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 that can be caused by gain-of-function mutations in superoxide dismutase 1 (SOD1). Despite its severity, there remains no cure for ALS and current therapies provide only modest benefit to patients, underscoring the critical need for new, effective strategies for the disorder. 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 single-base substitutions in DNA, but without the requirement for a mutagenic DNA break, thus overcoming a major safety hurdle facing the implementation of traditional CRISPR nuclease-based gene-editors. Through the support of the CREATE Bio Optimization Track, we have undertaken a milestone-driven research plan that has enabled us to refine not only the editing capabilities but also the specificity and tolerability of our SOD1-targeting CRISPR gene therapy. As a next step in this project, we will determine the properties of a new SOD1-targeting platform in a non- human primate, which, compared to most pre-clinical animal models, has greater anatomical, physiological, and immunological similarities with humans. However, a worsening shortage of NHPs, alongside increases in their demand for research, has resulted in unanticipated and hefty cost increases for an already limited domestic supply. To this end, this Administrative Supplement will contribute to: (1) the acquisition of three rhesus macaques, (2) their intrathecal dosing with an adeno-associated virus (AAV) vector formulation encoding a new, thoroughly optimized CRISPR-based gene therapy with increased safety, (3) their housing, and (4) the isolation of nervous system tissue and peripheral tissues. This Supplement will thus ensure that our study not only carries sufficient statistical power to most accurately quantify the effects for our therapeutic agent but that it also can be compared to a previously benchmarked version of our technology. As such, this Supplement is critical to the completion of the milestone goals for the project, “Optimization of an in vivo base editing strategy to treat SOD1-linked ALS” (5U01NS122102). In sum, by providing a means to increase the statistical power of our study to most accurately quantify the efficacy, tolerability, and immunological responses of a novel CRISPR gene therapy, this Administrative Supplement will ensure that our project exerts a maximum impact in the field of ALS therapeutics development and the field of gene therapy at large.