Correcting a Dravet syndrome-causing dominant negative mutation in GABRG2 with in vivo CRISPR gene editing

点击翻译按钮获取中文摘要

Dominant negative mutations are heterozygous mutations that produce gene products that are not just pathogenic, but also override the normal function of the healthy allele. Diseases caused by dominant negative mutations have historically been difficult to target with gene therapies due to the dominant effect of the pathogenic allele. Correcting dominant negative mutations by genome editing using a CRISPR/Cas9 strategy represents the ideal gene therapy approach and has been accomplished in mitotic cells. However, this approach has not been achieved in the CNS because of the relative inactivity of the homology directed DNA repair pathway in neurons. Recently, gene insertion in neurons has been achieved using a CRISPR/Cas9 paradigm called Homology Independent Targeted Integration (HITI), but this has not been applied to a dominant-negative disease of the CNS.In the gene GABRG2, which encodes the y2 subunit of the GABAA receptor, the dominant negative mutation Q390X in the final exon yields a truncated y2 subunit that reduces GABAA receptor membrane expression and causes Dravet syndrome. Dravet syndrome is an early-onset epileptic encephalopathy associated with intractable seizures, cognitive and developmental impairments, and high mortality. While most cases of Dravet syndrome are caused by mutations in SCNA1, around 20% of cases are caused by mutations in other genes, such as GABRG2, which is also associated with milder forms of childhood epilepsy. In a Gabrg2+/Q390X mouse model, the mutation has been shown to recapitulate severe epileptic and neurobehavioural comorbidities, as well as causing marked chronic neurodegeneration. Because Dravet syndrome is one of the most-drug resistant forms of epilepsy, there are currently poor clinical outcomes for patients and a need for new therapies. We aim to use HITI to correct Q390X in the Gabrg2+/Q390X mouse model. Because Q390X is in the last exon of the gene, we will insert a wild-type copy of the exon followed by a stop codon in front of the faulty exon in order to correct the mutation and allow normal functional expression of the GABAA y2 subunit. If successful, this will be the first demonstration that a dominant negative CNS disease can be treated with gene editing.