摘要:Dravet syndrome是小兒嚴重型嬰兒肌躍震顫癲癇(Severe Myoclonic Epilepsy in Infancy, SMEI),病患通常在出生一歲間發生熱痙攣(Febrile Seizure),此後病徵隨年齡加劇而多樣化,且致死率達20%。目前治療Dravet syndrome藥物大多為提升下游的effector, GABA (gamma-aminobutyric acid),的釋放,但因為是廣泛且非特異性的刺激GABA神經元的釋放,除了藥物無法達到有效濃度外,常會造成至今原因未明的抗藥性而降低治療效果導致預後極差。因此針對Dravet的神經元的治療才能解決問題。文獻顯示大部份Dravet病人具有Sodium channel protein type I subunit alpha (SCN1A)基因突變造成第一型鈉離子通道(Nav1.1 channel)異常且大部份的是原生性的(de novo)異型合子突變 (heterozygous mutation)。包括在台灣也有相關病例,且有多達百種以上的不同突變型。據統計有70%病人因SCN1A片段刪除(deletion)或stop-codon mutation造成SCN1A蛋白質截斷(protein truncation)或降解。由於目前治療Dravet syndrome藥物皆非針對表現SCN1A蛋白質的神經元,因此本計畫的目標在尋找針對表現SCN1A的神經元可提升其SCN1A的量及其功能的藥物,期望達到可單獨使用或合併使用於治療Dravet syndrome。本計畫的獨特性是能利用本團隊最近成功產製Dravet小鼠模型(Scn1aE1130X knock-in mice,造成E1130為TAA終止碼)作為藥物篩選平台,此小鼠具有自發性的癲癇及較高的致死率,非常近似人類的Dravet syndrome。另外,有別於目前的治療Dravet藥物,本計畫特別針對可調控SCN1A表現的藥物為測試療效的方向,盡可能不影響其他基因表現或是神經傳導物質分泌,進而降低影響其他神經網路及減少副作用。 本計畫選擇的藥物其標的理論基礎有四個方向:第一為可克服stop-codon mutation並產生全長具功能性蛋白質產物的藥物, PTC124。PTC124在Duchenne muscular dystrophy (DMD)及cystic fibrosis的臨床試驗顯示無顯著的副作用且有良好的安全性和耐受性,PTC124目前用於cystic fibrosis疾病治療上已進入第三期臨床試驗,因此極具潛力。第二為調高病人另一半正常染色體的SCN1A表現。由於Dravet病人皆為heterozygous,文獻顯示許多病人不會造成dominant-negative [1],因此可藉由NRPB核心設施<藥物化學樣品庫與超高速藥物篩選>的資源,篩選出這類具有治療潛力的藥物。第三為搜尋可克服其它2種stop-codon mutation的藥物。PTC124最佳的結合碼為TGA,對TAG及TAA的結合力較低,差距3-6倍,因此本計畫是希望藉由NRPB核心設施<藥物化學樣品庫與超高速藥物篩選>的資源,尋找類似PTC124但有更強結合力的藥物。第四為尋找修飾基因與藥物。Dravet症候群病人具有極大的異質性,即便是相似位置的大段基因缺失,症狀皆可能因為各別基因特異性而嚴重度不同,繼而藥物反應不同而使治療無效。本計畫目前由觀察Dravet小鼠得到類似於病人的現象,即使是相同的突變及類似的基因背景,在7週齡前有50%的致死率,但另外50%可存活>24週齡,因此本計畫的Dravet小鼠非常適合找出SCN1A修飾基因並確定其與SCN1A及癲癇的關係為加重病情或是保護性,並能幫助搜尋可調控修飾基因表現的藥物,進而與目前的藥物或是PTC124等合併使用,藉此觀察其療效。因此本計畫逐年要完成的目標如下:1.測試可克服提早形成終止密碼子(premature-stop-codon)的藥物, PTC124,之治療潛力(第一年)。2.測試其他新型藥物治療Dravet syndrome之潛力(第二年)。3.找出Dravet syndrome的修飾基因並驗證其與此病之相關性(第二、三年)。4.測試PTC124或其他抗癲癇藥物與可調控修飾基因表現量的新型藥物合併使用之治療潛力(第二、三年)。
Abstract: Dravet syndrome, also known as Severe Myoclonic Epilepsy In Infancy (SMEI), is a rare disease. The onset of the disease occurs within the first year after birth typically exhibiting febrile seizure. The severity of the disease gradually reduces throughout the lives of patients with a mortality of approximately 20%. Most medications for treating Dravet patients aim at promoting the release of the downstream effector, gamma-aminobutyric acid (GABA). Due to nonspecific stimulation, limited local concentration of the drugs, and other, as yet unknown reasons, patients become refractory to current antiepileptic medications and suffer from poor treatment outcome. We reason that targeting defective neurons is a plausible new strategy for the treatment of Dravet syndrome. Studies indicate that most Dravet patients harbor mutations in the sodium channel protein type I subunit alpha (SCN1A) gene, which encodes Nav1.1 channels. The mutations are de novo and heterozygous. More than one hundred mutations have been identified worldwide, including cases in Taiwan. Among these, approximately 70% patients carry SCN1A deletions and nonsense mutations that lead to truncated or diminished protein products. Most conventional antiepileptic drugs (AEDs) do not target the SCN1A protein product. The goal of this proposal is, thus, to search for novel therapeutic compounds that specifically increase protein levels and function of SCN1A. These drugs can be used alone or as add-ons to treat Dravet syndrome. This proposal takes advantage of our recent success in creating Dravet syndrome mice (Scn1aE1130X knock-in mice, carrying TAA nonsense codon at E1130) that will allow us to build up a drug testing platform. The mice demonstrate spontaneous seizure and lethality phenotypes that mimic Dravet patients. Because we aim to increase SCN1A protein levels in mice that express the gene, we might also decrease or eliminate the side-effects seen with current drugs. We propose 4 different rationales and approaches to reach our goal. First, we will focus on candidate drugs that can read-through the stop-codon-mutations and produce a full-length SCN1A functional protein. One available drug we can test is PTC124. PTC124 has been shown to be successful in the treatment of Duchenne muscular dystrophy (DMD) and cystic fibrosis. PTC124 is currently entering phase III clinical trials for cystic fibrosis. Secondly, we will look for candidate drugs that can upregulate wild-type (WT) SCN1A expression. Dravet syndrome patients are heterozygous and the literature suggests that most of their SCN1A mutations are not dominant-negative [1]. We propose to screen the National Research Program for Biopharmaceuticals (NRPB) core for candidate drugs that can increase SCN1A expression. Thirdly, we will look for candidate drugs that can read-through the other stop-codon mutations. PTC124 binds preferentially to TGA over TAG or TAA with 3-6 times greater affinity. We will screen the NRPB core for candidate drugs that can read-through TAG and TAA. Fourth, we will search for modifiers of SCN1A and their related drugs. Because of the great heterogeneity in the Dravet syndrome patients, the severity of the disease is highly dependent on the genetic backgrounds of the patients. According to the preliminary data derived from our Dravet model mice, the death rate of the heterozygous mice with the same mutation is 50% at 7 weeks of age; the other 50% of mice survive > 24 weeks. Our Dravet mice are thus suitable to search for modifier genes that affect survival and also to study treatment variability. Our specific aims for each fiscal year are as follows: Specific Aim 1: To test the therapeutic potential of premature-stop-codon read-through compound, PTC124, in the treatment of Dravet syndrome. (first year)Specific Aim 2: To test the therapeutic potential of other novel compounds in the treatment of Dravet syndrome. (second year)Specific Aim 3: To identify the loci of genetic modifiers of Dravet syndrome and verify their associations. (second to third year)Specific Aim 4: To test the therapeutic effects of PTC124 or anti-epileptic drugs (AEDs) in combination with novel compounds that can regulate the expression of the modifier genes. (second-third year)