieve 15-30% editing, and in some cases up to 75% editing in vitro [9-13]. Two-thirds of all genetic diseases are caused by point mutations [1] and base editors have now been developed that can target more than 68% of all disease-causing point mutations [8]. Despite the growing potential of base editing, limitations in the ability to quantify gene editing quickly and precisely have resulted in a lack of robust high throughput studies to compare and optimize base editors. We hypothesized that developing base editor reporter model systems would enable us to efficiently test and optimize the safety and effectiveness of base editing. To this end, we successfully developed both in vitro and in vivo reporter models and utilized these models to test the safety, delivery, and effectiveness of base editing. This series of novel model systems address a critical gap in the field of CRISPR/Cas9 gene editing. Using these, we were able to significantly increase the efficiency of base editing in vitro. We were also able to compare the safety of different base editors using a new application of my in vitro reporter model. We also demonstrated the therapeutic feasibility of our base editing approach in vitro to repair three prevalent mutations that cause a debilitating genetic disease: Lipoprotein Lipase Deficiency. Lastly, using the in vivo reporter model, we were able to demonstrate sustained correction and efficient delivery of base editors using lipid nanoparticles. Overall, these experiments have demonstrated critical steps towards the implementation of new treatment options for patients with a wide variety of genetic diseases.

Text

2024-05-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/84719

Medical Genetics

University of British Columbia

UBCV

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