CRISPR Technology to Treat Complex Genetic Disorders

Abstract

Despite enormous advances in the gene editing and gene therapy fields, there remains significant challenges to translating these novel therapeutics to the clinic. In this dissertation, I will address two major challenges: the lack of 1) preclinical models that better predict therapeutic outcomes in patients and 2) development of gene editing strategies that correct causal mutations, vs deleting a part of or the entire gene.First, we demonstrate the utility of novel 3D human skeletal muscle microphysiological tissues (“myobundles”) to evaluate the safety and efficacy of AAV-delivered gene editing therapeutics in the context of mutations that cause Duchenne muscular dystrophy. Using an established exon deletion strategy, we first showed rescue of the dystrophic phenotype in uniformly corrected monoclonal myobundle model compared to an uncorrected model. Following this proof-of-concept study, wedetermined whether AAV could be used to deliver the CRISPR therapeutic to myobundles at the time of tissue formation. AAV6 led to the highest transduction rates out of the serotypes tested, which was then used to deliver the CRISPR therapeutic. We achieved exon deletion in 13% of alleles, which resulted in 36% of transcripts being corrected, and 12% of wild-type dystrophin protein levels restored. Additional functional measurements were performed to evaluate the effect of AAV and gene editing on overall muscle heath and function. Finally, we tested two additional gene editing strategies in a myobundle model with a different DMD-causal mutation to demonstrate the broad utility of this technology. This study lays the groundwork for the use of these novel human microphysiological tissue models to address the safety and efficacy concerns that are at the forefront of the gene therapy field.Second, we developed a base editing strategy to correct Neurofibromatosis Type I (NF1)-causal mutations. NF1 is a tumor predisposition syndrome caused by mutations in the NF1 gene, leading to unchecked cellular growth. To test gene correction strategies to repair the gene, we first generated a mouse embryonic fibroblast (MEF) cell line that had a nonsense mutation in one of the NF1 alleles that could be corrected using adenine base editing. We then tested two different gRNAs and found that we were able to achieve modest correction (22%) of the nonsense mutation, which led to an increase in NF1 transcripts. Excitingly, neurofibromin (the protein encoded by the NF1 gene) was restored in edited cells and was proven functional based on a decrease in phosphorylated ERK. Edited cells also displayed a clear growth disadvantage compared to unedited cells, supporting the finding that functional neurofibromin was restored. This study represents the first application of gene editing for NF1 and findings from this work will not only inform future gene therapy and gene editing strategies for NF1, but alsoother tumor predisposition disorder and dominant genetic diseases that require correction of the causal mutation.Collectively, this work addresses two major limitations in the gene therapy and gene editing fields and establishes novel techniques and technologies that could be applied to a wide range of diseases.