Development of a novel therapeutic strategy for treatment of SOD1-linked ALS by CRISPR/Cas9-mediated SOD1 promoter editing

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Amyotrophic lateral sclerosis (ALS) is a fatal disorder caused by the degeneration of motor neurons in the brain and spinal cord, usually resulting in death within five years after disease onset. Although Riluzole and Edaravone have been approved for the treatment of ALS, their therapeutic efficacies appear to be very limited, and more effective therapies are not currently available. Mutations of SOD1 have been linked to ~20% of familial and ~1% of sporadic ALS patients. Previous studies have demonstrated that mutant SOD1 causes motor neuron degeneration through a “gain-of-function” mechanism. Over the past two decades, a variety of therapeutic approaches to decrease SOD1 levels by small interfering RNA, short hairpin RNA, microRNA, antisense oligonucleotides or small molecules have been tested in SOD1-ALS mouse models, with a variety of therapeutic effects observed. Some of these approaches are now being tested in clinical trials. However, the efficacy of these candidate drugs in reducing SOD1 in the cerebrospinal fluid (CSF) appeared to be limited (~10%), and preliminary data from these clinical trials failed to show significant therapeutic benefits. For this reason, alternative and more effective strategies need to be explored and tested in preclinical studies. To target SOD1 more effectively, we designed a transgenic strategy to edit the human SOD1 coding sequence (exon2) using CRISPR-Cas9. We found that CRISPR-Cas9-mediated editing of SOD1-exon2 prevented the development of a clinical ALS phenotype and pathology in two SOD1-ALS mouse models. All of the SOD1-G93A transgene copies were effectively edited and inactivated. Although very effective, this coding sequence editing may have some safety concerns, as a complete loss of SOD1 may lead to progressive spastic tetraplegia and axial hypotonia. An optimal therapeutic strategy would be to effectively remove the majority of SOD1 protein to reach significant therapeutic efficacy, while maintaining a small fraction of the functional SOD1 to avoid adverse effects caused by its complete loss. To reach this goal, we designed a strategy to target/edit the TATA box, the major core promoter element of the human SOD1 (hSOD1) gene using CRISPR-Cas9. We tested this strategy in vitro using a cell culture system, and we found that the TATA box-edited alleles lost over 70% of hSOD1 protein. In this application, we propose three specific aims to test this TATA box editing strategy in two SOD1-ALS mouse models. In aim 1, we will develop the hSOD1-TATA-Cas9 single and hSOD1-TATA-Cas9/SOD1-G93A double transgenic mice. In aim 2, we will characterize the phenotype and pathology of the TATA box-edited SOD1-ALS mice. In aim 3, we will study the efficiency and safety profiles in the TATA box-edited SOD1-ALS mice and in human cells. Promoter editing represents a novel and more optimal therapeutic strategy for SOD1-ALS. If the concept is proven, a similar strategy may be adapted to a broad spectrum of diseases, as long as the core promoter elements of the relevant genes, such as TATA boxes, CAT boxes, GC boxes and other key regulatory elements in the promoters could be identified and appropriate PAMs are available.