Optimization of an in vivo base editing strategy to treat SOD1-linked ALS

PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, paralytic and ultimately fatal disease characterized by the selective loss of motor neurons in the spinal cord and brain. The overarching objective of this application to the Optimization Track of the CREATE Bio Program is to refine the safety and efficacy of a gene therapy that we developed for forms of ALS caused by toxic, gain-of-function mutations in superoxide dismutase 1 (SOD1), which account for up to 20% of all familial cases of the disease. Specifically, we have developed an approach to inactivate the production of the mutant SOD1 protein in vivo using CRISPR base editing, a gene- editing modality capable of introducing precise base substitutions in DNA, but without the requirement for a mutagenic DNA break, thereby overcoming a major safety hurdle facing the implementation of traditional gene- editing nucleases. In particular, when delivered to the spinal cord via adeno-associated virus, our SOD1- targeting base-editing platform prolonged survival and markedly slowed the progression of disease in a highly aggressive mouse model of SOD1-linked ALS. Importantly, as opposed to current strategies for silencing SOD1, which target SOD1 mRNA and can have a transient effect that requires a lifetime of redosing or can risk saturating endogenous RNA processing pathways, which could then lead to adverse effects, our approach harnesses a highly precise DNA editing pathway to permanently turn-off the production of mutant SOD1 and involves only a single treatment. Thus, because of its strengths and in vivo efficacy, we now aim to refine this strategy for the ultimate goal of developing a gene therapy for ALS. Specifically, by optimizing its targeting specificity, its editing capabilities, its pharmacokinetics and its safety, which we will ensure via the introduction of a self-inactivating functionality that facilitates its clearance from cells, we will develop a highly optimized SOD1-targeting CRISPR base editing platform that can be used to permanently and effectively treat SOD1-linked ALS. Thus, by capitalizing on: (1) a highly innovative DNA editing technology that has the capabilities to overcome the limitations of gene-silencing and (2) a multidisciplinary research team with complementary expertise in ALS, AAV delivery, gene-editing and immunology, this project will result in not only an optimized therapeutic candidate for ALS, a devastating, debilitating and currently incurable disorder with few effective treatment options, but also lay the foundation for using base editing to safely treat neurological disorders.