Therapeutic Genome Editing for Amyotrophic Lateral Sclerosis with a Compact, Hyper-accurate, and Versatile Cas9

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Project Summary Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease in which the average survival rate from onset is 2-4 years, and only 10% of individuals live past 10 years. There is currently no cure for ALS, and available medications do not significantly prolong survival. Certain forms of ALS arise from gene abnormalities, such as a dominant, gain-of-function point mutation in superoxide dismutase 1 (SOD1G93A) and an intronic repeat expansion in C9ORF72 (chromosome 9 open reading frame 72). These mutations ultimately lead to neuronal death, decreased muscle mass, and loss of motor control. Gene editing approaches that eliminate ALS-causing mutations may prevent disease progression and improve the quality of life of some patients. Therapeutic gene editing has become a possible reality with the advent of CRISPR-Cas9 editing, which uses guide RNA (gRNA) and a bacterial Cas9 nuclease to recognize a specific DNA sequence, create a double- stranded break, and inactivate a target gene. However, currently-available Cas9 proteins are too large for efficient in vivo delivery and often induce unwanted gene editing at off-target sites. The goal of this project is to develop efficient and safe Cas9-based gene editing approaches to treat genetic cases of ALS. These approaches will use a recently-characterized Cas9 from a strain of Neisseria meningitidis (Nme2Cas9). Nme2Cas9 is compact, so it can be packaged with gRNA into a single vector (adeno-associated virus, AAV) for in vivo delivery. It is also hyper-accurate, with negligible off-target editing. Most importantly, Nme2Cas9 targeting relies on a unique DNA binding signal with a flexible sequence motif that provides a larger selection of potential target sites in the genome than what is available for other Cas9s. Using the SOD1G93A mouse model of ALS, Aim 1 will determine whether Nme2Cas9 can specifically target the mutant allele of the SOD1G93A gene while leaving the wild-type allele unscathed. To test this, Nme2Cas9 and a gRNA targeting SOD1G93A will be packaged into an all-in-one AAV9 vector, which targets the central nervous system (CNS), and injected via facial vein into SOD1G93A mice. This approach should knockout mutant SOD1 and reduce ALS phenotypes without inducing potential side effects associated with loss of normal SOD1 function. Aim 2 will use C9BAC transgenic mice expressing C9ORF72 expanded repeats to determine whether Nme2Cas9 can excise the intronic expansion in C9ORF72 without breaching the boundaries of flanking exons. An all-in-one AAV9 vector containing Nme2Cas9 and two gRNAs targeting each end of the repeat will be delivered by facial vein injection into C9BAC mice. Nme2Cas9-mediated excision of C9ORF72 repeats should ameliorate pathogenic biomarkers of ALS without altering normal C9ORF72 expression. Completion of the proposed aims will improve the potential of Cas9-based gene editing approaches for ALS treatment, and will guide the future development of therapies for genetically-defined neurological disorders.