SCGE Disease Models Studies Supplement: Allele-specific Genome Editing in Two Mouse Models of Charcot-Marie-Tooth Disease

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ABSTRACT We propose to use somatic cell genome editing by CRISPR/Cas9 approaches in vivo to treat two mouse models of inherited peripheral neuropathy (Charcot-Marie-Tooth disease, CMT) in a collaboration between the Gladstone Institutes (Conklin, U01-ES032673) and The Jackson Laboratory (JAX, Murray and Lutz, U42- OD026635). CMT is a collection of incurable crippling peripheral neuropathies with overall prevalence of 1:2500 people. Certain CMT mutations result in dominant-negative alleles that could be corrected by allele- specific silencing with CRISPR/Cas9. CMT type 2E (CMT2E) is caused by dominant mutations in the neurofilament light chain gene (NEFL). We have used allele-specific genome editing to silence the severe NEFLN98S allele in patient-derived iPSCs, reversing the disease phenotype. In Aim 1 we will extend our in vitro studies to an existing NeflN98S mouse model of CMT2E. Similarly, CMT type 2D (CMT2D) is caused by dominant negative mutations in glycyl tRNA-synthetase (GARS). We have shown that AAV9 delivery of allele- specific RNAi targeting the mutant Gars mRNA was able to effectively prevent disease in two mouse models of CMT2D including the GarsDETAQ allele. In Aim 2, we will test in vivo allele-specific genome editing in mice targeting the GarsDETAQ allele. Together, these studies will establish the levels of genome editing that improve the neuropathy phenotypes by measuring the editing efficiency, testing clinically relevant phenotypic outcomes, and profiling toxicological and off-target effects. Vector design and packaging, as well as evaluation of editing efficiency will be done at Gladstone. In vivo preclinical studies will be done at JAX.