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

通过体内 CRISPR 基因编辑纠正导致 Dravet 综合征的 GABRG2 显性失活突变

• 批准号: 2076908
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2076908
• 关键词: Correcting; Dravet; syndrome; causing; dominant

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.

显性失活突变是杂合突变，产生的基因产物不仅具有致病性，而且还覆盖健康等位基因的正常功能。由于致病等位基因的显性作用，由显性失活突变引起的疾病历来很难用基因疗法来靶向。使用 CRISPR/Cas9 策略通过基因组编辑来纠正显性失活突变是理想的基因治疗方法，并且已在有丝分裂细胞中实现。然而，由于神经元中同源定向 DNA 修复途径相对不活跃，这种方法尚未在 CNS 中实现。最近，使用称为同源独立靶向整合（HITI）的CRISPR/Cas9范例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失活疾病。在编码GABAA受体的y2亚基的基因GABRG2中，最终外显子中的显性失活突变Q390X产生截短的y2亚基， 降低 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，但大约 20% 的病例是由其他基因突变引起的，例如 GABRG2，它也与较轻微的儿童癫痫有关。在 Gabrg2+/Q390X 小鼠模型中，该突变已被证明可重现严重的癫痫和神经行为合并症，并导致明显的慢性神经退行性变。由于 Dravet 综合征是耐药性最强的癫痫形式之一，目前患者的临床结果不佳，需要新的治疗方法。我们的目标是使用 HITI 来校正 Gabrg2+/Q390X 鼠标模型中的 Q390X。由于 Q390X 位于该基因的最后一个外显子中，因此我们将在有缺陷的外显子前面插入该外显子的野生型副本，并在其后插入终止密码子，以纠正突变并允许 GABAA y2 亚基正常功能表达。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。