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

显性负突变是杂合突变，它产生的基因产物不仅具有致病性，而且还覆盖了健康等位基因的正常功能。由于致病等位基因的显性作用，由显性负突变引起的疾病在历史上很难用基因治疗来靶向。使用CRISPR/Cas9策略通过基因组编辑纠正显性负突变代表了理想的基因治疗方法，并已在有丝分裂细胞中完成。然而，由于神经元中同源定向DNA修复途径的相对不活跃，这种方法尚未在中枢神经系统中实现。最近，使用一种称为同源性独立靶向整合（HITI）的CRISPR/Cas9范式实现了神经元中的基因插入，但这尚未应用于中枢神经系统的显性阴性疾病。在编码GABAA受体y2亚基的GABRG2基因中，最后外显子的显性负突变Q390X产生截断的y2亚基，从而减少GABAA受体膜表达并导致Dravet综合征。Dravet综合征是一种早发性癫痫性脑病，伴有顽固性癫痫发作、认知和发育障碍以及高死亡率。虽然大多数Dravet综合征病例是由SCNA1突变引起的，但约20%的病例是由GABRG2等其他基因突变引起的，GABRG2也与较轻形式的儿童癫痫有关。在Gabrg2+/Q390X小鼠模型中，该突变已被证明可再现严重的癫痫和神经行为合并症，并引起显著的慢性神经退行性变。由于Dravet综合征是最耐药的癫痫形式之一，目前患者的临床结果很差，需要新的治疗方法。我们的目标是在Gabrg2+/Q390X小鼠模型中使用HITI来纠正Q390X。由于Q390X位于基因的最后一个外显子，我们将在错误的外显子前插入一个野生型外显子，然后插入一个停止密码子，以纠正突变并允许GABAA y2亚基的正常功能表达。如果成功，这将首次证明显性阴性中枢神经系统疾病可以通过基因编辑来治疗。

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.