Directing Splicing of SCN8A to Treat Dravet Syndrome

 描述(由申请人提供)：由SCN1A功能丧失突变引起的Drave谱疾病包括发热性癫痫、全身性癫痫伴发热性发作加 (GEFS+)和Dravet综合征(婴儿期严重肌阵挛癫痫或SMEI)，按严重程度排列。在最严重的病例中，进行性发育和行为障碍伴随着反复和多样的癫痫发作，到2岁时进展到包括多种癫痫类型。在许多患者中，癫痫发作对目前可用的抗癫痫药物具有抵抗力。因此，迫切需要开发新的治疗方法。抑制中的SCN1通道功能与相关的SCN8通道功能相反。因此，为了重新平衡由SCN1A功能缺失突变引起的抑制输入的缺陷，一个有效和合理的策略是专门减少SCN8介导的兴奋。VGS通道α亚基经历几种选择性剪接事件，调节中枢神经系统钠电流的抑制性和兴奋性平衡。SCN8A亚基自然地交替地在控制通道功能或动力学的两个特定位置上剪接。一种新的策略来减少SCN8A介导的兴奋和与SCN1A功能丧失突变相关的癫痫发作作为治疗DS的方法是通过开发称为剪接调节寡核苷酸(Smos)的化合物来在每个位置直接剪接。SMO是一类基于合成RNA的化合物，可以立体地阻断或减弱剪接机械元件和前mRNA之间的相互作用。SMO是理想但不发达的候选药物，因为它们以非凡的效力、特异性和可以忽略的脱靶效应结合到他们的靶标上。针对SCN8A剪接的SMO将以硅胶设计，并在体内针对效力和特异性进行精炼(目标1)，从而为每个剪接位置提供SMO候选药物。每个SMO候选者将在P10-42天在正常小鼠中以剂量反应为特征，并在正常小鼠中评估SMO对氟乙基诱发癫痫阈值的剂量效应(目标2)。最后，将评估SMOS在SNN1AR1648H突变小鼠(AIM 3)中的治疗作用，以观察其对长寿、运动功能(共济失调、震颤)和氟乙基诱发癫痫敏感性的影响。SMO治疗的安全性和有效性将进一步评估对成年杂合SCN1AR1648H小鼠行为的积极和不利影响，这些小鼠的寿命正常。与使用钠通道阻滞剂对抗多个VGS通道相比，仅减少抵消SCN1A输入的Na+通道(SCN8A)的策略应该更有效，且不太可能引起不良影响。最终目标是开发这些SMO作为潜在的治疗方法，用于治疗对目前可用的药物治疗无效的患者的Drave氏综合症和相关疾病。

 DESCRIPTION (provided by applicant): Dravet Spectrum disorders resulting from SCN1A loss-of-function mutations include febrile seizures, generalized epilepsy with febrile seizure plus (GEFS+), and Dravet syndrome (severe myoclonic epilepsy of infancy or SMEI), in order of increasing severity. In the most severe cases, progressive developmental and behavioral impairments manifest along with the recurrent and varied seizure episodes that advance to include multiple seizure types by age 2. In many patients, seizures are resistant to currently available antiepileptic drugs. Thus, there is a significant and urgent need for the development of novel approaches to therapy. SCN1 channel function in inhibition is functionally opposed by the related SCN8 channel. Thus, a validated and logical strategy for rebalancing the deficit of inhibitory input caused by SCN1A loss-of-function mutation is to specifically reduce SCN8-mediated excitation. VGS channel alpha subunits undergo several alternative splicing events which regulate the inhibitory and excitatory balance of sodium currents in the CNS. SCN8A subunits are naturally alternatively spliced at two specific sites of interest which control channe function or kinetics. A novel strategy to reduce SCN8A- mediated excitation and seizures associated with SCN1A loss-of-function mutations as a treatment for DS is to direct splicing at each of these sites by developing compounds called splice modulating oligonucleotides (SMOs). SMOs are a class of synthetic RNA based compounds that sterically block or weaken interactions between elements of the splice machinery and the pre-mRNA. SMOs are ideal but under-developed drug candidates as they bind to their targets with exceptional potency, specificity, and negligible off-target effects. SMOs targeting SCN8A splicing will designed in silico and refined for potency and specificity in vivo (Aim 1) leading to an SMO drug candidate for each splice site. Each SMO candidate will be characterized by dose-response in normal mice at P10-42 days, and SMO dose-effect on flurothyl-induced seizure threshold evaluated in normal mice (Aim 2). Finally, SMOs will be evaluated as therapeutics in SNN1A R1648H mutant mice (Aim 3), for effects on longevity, motor function (ataxia, tremor) and flurothyl-induced seizure susceptibility. Safety and efficacy of SMO treatment will further be assessed for both positive and adverse effects on behavior in adult heterozygous SCN1A R1648H mice, which have a normal lifespan. The strategy of specifically reducing only the Na+ channel (SCN8A) that counterbalances SCN1A input should be more efficacious and be much less likely to cause unwanted effects than using sodium channel blockers which antagonize multiple VGS channels. The ultimate goal is to develop these SMOs as potential therapeutic for the treatment of Dravet Syndrome and related disorders in patients resistant to currently available pharmacotherapies.