Advanced Base and Prime Editing Strategies to Correct Common ALS-causing SOD1 Mutations

Amyotrophic lateral sclerosis (ALS) is a devastating degenerative motor neuron disease that is largely untreatable and leads to death within 5 years of diagnosis. ~10% of ALS cases are familial and caused by mutations in various ALS genes. Ultimately, the ideal treatment for genetic diseases such as ALS is somatic gene correction. Recently, advances in CRISPR/Cas systems have shown considerable promise for precise editing of disease loci using base and prime editing systems delivered by AAV. The second-most prevalent cause of familial ALS are mutations in the SOD1 gene. These mutations confer multiple toxic properties onto the protein. This project proposes to develop treatment to achieve somatic gene correction for common missense mutations in SOD1. The aims of this proposal are: (1) To develop AAV-mediated base editing gene correction strategies for the SOD1 A5V mutation in vitro. We will create next-generation base editors with a compact size, increased efficiency, and greater control over bystander editing. (2) To develop AAV-mediated prime editing gene correction strategies for the SOD1 A5V and G94A mutations in vitro. Different prime editor systems will be tested for optimal editing efficiencies and low off-target editing. (3) In in vivo studies, examine and optimize AAV- mediated base and prime editing gene correction strategies for the A5V and G94A mutations in A5V and SOD1G93A mouse models. Mice will receive AAV-mediated base and prime editors through an intracerebroventricular injection. Base and prime editor strategies will first be screened in mutation carrying HEK293T cells and then optimized in patient fibroblasts and mouse models. The effects of gene correction on gain- and loss-of-function molecular and motor phenotypes will next be evaluated. The fundamental hypothesis driving this proposal is that AAV-mediated somatic gene correction strategies, using base editing or prime editing to target the SOD1 mutations A5V and G94A, will decrease toxic GOF pathology and increase WT SOD1 protein levels in vivo, resulting in a balanced treatment for SOD1-ALS and a rescue of motor phenotype. In addition, with mentorship from experts in ALS and gene editing and the wealth of resources available at UMASS Chan, these studies will provide extensive training in gene editing for CNS diseases and project development that will be an essential foundation for a future career as an independent researcher developing gene therapies for a range of genetic CNS diseases.