Development of a CRISPR-Cas13 Gene Therapy for SOD1-Linked ALS - PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, paralytic disorder characterized by the selective loss of motor neurons in the spinal cord and brain. While most cases of ALS are sporadic, toxic gain-of-function mutations in superoxide dismutase 1 (SOD1) are responsible for ~20% of all inherited forms of the disease. Given its causative role in ALS, antisense oligonucleotides (ASOs) and RNA interference (RNAi) have been used to silence the expression of the mutant SOD1 protein. However, owing to their transient lifecycle, ASOs will require a lifetime of costly, invasive administrations, while RNAi is prone to inducing off-target effects. Conversely, while gene-editing technologies, such as CRISPR-Cas9, can be used be used to genetically inactivate mutant SOD1, the implementation of these strategies for gene therapy could prove challenging, as DNA editors can introduce off-target mutations and inadvertently create new, mutant SOD1 protein variants that can compromise their safety. Thus, there remains a crucial need for therapies that can safely and efficiently lower SOD1 for treatment of ALS. An alternative technology that holds little risk for inducing DNA damage within a cell but could still be used to efficiently lower SOD1 are RNA-targeting CRISPR-Cas13 effectors. CRISPR-Cas13 systems possess the programmability and versatility characteristic of DNA-editing CRISPR-Cas nucleases but pose limited risk for inducing genotoxicity since they are unable to cleave DNA. Moreover, Cas13 proteins display favorable specificity compared to gene silencing technologies and many are compact enough to fit within a single adeno- associated virus (AAV) vector, a clinically promising gene delivery vehicle that can mediate long-term, cell-type specific gene expression in the nervous system. Thus, CRISPR-Cas13 has the potential to safely and persistently silence mutant SOD1 following just a single administration of an engineered viral vector. However, it remains unknown whether Cas13 can be harnessed to reduce SOD1 in vivo and treat the disease. The overarching objective of this proposal is to develop a gene therapy for ALS. Specifically, we propose to harness CRISPR-Cas13d technology to lower mutant SOD1 in vivo for treatment of SOD1-linked ALS. In support of the feasibility of this objective, our preliminary studies have demonstrated that Cas13 proteins are more active and specific than a preclinically promising shRNA, that they can be delivered at high efficiencies by AAV9 to spinal cord astrocytes, that they can efficiently lower mutant SOD1 protein throughout the spinal cord, and that they can provide therapeutic benefit. We now aim to optimize the performance of this platform (Specific Aim 1) for the goal of testing its efficacy in mouse models of ALS (Specific Aim 2) and to determine its safety as a gene therapy agent (Specific Aim 3). Thus, by harnessing an innovative technology for transcriptional engineering that can overcome the limitations of traditional gene-silencing, we will develop a new therapy for ALS, a debilitating and currently incurable disorder with few effective treatment options.
