Authors :
Presenting Author: Wenxi Yu, PhD – University of Michigan
Sophie Hill, PhD – University of Michigan; Yumei Huang, PhD – Greater Bay Area Institute of Precision Medicine; Julie Ziobro, MD, PhD – University of Michigan; Faith Reger, BS – University of Michigan; Xiaoyan Jia, PhD – Greater Bay Area Institute of Precision Medicine; Miriam Meisler, PhD – University of Michigan
Rationale:
DEEs (Developmental and Epileptic Encephalopathies) are rare genetic disorders characterized by refractory seizures, developmental delay, and impaired movement. De novo mutations of SCN8A encoding the voltage-gated sodium channel Nav1.6 have been identified in >800 cases of DEE. Affected individuals are heterozygous for wildtype and mutant alleles. Mutant alleles exhibit gain-of-function changes such as premature channel opening and impaired channel inactivation. Long-term reduction of Scn8a mRNA using ASO or shRNA protects against seizures in mouse models of DEE (Lenk et al Ann Neurol 2020, and submitted). These ASO and shRNA treatments reduce both wildtype and mutant transcripts. Specific reduction of only the pathogenic transcript would be preferable, in order to retain at least 50% of wildtype mRNA abundance and to minimize potential adverse effects.
Methods:
We targeted the mutant allele in the D/+ mouse line carrying the patient Scn8a mutation p.Asn1768Asp (N1768D). In addition to N1768D, this mutant allele contains 8 nearby silent base substitutions (Jones and Meisler, Genesis 2014). Experimental D/+,Cas9+ mice carried the ubiquitously expressed Cas9 gene from JAX line 026179. The allele-specific sgRNAs matched the mutant allele at 20/20 nucleotides but had 3/20 mismatches to the wildtype allele.
Results:
Four allele-specific sgRNAs were tested in an in vitro Cas9 digestion assay and in embryonic fibroblasts from D/+ mice. The most effective sgRNA was cloned into an AAV9 vector and administered to D/+,Cas9+ mice on postnatal day 1. Mice received 2 µL of virus (1x1013 vg/mL) by intracerebroventricular injection. One to three months later, Scn8a cDNA was amplified from brain and subjected to next-gen sequencing. Out-of-frame indels were detected in approximately 25% of mutant transcripts but were not present in wildtype transcripts. Untreated D/+ animals develop convulsive seizures at two to three months of age and survive for three to five months. The sgRNA treated D/+,Cas9+ mice did not develop convulsive seizures and have survived beyond eight months of age (n=12, p< 0.01). The sgRNA treated mice also exhibited normal performance in open field tests and normal EEGs.
Conclusions: Allele-specific CRISPR-Cas9 inactivation of 25% of mutant
Scn8a alleles is attainable in brain of heterozygous mutant mice and is sufficient to rescue seizures and survival in a mouse model of
SCN8A-DEE.
Funding:
Supported by NS R01 NS34509