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UBC Theses and Dissertations

Development of reporter mouse models to evaluate and optimize crispr/cas9 base editing therapeutics Yu, Siyue


Genetic diseases are a leading cause of death and disability worldwide with immense economic and societal burdens. However, currently fewer than 5% of genetic diseases have approved treatments, often with limited therapeutic benefit to patients. Genome editing provides a new therapeutic strategy to treat genetic diseases. In the last few years, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has emerged as a promising gene editing approach due to its ease in design and flexibility to target different genetic diseases. One factor limiting its broader application is that it will introduce double-stranded deoxyribonucleic acid (DNA) breaks (DSBs). New advances in the CRISPR/Cas9 system are “base” and “prime” editing, which do not generate DSBs. Base editing has shown great potential to precisely repair the majority of pathogenic point mutations in the patient’s DNA. Prime editing, while currently less efficient, can introduce a broader range of possible edits. However, there remains a major challenge: delivery of genome editors to the affected tissues. To begin to address this challenge, two reporter gene mouse models were developed. Both models introduce a point mutation to knockout the reporter gene activity. Upon gene editing, the mutation is corrected back to the wildtype sequence, thereby restoring the reporter gene activity. The efficiency of gene editing can be visualized and measured with an in vivo whole animal imaging system, which permits repeated and long-term assessments of gene editing. To validate both mouse models, base editor mRNA and a mutation-specific single guide RNA (sgRNA) were encapsulated with lipid nanoparticles (LNPs) and intravenously administered into the reporter mice. This resulted in 83.5% restoration of wildtype reporter gene activity in the liver. One major application of these mouse models will be to evaluate and optimize gene editor delivery systems targeting various tissues. As an example, intramuscular administration of LNPs encapsulated with base editing components led to significant genome editing in the injected muscle, which could be readily visualized in the live imager. These novel reporter gene mouse models will provide critical tools for the in vivo evaluation and optimization of genome editor delivery and improved genome editor formulations.

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