CN112955169A - 用于肌萎缩性侧索硬化疗法中的胆固醇24-水解酶的表达载体 - Google Patents
用于肌萎缩性侧索硬化疗法中的胆固醇24-水解酶的表达载体 Download PDFInfo
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Abstract
本发明涉及用于治疗肌萎缩性侧索硬化的载体,所述肌萎缩性侧索硬化与额颞痴呆和相关运动神经元紊乱有关联或无关联,所述载体包含编码胆固醇24‑羟化酶的核酸。
Description
技术领域
本发明涉及肌萎缩性侧索硬化(amyotrophic lateral sclerosis)的治疗。
背景技术
肌萎缩性侧索硬化(ALS)是属于运动神经元紊乱组的罕见神经疾病,所述运动神经元紊乱主要涉及负责控制自主肌肉运动(如咀嚼、行走和说话)的神经元(Zarei S等,2015)。ALS的特征在于运动神经元逐渐退化和死亡,从脑延伸至脊髓,并延伸至整个身体的肌肉。ALS涉及上运动神经元(来自脑中的运动神经元的信息传递至脊髓)和下运动神经元(脑中的运动核)二者,所述神经元退化并因此停止向肌肉发送信息,肌肉逐渐变弱、开始抽搐并变得萎缩(Rowland LP等,2001)。
患有ALS的大多数人在首次症状出现后3年至5年内死亡,通常是由于呼吸衰竭。症状发作后,仅10%的患有ALS的人生存超过10年。
在ALS中,数个潜在的风险因素已被关联:年龄,症状通常出现在55岁至75岁之间;性别,ALS在男性中稍微更普遍;以及种族和民族,高加索人和非西班牙裔最有可能发展为ALS。
但是,绝大多数ALS病例(90%)是散发性的。相对地,约10%的ALS病例是家族性的(FALS)。对于这些家族性病例,涉及了数个基因:C9ORF72、SOD1、FUS、TARDBP和更近期的SMCR8(Greenway等2006;Kabashi等,2008;Kaur等,2016;Turner MR等,2017;Ticozzi等,2011;Valdmanis PN等,2008)。
关于ALS的病理生理学,在考虑之中的有数个方面,主要理论中的一种与RNA加工有关,特别是基于以下的发现:在多数ALS病例中包含TDP-43以及诸如TARDBP和FUS的基因作为ALS的遗传原因。这两个基因均与前体mRNA(pre-mRNA)剪接、RNA转运和翻译有关。包含C9orf72重复序列的前体mRNA可隔绝核RNA结合蛋白,从而使其无法正确剪接其它mRNA。ALS的另一个公认的方面是蛋白质聚集,包括SOD1、TDP43和FUS,这可以在ALS患者和动物模型二者中观测到。已提出,聚集体干扰正常的蛋白质稳态并诱导细胞应激。此外,蛋白质聚集体可隔绝正常细胞功能必需的其它蛋白质或RNA。
到目前为止,尚无已知的ALS治愈方法。仅有两种药物经FDA批准用于ALS:利鲁唑和依达拉奉。据信利鲁唑通过降低谷氨酸水平起作用来减少对运动神经元的损害。利鲁唑将生存期延长了数个月,但并没有逆转神经元中已经存在的损害(Zoccolella等,2007)。关于依达拉奉,仅证实其使日常损害的临床评估下降(Brooks等,2018)。因此,存在开发用于ALS疗法的新策略的迫切需要。
发明内容
本发明人现在提出通过调节胆固醇代谢途径来对抗ALS,更具体而言,通过包含编码胆固醇24-羟化酶的核酸的载体的方式,所述载体在靶细胞中表达胆固醇24-羟化酶。
因此,本发明的目的是提供用于治疗肌萎缩性侧索硬化的载体,所述载体包含编码胆固醇24-羟化酶的核酸。
在实施方式中,所述载体包含编码氨基酸序列SEQ ID NO:2的核酸序列。或者,所述载体包含核酸序列SEQ ID NO:1。
在实施方式中,所述载体选自于由腺病毒载体、慢病毒载体、逆转录病毒载体、疱疹病毒载体和腺相关病毒(AAV)载体所组成的组,优选AAV载体,更优选AAV9、AAV10(AAVrh.10)或AAVPHP.eB载体,甚至更优选AAVPHP.eB。
在实施方式中,将所述载体直接给予至患者的脑和/或脊髓中,优选给予至脊髓和/或运动皮层。
本发明的另一目的是提供用于治疗肌萎缩性侧索硬化的药物组合物,所述药物组合物包含含有编码胆固醇24-羟化酶的核酸的载体。
附图说明
图1-WT和SOD1G93A动物的腰脊髓中在第8周时参与胆固醇代谢的基因的mRNA水平和WT和SOD1G93A动物的脊髓中在第15周时24-OH胆固醇的水平。(A)从8周龄的WT和SOD1G93A小鼠的腰脊髓中提取mRNA。将mRNA水平归一化至肌动蛋白看家基因。数据表示为平均值±SEM(每组n=4-5)。(B)通过UPLC-HRMS评估的SOD1G93A小鼠中的腰脊髓24S-羟基胆固醇含量。数据表示为平均值±SEM(每组n=7-9)。统计学分析:学生t检验。*p<0.05,**p<0.01。
图2-在WT动物中在8周静脉内递送AAVPHP.eB-CYP46A1-HA后评价CYP46A1-HA的表达以及在低剂量、中剂量或高剂量注射后3周进行的分析。脊髓的颈、胸和腰切片上的HA染色。
图3-在WT动物中在8周静脉内递送AAVPHP.eB-CYP46A1-HA后评价炎症以及在低剂量、中剂量或高剂量注射后3周进行的分析。脊髓的腰切片上的GFAP染色。NI:未注射
图4-在预防性阶段(3周)静脉内给予AAVPHP.eB-CYP46A1后SOD1小鼠的行为评价。(A)重量跟踪,(B)抱握测试,和(C)倒置测试。结果表示为平均值±SEM。*p<0.05,**p<0.01,***p<0.001,****p<0.0001。黑色*对应于相对于WT动物的p值,灰色为相对于SOD1动物。
图5-在SOD1动物中进行预防性处理后AAVPHP.eB-CYP46A1的生物分布,以及在预防性治疗后在15周龄时通过24OH胆固醇水平定量进行的靶标参与评价。(A)中枢神经系统和(B)周围器官中的生物分布、以及(C)通过UPLC-HRMS评估的SOD1G93A小鼠中的腰脊髓24S-羟基胆固醇含量。结果表示为平均值±SEM。
图6-静脉内递送AAVPHP.eB-CYP46A1作为预防性处理后,15周龄时的CYP46A1-HA表达和脊髓的组织学分析评价。(A)经AAV处理的SOD1动物的脊髓的颈、胸和腰切片上的HA染色;(B)在WT、SOD1和SOD1AAV动物的脊髓的颈、胸和腰切片上针对HA和VachT的共染色;(C)对运动神经元数的定量;(D)脊髓的腰切片的Luxol染色,以及(E)髓鞘质百分比的评价。结果表示为平均值±SEM。*p<0.05,**p<0.01。黑色*对应于相对WT动物的p值,灰色为相对于SOD1动物。
图7–预防性处理后,经AAV处理的SOD1动物的肌肉表型改善。(A)氧化苏木精伊红着色以分析肌肉中的纤维大小。表示胫骨肌(B)、腓肠肌(D)和四头肌(F)的平均纤维面积,并根据其在胫骨肌(C)、腓肠肌(E)和四头肌(G)中的横截面大小再分隔纤维百分比。结果表示为平均值±SEM。*p<0.05,**p<0.01。
图8-预防性处理后,经AAV处理的SOD1动物中神经肌肉接头的保留。(A)在静脉内递送AAVPHP.eB-CYP46A1后,对15周龄的WT、SOD1和SOD1经处理的动物中的神经丝(neurofilament,NF)和银环蛇毒素(BTX)共染色。(B-C)根据NMJ在15周龄时的神经支配状态和完整性对其的评分。(D)使用pan NF、银环蛇毒素和VachT的三重染色对WT和SOD1动物在3周时的NMJ形成的评价。(E)根据3周龄时NMJ的M1至M4的成熟状态对NMJ进行量化。
图9-预防性处理中在15周时NMJ的分子分析。分析胫骨肌(A)和腓肠肌(B)中的Musk表达以及胫骨肌(C)和腓肠肌(D)中的nAchR表达定量。
图10-在治疗性阶段(8周),静脉内给予AAVPHP.eB-CYP46A1后SOD1小鼠的行为评价。(A)重量跟踪,(B)抱握测试,(C)倒置测试,以及(D)生存。结果表示为平均值±SEM。*p<0.05,**p<0.01,***p<0.001,****p<0.0001。黑色*对应于相对WT动物的p值,灰色为相对于SOD1动物。
图11-预防性处理后AAVPHP.eB-CYP46A1在SOD1动物中的生物分布,以及治疗性处理后在15周龄时通过24OH胆固醇水平定量进行的靶标参与评价。(A)中枢神经系统和(B)周围器官中的生物分布,以及(C)通过UPLC-HRMS评估的SOD1G93A小鼠中腰脊髓24S-羟基胆固醇含量。结果表示为平均值±SEM。
图12-CYP46A1-HA表达在15周龄时的评价以及静脉内递送AAVPHP.eB-CYP46A1作为治疗性处理后的脊髓的组织学分析。(A)在WT、SOD1和SOD1AAV动物的脊髓的腰切片上针对HA和VachT的共染色。(B)腰脊髓切片上的运动神经元数的量化,以及(C)在脊髓的腰切片上的Luxol染色。结果表示为平均值±SEM。*p<0.05,**p<0.01。黑色*对应于相对WT动物的p值,灰色为相对于SOD1动物。
图13-治疗性处理后,经AAV处理的SOD1动物的肌肉表型的部分修正。表示了胫骨肌(A)、腓肠肌(C)和四头肌(E)的平均纤维面积,以及根据胫骨肌(B)、腓肠肌(D)和四头肌(F)中纤维的横截面大小的再分隔纤维百分比。结果表示为平均值±SEM。*p<0.05,**p<0.01。
图14-治疗性处理后,经AAV处理的SOD1动物中神经肌肉接头的保留。(A)在治疗性静脉内递送AAVPHP.eB-CYP46A1后,15周龄的WT、SOD1和SOD1经处理动物的神经丝(NF)和银环蛇毒素(BTX)共染色。(B-C)根据NMJ在15周龄时的神经支配状态和完整性对其的评分。
图15-在治疗性处理中15周时NMJ的分子分析。分析胫骨肌(A)和腓肠肌(B)中的Musk表达以及胫骨肌(C)和腓肠肌(D)中的nAchR表达定量。
图16-在治疗性阶段(8周)以高剂量静脉内给予AAVPHP.eB-CYP46A1后,对SOD1小鼠的行为评价。抱握测试。结果表示为平均值±SEM。*p<0.05,**p<0.01,***p<0.001。黑色*对应于相对WT动物的p值,灰色为相对于SOD1动物。
图17-在预防性阶段(4周)以高剂量静脉内给予AAVPHP.eB-CYP46A1后,对C9ORF72小鼠的行为评价。(A)重量跟踪和(B)缺口棒表现。结果表示为平均值±SEM。*p<0.05,**p<0.01,***p<0.001。黑色*对应于相对于WT动物的p值,灰色为相对于C9ORF72动物。
图18–(A)在SMCR8 WT或突变体的过表达和CYP46A1的过表达后细胞中p62聚集体的评价。(B)反映在不同条件下神经元存活的阳性经转导细胞(HA)的总数的定量。
具体实施方式
本发明人证明在ALS的小鼠模型中静脉内递送表达CYP46A1基因的载体能够预防/修正运动损伤的发展。更具体而言,本发明人证明将表达CYP46A1基因的质粒递送到模拟肌萎缩性侧索硬化病状的原代纹状体神经元(striatal neurons)中引起p62聚集体(其为SMCR8功能障碍的标志,并通过自噬这一损伤来参与ALS)的显著减少,并改善神经元的存活。
在此基础上,本发明人提供了用于治疗ALS的病毒载体,其中,所述载体在中枢神经系统的细胞中(特别是运动神经元和运动皮层内的神经元中)表达CYP46A1。该策略在治疗任何运动神经元紊乱中有用,所述运动神经元紊乱与主要通过共同的突变基因形成的一些ALS作为连续统一体(continuum)。特别是,本发明人提供了用于治疗与ALS相关的额颞痴呆(FTD)的病毒载体。
肌萎缩性侧索硬化
本发明具体涉及肌萎缩性侧索硬化的治疗。优选地,本发明涉及由散发性因素引起和/或当疾病相关蛋白(即,SOD1、FUS、C9ORF72、TDP43…)包含功能获得性突变时的ALS的治疗。在实施方式中,本发明涉及与至少一种运动神经元相关紊乱有关联的ALS的治疗。此类相关的运动神经元紊乱优选选自下运动神经元紊乱或上运动神经元紊乱,包括痉挛性截瘫。在具体实施方式中,本发明涉及与额颞痴呆(FTD)有关联的ALS的治疗。FTD是致命的神经退行性疾病,其特征在于额叶和颞叶的神经元退化,导致语言、性格和行为的逐步恶化。至少有15-20%的ALS患者收到FTD的伴随诊断(Prudencio等,2015年)。在另一具体实施方式中,本发明涉及无额颞痴呆的ALS的治疗。
在本发明的上下文中,术语“治疗(treatment/treat/treating)”在本文中用于表征旨在以下的治疗性方法或过程:(1)减慢或阻止这样的术语所应用至的病症或疾病状态的症状的进展、加重或恶化;(2)减轻这样的术语所应用至的病症或疾病状态的症状或者给这样的术语所应用至的病症或疾病状态的症状带来改善;和/或(3)逆转或治愈这样的术语所应用至的病症或疾病状态。
本文所使用的术语“受试者”或“患者”是指动物,优选地指哺乳动物,甚至更优选地指人,包括成人和儿童。然而,术语“受试者”还可以指非人类动物,哺乳动物(如小鼠)和非人灵长类动物。
CYP46A1序列
本发明的第一个目的涉及用于治疗肌萎缩性侧索硬化的载体,所述载体包含编码胆固醇24-羟化酶的核酸的完整序列。
本文所使用的术语“基因”是指含有至少一个开放阅读框的多核苷酸,其在被转录或翻译后可编码特定的多肽或蛋白质。
本文所使用的术语“编码序列”或“编码特定蛋白质的序列”是指当将其置于适当的调控序列的控制下时在体外或体内被转录(对于DNA而言)并翻译(对于mRNA而言)为多肽的核酸序列。编码序列的边界由5'(氨基)末端的起始密码子和3'(羧基)末端的翻译终止密码子确定。编码序列可包括但不限于:来自原核生物mRNA或真核生物mRNA的cDNA、来自原核生物DNA或真核生物DNA的基因组DNA序列、以及甚至合成的DNA序列。
CYP46A1基因编码胆固醇24-羟化酶。这种酶是细胞色素P450酶超家族的成员。该酶将胆固醇转化为可动态地穿过BBB的24S-羟基胆固醇(24S-OH-Chol),完成外周循环以从体内排出(等,1998),从而保持胆固醇的体内平衡。CYP46A1的cDNA序列公开于Genbank登录号AF094480(SEQ ID NO:1)。氨基酸序列在SEQ ID NO:2中示出。
本发明利用包含序列SEQ ID NO:1或其变体的核酸构建体来治疗肌萎缩性侧索硬化和任选的相关的运动神经元紊乱(例如FTD)。
变体包括例如由于个体之间的等位基因变异(例如多态性)、替代的剪接形式等而来的天然存在的变体。术语变体还包括来自其它来源或有机体的CYP46A1基因序列。变体优选与SEQ ID NO:1基本上同源,即,与SEQ ID NO:1表现出通常至少约75%、优选至少约85%、更优选至少约90%、更优选至少约95%的核苷酸序列同一性。CYP46A1基因的变体还包括在严格杂交条件下与上文所定义的序列(或其互补链)杂交的核酸序列。典型的严格杂交条件包括高于30℃、优选高于35℃、更优选超过42℃的温度,和/或盐度小于约500mM、优选小于200mM。技术人员可通过改变温度、盐度和/或其它试剂(例如SDS、SSC等)的浓度来调节杂交条件。
非病毒载体
在实施方式中,本发明所使用的载体为非病毒载体。通常,非病毒载体可为编码CYP46A1的质粒。这种质粒可直接进行给予或通过脂质体、外来体或纳米颗粒进行给予。
病毒载体
可利用分子生物学领域公知的方法学来构建在本发明的实践中有用的基因递送病毒载体。通常,携带转基因的病毒载体由编码该转基因的多核苷酸、合适的调控元件、和产生介导细胞转导的病毒蛋白所必需的元件组装而成。
术语“基因转移”或“基因递送”是指用于将外源DNA可靠地插入宿主细胞中的方法或系统。此类方法可引起非整合的转移DNA的瞬时表达、转移的复制子(例如附加体)的表达和染色体外复制、或转移的遗传材料整合到宿主细胞的基因组DNA中。
病毒载体的实例包括腺病毒载体、慢病毒载体、逆转录病毒载体、疱疹病毒载体和腺相关病毒(AAV)载体。
此类重组病毒可通过本领域已知的技术产生,例如通过转染包装细胞或通过用辅助质粒或病毒进行瞬时转染。病毒包装细胞的典型实例包括PA317细胞、PsiCRIP细胞、GPenv+细胞、293细胞等。生产此类复制缺陷型重组病毒的详细方案可见于例如WO95/14785、WO96/22378、US5,882,877、US6,013,516、US4,861,719、US5,278,056和WO94/19478。
在优选的实施方式中,使用腺相关病毒(AAV)载体。
“AAV载体”所指的是由腺相关病毒血清型衍生而来的载体,包括但不限于AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV8、AAV9、AAV10(例如AAVrh.10)、AAVPHP.eB等。AAV载体可具有全部或部分删除的一个或多个AAV野生型基因(优选rep基因和/或cap基因),但保留功能性侧翼ITR序列。功能性ITR序列对于AAV病毒粒子的挽救、复制和包装是必需的。因此,本文中AAV载体定义为至少包括顺式的病毒复制和包装所需的那些序列(例如功能性ITR)。ITR不必是野生型核苷酸序列,并且可以改变(例如通过核苷酸的插入、删除或置换),只要该序列提供功能性的挽救、复制和包装。使用已知技术构建AAV表达载体,以至少提供在转录方向上的可操作连接的组分、包含转录起始区的控制元件、感兴趣的DNA(即CYP46A1基因)和转录终止区。可以包含感兴趣的DNA的两个拷贝作为自互补的构建体。
在更优选的实施方式中,AAV载体为AAV9、AAV10(优选AAVrh.10)、AAVPHP.B或AAVPHP.eB载体,或由这些血清型中的一种衍生而来的载体。在最优选的实施方式中,AAV载体为AAVPHP.eB载体。AAVPHP.eB载体为经演化的AAV-PHP.B变体,其有效转导CNS神经元(Chan KY等,2017;WO2017100671)。也可使用其它载体,例如在WO2015038958和WO2015191508中描述的载体。
选择控制元件以在哺乳动物细胞中具有功能性。包含可操作连接的组分的所得构建体与功能性AAV ITR序列结合(5'和3')。“腺相关病毒反向末端重复序列”或“AAV ITR”是指存在于AAV基因组的各个末端的本领域公认的区域,它们顺式地一起发挥作用作为DNA复制的起点以及作为病毒的包装信号。AAV ITR与AAV rep编码区一起提供了如下:从中有效切除和挽救,以及将两个侧翼ITR之间插入的核苷酸序列整合到哺乳动物细胞基因组中。AAV ITR区的核苷酸序列是已知的。对于AAV-2序列(参见例如Kotin,1994;Berns,KI,"Parvoviridae and their Replication",Fundamental Virology,第二版(B.N.Fields和D.M.Knipe编著))。本文所使用的“AAV ITR”不必需包含野生型核苷酸序列,而可经改变(例如通过核苷酸的插入、删除或置换)。此外,AAV ITR可衍生自数种AAV血清型中的任一种,包括但不限于AAV1、AAV2、AAV3、AAV4、AAV5、AAV6等。此外,在AAV载体中位于所选择的核苷酸序列侧翼的5'和3'ITR不需要必须与相同的AAV血清型或分离株统一,或由相同的AAV血清型或分离株衍生而来,只要它们如预期地发挥作用即可,即允许从宿主细胞基因组或载体中切除和挽救目的序列,并且当AAV Rep基因产物存在于细胞中时允许异源序列整合到受体细胞基因组中。
特别优选的是由AAV血清型衍生而来的载体,其对于哺乳动物中枢神经系统(CNS)的细胞(特别是神经元)具有嗜性并在其中具有高转导效率。在Davidson等,2000中提供了对不同血清型的转导效率的综述和比较。在一个优选的实例中,已证实基于AAV2的载体指导CNS中转基因的长期表达,优选转导神经元。在其它非限制性实例中,优选的载体包括由AAV4和AAV5血清型衍生而来的载体,它们也已被证明转导CNS的细胞(Davidson等,同上)。特别是,载体可为包含由AAV5衍生来的的基因组(特别是ITR为AAV5ITR)和由AAV5衍生而来的衣壳的AAV载体。
在本发明的具体实施方式中,所述载体为假型AAV载体。具体而言,假型AAV载体包含由第一AAV血清型衍生而来的AAV基因组和由第二AAV血清型衍生而来的衣壳。优选地,所述AAV载体的基因组由AAV2衍生而来。此外,衣壳优选由AAV5衍生而来。假型AAV载体的具体非限制性实例包括:在由AAV5衍生而来的衣壳中包含由AAV2衍生而来的基因组的AAV载体、在由AAVrh.10衍生而来的衣壳中包含由AAV2衍生而来的基因组的AAV载体等。
所选择的核苷酸序列可操作地连接至控制元件,所述控制元件指导其在受试者体内的转录或表达。此类控制元件可包含通常与所选基因相关的控制序列。特别是,此类控制元件可包括CYP46A1基因的启动子、特别是人CYP46A1基因的启动子(Ohyama Y等,2006)。
或者,可以采用异源控制序列。有用的异源控制序列通常包括由编码哺乳动物或病毒基因的序列衍生而来的那些控制序列。实例包括但不限于:磷酸甘油酸激酶(PGK)启动子、SV40早期启动子、小鼠乳腺肿瘤病毒LTR启动子;腺病毒主要晚期启动子(Ad MLP);单纯疱疹病毒(HSV)启动子、巨细胞病毒(CMV)启动子(如CMV即刻早期启动子区域(CMVIE))、劳斯肉瘤病毒(RSV)启动子、合成启动子、杂合启动子等。另外,还将在本文中发现使用由非病毒基因(如鼠类金属硫蛋白基因)衍生而来的序列。此类启动子序列可商购自例如Stratagene(San Diego,CA)。为了本发明的目的,异源启动子和其它控制元件(例如CNS特异性和诱导型启动子、增强子等)均将特别有用。
异源启动子的实例包括CMV启动子。CNS特异性启动子的实例包括从来自以下的基因中分离的启动子:髓磷脂碱性蛋白(MBP)、胶质纤维酸性蛋白(GFAP)、突触蛋白(synapsins)(例如人突触蛋白1基因启动子)和神经元特异性烯醇化酶(NSE)。
诱导型启动子的实例包括用于蜕皮激素、四环素、hypoxia andaufin的DNA响应元件。
可通过将所选择的序列直接插入已从中切除了主要AAV开放阅读框(“ORF”)的AAV基因组中,来构建带有被AAV ITR结合的感兴趣的DNA分子的AAV表达载体。只要保留足以允许复制和包装功能的ITR部分,AAV基因组的其它部分也可以删除。可以使用本领域熟知的技术来设计此类构建体。参见例如,美国专利号5,173,414和5,139,941;国际公布号WO 92/01070(1992年1月23日公布)和WO 93/03769(1993年3月4日公布);Lebkowski等,1988;Vincent等,1990;Carter,1992;Muzyczka,1992;Kotin,1994;Shelling和Smith,1994;以及Zhou等,1994。或者,可从病毒基因组或从包含AAV ITR的AAV载体中切除所述AAV ITR,并使用标准连接技术将所述AAV ITR与存在于另一载体的所选择核酸构建体的5'和3'融合。包含ITR的AAV载体已描述于例如美国专利号5,139,941中。特别是,其中描述了可以从美国典型培养物保藏中心(“ATCC”)以登录号53222、53223、53224、53225和53226获得的数种AAV载体。此外,可合成产生嵌合基因以包括排列于一个或多个所选择的核酸序列的5'和3'的AAVITR序列。可以使用在哺乳动物CNS细胞中对于嵌合基因序列表达而言优选的密码子。完整的嵌合序列由通过标准方法制备的重叠寡核苷酸组装而成。(参见例如,Edge,1981;Nambair等,1984;Jay等,1984)。为了产生rAAV病毒粒子,使用已知技术(例如通过转染)将AAV表达载体引入合适的宿主细胞中。很多转染技术通常都是本领域公知的。(参见,例如,Graham等,1973;Sambrook等(1989)Molecular Cloning,a laboratory manual,ColdSpring Harbor Laboratories,New York,Davis等,(1986)Basic Methods in MolecularBiology,Elsevier;以及Chu等,1981)。特别合适的转染方法包括磷酸钙共沉淀(Graham等,1973)、直接显微注射到培养的细胞中(Capecchi,1980)、电穿孔(Shigekawa等,1988)、脂质体介导的基因转移(Mannino等,1988)、脂质介导的转导(Felgner等,1987)和使用高速微弹丸(microprojectiles)的核酸递送(Klein等,1987)。
例如,优选的病毒载体(例如AAVPHP.eB)除了包含编码胆固醇24-羟化酶的核酸序列外,还包含具有由AAV-2衍生而来的ITR的AAV载体的骨架、启动子、或任何神经元启动子,所述启动子例如小鼠PGK(磷酸甘油酸激酶)基因或巨细胞病毒/β-肌动蛋白杂合启动子(CAG),所述CAG由来自巨细胞病毒即刻基因的增强子,来自鸡β-肌动蛋白基因的启动子、剪接供体和内含子,来自兔β-珠蛋白的剪接受体所组成;所述神经元启动子例如多巴胺1受体或多巴胺2受体的启动子,所述多巴胺1受体或多巴胺2受体的启动子具有或不具有土拨鼠肝炎病毒转录后调控元件(WPRE)的野生型或突变形式。
载体的递送
公开了治疗肌萎缩性侧索硬化的方法,所述方法包括向有需要的患者给予包含编码胆固醇24-羟化酶的核酸的载体。所述载体可直接递送到受试者的脑和/或脊髓中,或者通过血管内、静脉内、鼻内、脑室内或鞘内注射进行递送。在具体实施方式中,所述载体为AAVrh10或AAVPHP.eB,并通过静脉内注射进行递送。
在具体实施方式中,提供了用于治疗受试者的肌萎缩性侧索硬化(ALS)的方法,所述方法包括:
(a)提供如上所定义的载体,所述载体包含编码胆固醇24-羟化酶的核酸;以及
(b)将所述载体递送至受试者的脑和/或脊髓,由此所述载体转导脑和/或脊髓中的细胞,并且由此经转导的细胞以治疗有效水平表达胆固醇24-羟化酶。
有利地,所述载体为病毒载体,更有利地为AAV载体,甚至有利地为选自于由AAV9、AAV10或AAVPHP.eB所组成的组中的AAV载体。
在具体实施方式中,所述载体被递送至大脑(特别是运动皮层)和/或脊髓。在具体实施方式中,所述载体仅被递送至脊髓。
在另一具体实施方式中,通过静脉内注射给予所述载体。
将病毒载体递送或给予至神经元和/或星形胶质细胞和/或少突胶质细胞和/或小胶质细胞的方法通常包括适合于(直接或通过造血细胞转导)将载体递送至所述细胞的任何方法,使得所选择的突触连接的细胞群中至少部分的细胞被转导。可将所述载体递送至中枢神经系统的任何细胞、外周神经系统的细胞或两者皆有。优选地,将所述载体递送至脑和/或脊髓的细胞。通常,将所述载体递送至脑(包括例如运动皮层的细胞)、脊髓或它们的组合的细胞,或其任何合适的亚群。
为了将所述载体特异性地递送至大脑或脊髓的特定区域和特定细胞群,可通过立体定向的(stereotaxic)显微注射来给予所述载体。例如,患者具有固定到位(拧入头骨)的立体定位(stereotactic)框架基部。使用高分辨率MRI对具有立体定位框架基部的脑(与置信标记兼容的MRI)进行成像。然后,将MRI图像传输到运行立体定位软件的计算机。一系列冠状、矢状和轴向图像用于确定靶标(载体注射的位点)和轨迹。该软件将轨迹直接转换为适合立体定位框架的3维坐标。在进入位点上方钻了钻孔,并在给定深度用植入的针定位立体定位仪器。然后,将载体注射到靶位点,最终与造影剂混合。由于载体整合到靶细胞中,而不是产生病毒颗粒,因此载体的后续扩散很小,并且主要是整合前从注射位点被动扩散,当然还有期望的跨突触转运的作用。扩散程度可以通过调节载体与流体运载体的比例来控制。
给予的另外途径还可包括在直接可视化下对载体进行局部应用,例如,浅层皮层应用、鼻内应用或其它非立体定位的应用。
本发明的载体的靶细胞为患有ALS的受试者的脊髓(运动神经元)的细胞和脑(运动皮层)的细胞,优选神经细胞。
优选地,受试者为人类,通常为成年人,但是可以是儿童或婴儿。然而,本发明涵盖将所述载体递送至疾病的生物学模型。在此种情况下,生物学模型在递送时可为处于任何发育阶段的任何哺乳动物,例如胚胎阶段、胎儿阶段、婴儿阶段、青少年阶段或成年阶段,优选为成年。此外,所述靶细胞可基本上来自任何来源,尤其是非人灵长类动物以及啮齿目(小鼠、大鼠、兔、仓鼠)、食肉目(猫、狗)和偶蹄目(牛、猪、绵羊、山羊、马)的哺乳动物,以及任何其它非人系统(例如斑马鱼模型系统)。
优选地,本发明的方法包括通过立体定向的注射进行脑内给予。但是,根据本发明,也可以采用其它已知的递送方法。例如,为了使载体在脑中更广泛地分布,可将其注射到脑脊液中,例如通过腰椎穿刺、小脑延髓池(cisterna magna)或脑室穿刺。为了将载体引导至脑,可将其注射到脊髓或注射到周围神经节、或感兴趣的身体部位的肉(皮下或肌内)。在某些情况下,例如此处对于AAVPHP.eB,可通过血管内方法给予所述载体。例如,在血脑屏障被干扰的情况下,可动脉内(颈动脉)给予所述载体。此外,为了更全面地递送,可在通过输注包括甘露醇的高渗溶液或超声局部递送而实现的血脑屏障的“开放”期间给予所述载体。
可将本文使用的载体配制在任何合适的媒介物中以进行递送。例如,可将它们置于药学上可接受的悬浮液、溶液或乳液中。合适的介质包括盐水和脂质体制剂。更具体地,药学上可接受的运载体可包括无菌的水性或非水溶液、悬浮液和乳液。非水性溶剂的实例为丙二醇、聚乙二醇、植物油(例如橄榄油)和可注射有机酯(例如油酸乙酯)。水性运载体包括水、醇溶液/水性溶液、乳液或悬浮液,包含盐水和缓冲介质。静脉内媒介物包括液体和营养补充剂、电解质补充剂(例如基于林格氏右旋糖的补充剂)等。
也可存在防腐剂和其它添加剂,例如,如抗菌剂、抗氧化剂、螯合剂和非活性气体等。
胶体分散系统也可用于靶向基因递送。胶体分散系统包括基于大分子复合物、纳米胶囊、微球、珠和脂质的系统,包括水包油乳液、微胶粒(micelles)、混合微胶粒和脂质体或外来体。
优选的剂量和方案可以由医师确定,并取决于受试者的年龄、性别、体重,以及疾病的阶段。作为实例,对于使用病毒表达载体递送胆固醇24-羟化酶,每单位剂量的表达胆固醇24-羟化酶的载体可包含处于药学上可接受的流体中的2.5μL至100μL的包含病毒表达载体的组合物,并且其提供每mL组合物从1010个多至1015个表达胆固醇24-羟化酶的病毒颗粒。
所述载体可用于治疗性治疗和/或预防性治疗中。
因此,本发明的目的在提供用于预防性治疗ALS的载体,所述载体包含编码胆固醇24-羟化酶的核酸。
本发明的另一目的在于提供用于治疗性治疗ALS的载体,所述载体包含编码胆固醇24-羟化酶的核酸。
药物组合物
本发明的进一步目的涉及用于治疗ALS的药物组合物,所述药物组合物包含治疗有效量的根据本发明的载体。
“治疗有效量”是指足以以适用于任何医药治疗的合理的受益/风险比来治疗ALS的本发明载体的量。
应该理解的是,本发明的化合物和组合物的每日总剂量将由主治医师在合理的医学判断范围内决定。对于任何特定患者的具体治疗有效剂量水平将取决于多种因素,包括正在治疗的紊乱和紊乱的严重程度;所运用的具体化合物的活性;所运用的具体组合物,患者的年龄、体重、总体健康状况、性别和饮食;给药时间、给药途径和所运用的具体化合物的排泄速率;治疗的持续时间;与所运用的具体多肽组合使用或同时使用的药物;以及医药领域公知的因素等。例如,本领域技术人员公知,以低于实现期望的治疗效果所需剂量的水平开始化合物的剂量,并逐渐增加剂量直至实现期望的效果。但是,每位成人每天的产品每日剂量可能在宽的范围内变化。应当给予的根据本发明的载体的治疗有效量、以及用一定量的本发明的病毒或非病毒颗粒和/或药物组合物治疗病理性病症的剂量将取决于多种因素,包括患者的年龄和状况、干扰或紊乱的严重程度、给予方法和频率以及待使用的具体肽。
包含根据本发明的载体的药物组合物的呈递可为适合于肌内、脑内、鼻内、鞘内、脑室内或静脉内给予的任何形式。在用于肌内、鼻内、静脉内、脑内、鞘内或脑室内给予的本发明药物组合物中,活性成分可以单独或与另一活性成分组合以单位给药形式(如与常规药物支持物作为混合物)对动物和人进行给予。
优选地,所述药物组合物包含对于能够被注射的制剂而言药学上可接受的媒介物。这些媒介物可以特别是等渗的无菌盐水溶液(磷酸二氢钠或磷酸二氢钠、氯化钠、氯化钾、氯化钙或氯化镁等,或此类盐的混合物),或是干燥的(尤其是冻干的)组合物,其根据情况在添加无菌水或生理盐水时允许无菌注射液的构成。
配制后,将以与剂型相容的方式并以治疗有效的量给予溶液。可以容易地以各种剂型(例如可注射溶液的类型)给予制剂,但是也可以使用药物释放胶囊等。
也可以给予多剂量。
也能够将所述治疗性方法与能够激活CYP46A1的小分子(如依非韦伦)(Mast N等,2013;Mast N等,2017;Mast N等,2014;Mast N等,2017)或抗真菌药(Mast N等,2013;Fourgeux等,2014)关联起来,这可能转而抑制CYP46A1,因此在需要时可以作为终止基因治疗的方法。
将通过以下实施例进一步说明本发明。然而,该实施例和附图不应以任何方式解释为限制本发明的范围。
实施例
在此实验中,基于SMCR8 D928V(SMCR8的突变体)的过表达,建立了ALS的体外模型,所述SMCR8 D928V与C9ORF72和WDR1形成复合物,并充当RAB8和RAB39b的GDP/GTP交换因子,从而控制自噬通量(Sellier等,2016)。WT SMCR8或突变SMCR8(其随SMCR8的siRNA表达而改变自噬)均过表达。
材料和方法
动物
两只雄性B6SJL-Tg(SOD1*G93A)1Gur/J获取自Jackson实验室(货号002726),并与C57BL/6小鼠交配以产生集群。
三只雄性FVB/NJ-Tg(C9orf72)500Lpwr/J获取自Jackson实验室(货号029099),并与FVB/NJ小鼠交配以产生集群。
将小鼠置于温度受控的房间中,并维持12h明/暗周期。食物和水可随意获得。实验是根据关于护理和使用实验动物的欧洲共同体理事会准则(2010/63/EU)进行的。
AAV质粒设计和载体生产
通过Atlantic Gene therapies(INSERM U1089,Nantes,法国)生产和纯化AAV载体。载体的生产已在其它地方进行了描述(Hudry E等,2010)。AAVPHP.eB-CYP46A1-HA的病毒构建体包含由人Cyp46a1基因组成的表达盒,其由CMV早期增强子/鸡β-肌动蛋白(CAG)合成启动子(CAG)驱动,并被AAV2的反向末端重复序列(ITR)围绕。批次的最终滴度在1.5至2.1013vg/mL之间。
基因分型
根据Jackson实验室程序对小鼠进行分型:对于SOD1G93A小鼠种系(https://www2.jax.org/protocolsdb/f?p=116:5:0::NO:5:P5_MASTER_PRO TOCOL_ID,P5_JRS_CODE:24173,002726);对于C9ORF72 500r种系,https://www2.jax.org/protocolsdb/f?p=116:5:0::NO:5:P5_MASTER_PROTO COL_ID,P5_JRS_CODE:30889,029099。
静脉内注射
为了确定在ALS小鼠模型中注射的AAVPHP.eB-CYP46A1-HA的剂量,在8周龄向C57BL6动物注射了3种剂量,总计2.5.1011vg(低)、总计5.1011vg(中)和总计1.1012vg(高剂量)(每组n=3)。
关于预防性处理,用4%的异氟烷对三周龄的SOD1G93A或四周龄的C9ORF72 500r小鼠诱导麻醉,然后以2%的异氟烷与80%的空气和20%的氧气维持。通过眶后注射,SOD1动物接受总计2.5.1011vg(低剂量)或总计5.1011vg(中剂量)(100μL体积)的静脉内注射。WT和未注射的SOD1动物接受了100μL盐水溶液的注射。
预防性组的详细信息:
-SOD1研究:n=10WT;n=12SOD1以及n=14SOD1AAV,以总计2.5.1011vg
-C9研究:n=16WT;n=16C9以及n=17C9AAV,以总计5.1011vg
关于治疗性处理,用4%的异氟烷对8周龄的SOD1G93A或16周龄的C9ORF72 500r小鼠诱导麻醉,然后以2%的异氟烷与80%的空气和20%的氧气维持。通过眶后注射,SOD1动物接受总计2.5.1011vg(低剂量)或总计5.1011vg(中剂量)(100μL体积)的静脉内注射。WT和未注射的SOD1动物接受了100μL盐水溶液的注射。
治疗性组的详细信息:
-SOD1研究:
οn=24WT;n=27SOD1以及n=22SOD1AAV,以总计2.5.1011vg
οn=5WT;n=5SOD1以及n=7SOD1AAV,以总计5.1011vg
-C9研究:n=14WT;n=13C9以及n=12C9AAV,以总计5.1011vg
行为测试
体重跟踪
在注射前对所有动物称重,对于SOD1动物每周称重,对于C9ORF72动物每两周称重。
抱握测试(Clasping test)
抱握测试评价协调性,并且对于ALS评价而言是经典的。在注射之前对动物进行评分,然后从6周或8周每2周进行评分,直至至15周。从尾部维持动物,如果小鼠抽搐,则分数为1,然后针对每个后肢抱握来增加得分。每个测试的结果均以每组的平均值±SEM表示,并进行了t检验分析。
倒置测试
倒置测试评价力量,并且对于ALS评价而言是经典的。在注射之前对动物评分,然后从6周或8周每2周进行评分,直至15周。将动物放在栅格上,并将栅格倒置,立即对附着在栅格上的剩余的脚进行评分(短暂倒置)。每个测试的结果均以每组的平均值±SEM表示,并进行了t检验分析。
对于C9模型,通过3次5分钟的试验(每次试验之间15分钟的恢复),使小鼠甚至更多地挑战倒置测试,从而获得更稳健的结果。对于每个试验,对它们维持附着的时间进行评分,并为每只动物计算平均值。
缺口棒
如前所述(Piguet F等,2018),使用缺口棒测试(上肢或下肢掉落的评分数)来评价协调性。
生存评价
每天观测小鼠,如果它们达到终点标准(后肢拖拽或体重减轻≥20%),则立即进行安乐死。
组织收集
用戊巴比妥(Euthasol 180mg/kg)溶液麻醉小鼠,并用磷酸盐缓冲盐水(PBS)进行穿心灌注。收集脑、脊髓和坐骨神经以及周围器官(肝、心脏、肺、肾、脾),并在石蜡包裹之前在4%PFA中进行后固定(post-fix),用于组织学(用切片机切成6-10μm)或立即在液氮中冷冻用于生物分子分析。
解剖四肢肌肉,并在4%PFA/PBS中于4℃后固定过夜。将样品在PBS中冲洗3次,并在20%蔗糖/PBS中冷冻保护48小时。将组织包埋在cryomatrix(Thermoscientific)中,并使用冷冻切片机cryostat(Leica CM3050S)切割(20μm)。将冷冻切片在室温下干燥并在-20℃下保存。
解剖腓肠肌、胫骨肌、四头肌和脊髓的部分,并将其在液氮中冷冻。
将不同的组织在液氮中研磨/压碎,然后分离用于分析RNA或DNA表达。
原代纹状体细胞培养和转染
如先前所述(Charvin D等,2005;Deyts等,2009),从来自怀孕Swiss小鼠(Janvier)的第14天胚胎中解剖出原代纹状体神经元。体外7天后,用LipofectamineTM2000(Invitrogen)进行纹状体细胞的瞬时转染。在这一阶段,非常少观测到胶质细胞(55%;数据未示出)。在5种条件下转染细胞(CYP46A1-HA;SMWR8 WT-HA;SMCR8 D928V-HA;SMWR8 WT-HA+CYP46A1-HA;SMCR8 D928V-HA+CYP46A1HA),未转染的细胞用作对照。对每种条件进行N=6个重复。在细胞固定前,用Lipofectamine在8孔labtek室中转染每个质粒的100ng DNA进行16h。
一抗
下表1列出了用于免疫组化(IHC)分析的抗体。
表1:用于免疫荧光和免疫组化(IHC)分析的抗体
一抗 | 来源 | IF |
小鼠抗HA | Biolegend(901514) | 1:1000 |
小鼠抗p62 | Abcam(ab56416) | 1:250 |
兔抗HA | Cell signaling(#3724) | 1:500 |
兔抗Iba1 | Wako(019-19741) | 1:500 |
小鼠抗GFAP | Sigma(G-3893) | 1:500 |
小鼠抗Pan-NF | Biolegend(837904) | 1:1000 |
Alexa 488-αBTX | Lifesciences(B13422) | 1:2000 |
兔抗VachT | Sigma(SABA2000559) | 1:1000 |
免疫染色
在细胞上
通过将细胞在PFA中固定15分钟来启动免疫荧光程序。洗涤3次后,将细胞在PBS/0.3%Triton X-100中透化,然后在含有5%正常山羊血清(NGS,Gibco)的PBS/0.1%TritonX-100中在RT封闭1h。然后将细胞与相应的一抗在4℃孵育过夜。洗涤3次后,将细胞与在PBS/0.1%Triton X-100中稀释的对应的二抗(1:1000;Vector Laboratories Inc.,CA,USA)在RT孵育1h。洗涤三次后,将细胞干燥并用DAPI在封固介质中封固。
在石蜡切片上
肌肉:在加工之前,将肌肉冰冻切片用处于PBS中的0.1M甘氨酸处理30min。在PBS中洗涤后,将切片用PBS/0.3%TritonX-100透化10min,并用PBS/0.3%Triton/10%BSA饱和45min。将一抗稀释在饱和溶液中,并在4℃孵育O/N。在PBS/0.1%Triton中洗涤后,将二抗和α-银环蛇毒素在PBS/0.1%Triton/10%BSA中稀释,并在室温下添加1小时。在PBS/0.1%Triton中洗涤后,将载玻片用荧光水性封固介质(F4680,Sigma)进行封固。
免疫荧光的一抗是小鼠抗Pan-Neurofilament(1:1000;Biolegend,837904)、兔抗囊泡乙酰胆碱转运体(VAChT;1:1000;sigma,2000559)。二抗以1:1000稀释,并分别为驴抗兔/AlexaFluor 594、抗小鼠/AlexaFluor Cy3(Life Technologies,Carlsbad,CA,USA)。
将α-银环蛇毒素-Alexa594(1:2000;Life Technologies,B13422)与二抗一起孵育。用共聚焦SP8Leica DLS倒置显微镜(Carl Zeiss,Zaventem,比利时)拍摄照片。对于所有图像,采集后用ImageJ软件调节亮度和对比度,以与观测结果进行匹配。
脊髓:为了进行免疫荧光,将脊髓在95℃用10mM Tris/1mM EDTA/0.1%TweenpH8.75处理45min。在PBS中洗涤后,将切片与透化溶液(PBS/0.3%TritonX-100)孵育15min,然后与饱和溶液(PBS/0.1%TritonX-100/10%马血清)孵育1小时。将一抗稀释在10%马血清/PBS/0.1%Triton X-100中,并在组织切片上于4℃孵育过夜。在PBS/0.1%TritonX-100中洗涤后,将切片与二抗(驴抗兔Alexa 594和驴抗小鼠alexa488,1:1000,Life Technologies)孵育。
免疫组化标记使用ABC方法进行。简而言之,用过氧化物处理组织切片30分钟以抑制内源性过氧化物酶。在PBS中洗涤后,将切片在95℃用10mM Tris/1mM EDTA/0.1%TweenpH8.75处理45min(仅用于抗HA)。在PBS中洗涤后,将切片与封闭溶液(处于PBS/0.3%TritonX-100中的山羊血清或10%山羊血清)孵育1小时。将一抗在封闭溶液中稀释,并在组织切片上于4℃孵育过夜。在PBS中洗涤后,将切片依次与缀合至生物素的山羊抗兔或山羊抗小鼠抗体(Vector Laboratories)、随后为ABC复合物(Vector Laboratories)在室温下孵育30分钟。在PBS中洗涤后,使用二氨基联苯胺作为发色剂(Dako,Carpinteria,CA)检测过氧化物酶活性。在某些情况下,将玻片用苏木精复染。载玻片用Depex(VWRInternational)封固。
Luxol染色
用经典luxol染色对脊髓切片上的髓鞘质染色/可视化。
图像采集
用配备有Leica DFC310FX数码照相机的Leica DM 5000B显微镜,在室温下用macroscope(Leica)和LAS V3.8(Leica)软件采集免疫荧光载玻片的图像。用于比较的照片是在相同的图像采集条件下拍摄的,并且对用于细胞培养分析的所有图像统一应用亮度和对比度的所有调节。
用axioscan,Zeiss和ZEN 2.6软件或nanozoomer采集肌肉和VachT图片。对于所有图像,采集后用ImageJ软件调节亮度和对比度,以与观察结果进行匹配。
对于所有IHC和着色,使用Hamamatsu玻片扫描仪获取切片。
肌纤维分析
在15周时解剖胫骨前肌(TA)、腓肠肌(gastro)和四头肌(quadri)。将它们用福尔马林固定,石蜡包埋,切割(10mm)并用苏木精/伊红染色。使用axioscan,Zeiss和ZEN 2.6软件采集图片。使用ImageJ软件在每只动物(n=4-13)中的最少150条纤维上测量TA、Gastro、Quadri的肌纤维横截面积(CSA)。
神经肌肉接头分析
在来自3只不同动物的至少100个TA接头上,对NMJ形态进行了量化。终板分布可分为六类:(1)正常终板、(2)经修饰终板、(3)碎片化终板、(4)退化终板、(5)无孔终板和(6)异位终板。对每只动物分析了至少100个接头(n=3-4)。根据先前描述的标准(Audouard等),在P21小鼠中评价运动终板的成熟。
运动神经元量化
在关于VAChT的免疫组化后,对颈、胸和腰脊髓切片中运动神经元的数量进行了量化。在关于HA的免疫组化后,对经转导的运动神经元的数量进行量化。对于每只小鼠,在三种脊髓切片的左腹侧和右腹侧对运动神经元进行计数(n=3)。
Luxol定量
已使用Fiji软件在腰脊髓上对髓鞘质的总面积进行测量,并通过总脊髓面积进行归一化。然后,将WT中的髓鞘质百分比报告为100%,并且与WT动物相比来报告结果。
DNA提取
使用氯仿/苯酚方案从脑、脊髓和周围器官中提取DNA。
RNA提取
使用Trizol或TriReagent(Sigma)从小鼠中的腓肠肌、胫骨肌、四头肌和腰脊髓中的部分和患者的腰脊髓中提取总RNA。按照制造商的说明,用Transcriptor First StrandcDNA合成试剂盒(Roche)将一微克总RNA转录成cDNA。
q-PCR
用SyberGreen(Roche)扩增cDNA。用于RT-qPCR的引物在上表中。用于所有引物的扩增方案为热启动(95℃进行5min)、45个扩增循环(95℃进行15s,60℃进行1min)和熔解曲线分析。使用Lightcycler 480软件用关于每个基因的效率因子对数据进行分析,并归一化至肌动蛋白。
名称 | 引物5’->3’ |
肌动蛋白_126 For SEQ ID NO:3 | TCC TGA GCG CAA GTA CTC TGT |
<u>肌动蛋白</u>127 Rev SEQ ID NO:4 | CTG ATC CAC ATC TGC TGG AAG |
AchR alpha For SEQ ID NO:5 | AGA TCA TTG TCA CTC ACT TTC CCT |
AchR alpha Rev SEQ ID NO:6 | ACG AAG TGG TAG GTG ATG TCC AG |
MuSK For SEQ ID NO:7 | TCA TCAC CAC GCC TCT TGA AAC |
MuSK Rev SEQ ID NO:8 | CAT CAT CAC TGT CTT CCA CGC TC |
Cyp46A1小鼠For SEQ ID NO:9 | GGC TAA GAA GTA TGG TCC TGT TGT AAG A |
cyp46A1小鼠Rev SEQ ID NO:10 | GGT GGA CAT CAG GAA CTT CTT GAC T |
ApoE For SEQ ID NO:11 | GTC ACA TTG CTG ACA GGA TGC CTA |
ApoE Rev SEQ ID NO:12 | GGG TTG GTT GCT TTG CCA CTC |
Hmgcr For SEQ ID NO:13 | CCC CAC ATT CAC TCT TGA CGC TCT |
Hmgcr Rev SEQ ID NO:14 | GCT GGC GGA CGC CTG ACA T |
Srebp1 For SEQ ID NO:15 | GGT CCA GCA GGT CCC AGT TGT |
Srebp1 Rev SEQ ID NO:16 | CTG CAG TCT TCA CGG TGG CTC |
Srebp2 For SEQ ID NO:17 | TGT TGA CGC AGA CAG CCA ATG |
gadph人for SEQ ID NO:18 | CGC TCT CTG CTC CTC CTG TT |
gadph人Rev SEQ ID NO:19 | CCA TGG TGT CTG AGC GAT GT |
cyp46A1人For SEQ ID NO:20 | CGA GTC CTG AGT CGG TTA AGA AGT T |
cyp46A1人Rev SEQ ID NO:21 | AGT CTG GAG CGC ACG GTA CAT |
mADcK3 for SEQ ID NO:22 | CCA CCT CTC CTA TGG GCA GA |
mADCK3 rev SEQ ID NO:23 | CCG GGC CTT TTC AAT GTC T |
使用Light Cycler 480 SYBR Green I Master(Roche,法国),通过对于从DRG、脊髓(颈、胸和腰水平)、脑、小脑和外周器官提取的基因组DNA进行的qPCR测量载体基因组拷贝数。结果(每个细胞的载体基因组拷贝数)表示为转基因序列拷贝数相对于作为内标的Adck3基因拷贝的n倍差异(2N基因组的病毒基因组拷贝数)。
胆固醇和氧化固醇的测量
胆固醇和氧化固醇的分析遵循“金标准”方法25,以使自氧化产物的形成最小化。简而言之,将小鼠纹状体组织样品称重并用Tissue Lyser II仪器(Qiagen)在含有丁基羟基甲苯(BHT,50μg/mL)和EDTA(0.5M)的500μL溶液中匀质化。此时,添加内标的混合物[表粪甾烷醇(epicoprostanol)、2H7-7-烯胆烷醇(lathosterol)、2H6-链甾醇(desmosterol)、2H6-羊毛甾醇和2H7-24(R/S)-羟基胆固醇](Avanti Polar Lipids)。在室温下使用0.35M乙醇KOH在Ar下进行碱性水解2h。用磷酸中和该溶液后,在氯仿中萃取固醇。收集下层相,在氮气流下干燥,并将残留物溶解在甲苯中。然后,在100mg Isolute硅胶柱(Biotage)上从胆固醇及其前体中分离氧化固醇;将胆固醇洗脱在处于己烷中的0.5%丙-2-醇中,然后将氧化固醇洗脱在处于己烷中30%丙-2-醇中。如前所述,用+10%TMCS[双(三甲基甲硅烷基)三氟乙酰胺+10%三甲基氯硅烷](Regis Technologies)分别将固醇和氧化固醇级分甲硅烷基化26。在中极性毛细管柱RTX-65(65%二苯基35%二甲基聚硅氧烷,长30m,直径0.32mm,膜厚0.25μm;Restesk)中,通过气相色谱法(Hewlett-Packard 6890系列)分离固醇和氧化固醇的三甲基甲硅烷基醚衍生物。建立了与气相色谱仪串联的质谱仪(Agilent5975 inert XL),用于检测正离子。在70eV下以电子撞击模式产生离子。将它们通过扫描模式下的碎片谱进行鉴别,并通过用适当的内标和外标进行归一化和校准后选择性监测特定离子来进行量化[表粪甾烷醇m/z 370,2H7-7-烯胆烷醇m/z 465,2H6-链甾醇m/z 358,2H6-羊毛甾醇m/z 504,2H7-24(R/S)-羟基胆固醇m/z 553,胆固醇m/z 329,7-烯胆烷醇m/z,7-去氢胆固醇m/z 325,8-去氢胆固醇m/z 325,链甾醇m/z 343,羊毛甾醇m/z 393以及24(R/S)-羟基胆固醇m/z 413]。
神经元存活的免疫荧光定量分析
以在Leica显微镜上的10×放大倍率的等同条件(par condition),在5个孔的不同条件下通过直接定量HA阳性细胞HA来评价神经元存活。
统计学分析
使用未配对的学生t检验进行统计学分析。结果表示为平均值±SEM。如文中所定义,显著阈值设置为P<0.05、P<0.01和P<0.001。所有分析均使用GraphPad Prism(GraphPad Software,La Jolla,USA)进行。
结果
对SOD1动物中胆固醇途径的基础评价
在8周龄的SOD1G93A和WT动物的腰脊髓中已经量化了胆固醇途径的数个基因的表达水平(图1)。已观测到CYP46A1和ApoE的显著降低(图1A),以及SREBP1、SREBP2和HMGCR的趋势(图1A)。此外,在15周龄时对SOD1G93A和WT动物的腰脊髓中的24-羟基胆固醇进行了测量,表明与WT动物相比,SOD1G93A中其含量大大降低(约60%减少)(图1B)。这些结果证实了在ALS模型中调节CYP46A1也挽救ALS表型的假设。
验证AAVPHP.eB作为ALS的良好载体
本发明人首先研究了AAVPHP.eB是否是用于ALS的良好载体。为此目的,向8周龄WT动物注射了低剂量(2.5.1011vg)、中剂量(5.1011vg)或高剂量(1.1012vg)的编码CYP46A1-HA的AAVPHP.eB。已突出显示了该3种剂量对运动神经元的良好靶向(图2),低剂量和高剂量之间无重大差异,转导率为50%至60%(图2)。
如通过GFAP(图3)和Iba1(未呈现的数据)染色评价所评估的(不引起任何小胶质细胞增生或星形胶质细胞增生),即使在高剂量组中,这种大量的转导与脊髓的任何免疫应答都无关(图3)。
这些结果促使以低剂量的载体继续进行。
在ALS的SOD1G93A小鼠模型中预防ALS表型
CYP46A1过表达在预防性处理的ALS小鼠模型中防止行为改变。
本发明人研究了通过CYP46A1水平的增加,胆固醇代谢途径的上调是否能够改善ALS的体内模型(SOD1G93A模型)中的运动改变。通过在3周龄的小鼠中预防性静脉内给予AAVPHP.eB-CYP46A1-HA进行的CYP46A1过表达明显防止了小鼠模型中的运动改变。首先,经AAV处理的SOD1G93A小鼠具有改善的生长曲线(图4A)。预防性AAVPHP.eB-CYP46A1-HA的递送显著修正了通过抱握试验测量的运动损伤(图4B),并完全防止了通过倒置试验测量的改变(图4C)。
生物分布研究显示了脊髓的所有水平中约1个载体基因组拷贝(VGC),在脑中2个到4个之间的拷贝(图5A),以及周围组织的非常低的转导,心脏中最大为0.4VGC,其它周围器官中小于0.1VGC(图5B)。已经用动物腰脊髓中的24羟基胆固醇的定量验证了靶标的参与,显示经处理的动物与WT动物相比的3倍增加(图5C)。
24OH胆固醇的这种增加与CYP46A1-HA在脊髓的所有水平上(特别是在运动神经元(MN)中)的大量表达完全相关(图6A),这也证实了先前在WT动物中获得的结果(图2)。
通过对VachT和HA的共染色的定量进行的评估,在腰水平上证实了MN的部分保留(与WT相比约60%)(图6B和图6C),胸段中与WT相比MN的数量相近,以及在颈水平无差异(图6C)。这种60%的保留足以避免腰脊髓脱髓鞘,如Luxol染色和髓鞘质百分比定量所证明的(图6D和图6E)。经AAV处理的SOD1动物具有与WT动物相等的髓鞘质百分比,并且显着高于未经SOD1处理的动物。
本发明人想要研究的其它方面是肌肉表型,实际上,肌肉在ALS中受到严重影响,这主要是由于相继于MN丧失的神经支配和神经肌肉接头(NMJ)的丧失。
他们首先证明了肌肉结构的显著保留,通过测量平均纤维面积进行评估(图7A)。经SOD1处理的动物的胫骨肌显示出与未经处理的动物相比显著增加的平均纤维面积(图7B)以及根据纤维横截面积进行的再分隔(repartition),明显地证明了大横截面纤维的保留(图7B)。对于腓肠肌(图7D和图7E)和四头肌(图7F和图7G),也观测到了相似的结果。
然后,他们集中于NMJ表型,证明:与未经处理的SOD1动物相比,经处理的动物的受神经支配的接头数量增加(图8A和图8B);以及NMJ的结构改善,其中值得注意的是具有正常终板或较厚的终板的NMJ数量较多,异位和碎片化终板的数量减少(图8C)。
该结果特别重要且有显著治疗意义,因为当本发明人在3周时在WT和未经治疗的SOD1G93A动物中评价NMJ的状态时,NMJ在3周时已经显示病理性,与WT相比SOD1动物中未成熟接头的数量增加(图8D和图8E)。这意味着即使在NMJ未完全形成时注射AAV-CYP46A1,它仍然能够显著改善经处理动物的表型。
为了完成研究,本发明人还在15周龄定量了在胫骨肌中Musk(参与集聚蛋白(agrin)结合的受体)和nAchR(乙酰胆碱的受体)的水平,两者都参与突触的稳定和NMJ的维持。在胫骨肌和腓肠肌中,MuSK和nAchR的表达水平均改善(图9)。
CYP46A1过表达在治疗性处理后减轻ALS小鼠模型的行为改变
基于对动物的预防性处理,进行了研究以确定通过增加CYP46A1水平进行的胆固醇代谢途径的上调是否可改善在有症状后的ALS体内模型(SOD1G93A模型)中的运动改变。通过在8周龄小鼠中静脉内给予AAVPHP.eB-CYP46A1-HA进行的CYP46A1过表达明显减轻了小鼠模型中的运动改变。与未经处理的动物相比,小鼠具有改善的生长(图10A)。
治疗性AAVPHP.eB-CYP46A1的给予明显降低了通过抱握测试测量的运动损伤(图10B),并减轻了通过倒置测试测量的变化(图10C)。此外,与未经处理的动物相比,该处理引起了经处理小鼠的生存增加,预期寿命增加了平均14天(图10D)。
在15周龄时对小鼠实施安乐死以进行组织学和分子分析。
生物分布研究显示了脊髓的所有水平中约3个-4个载体基因组拷贝(VGC),在脑中10个到20个之间的拷贝(图11A),以及周围组织的非常低的转导,肝中最大为0.5VGC,其它周围器官中小于0.2VGC(图11B)。已经用对动物腰脊髓中的24羟基胆固醇的定量验证了靶标的参与,显示经处理的动物与WT动物相比的1.3倍增加(图11C)。
通过VachT和HA的共染色的量化进行评估,在腰水平上证实了MN的部分保留(与WT相比约60%)(图12A和图12B),与预防性处理的结果相似(图6C)。
此处这种60%的保留再次足以避免腰脊髓脱髓鞘,如Luxol染色和髓鞘质百分比定量所证明的(图12C)。经AAV处理的SOD1动物具有与WT动物相等的髓鞘质百分比,并且显着高于未经处理的SOD1动物。
通过测量平均纤维面积(图13)进行的肌肉结构评估显示,与未经处理的动物相比,经处理的动物中的表型明显改善。与未经处理的动物相比,经处理的SOD1动物的四头肌显示出平均纤维面积的显著增加(图13E和图13F)。对于胫骨肌(图13A和图13B)、腓肠肌(图13C和图13D),观测到了相似的结果。
然后,他们集中于NMJ表型,证明:与未经处理的SOD1动物相比,经处理的动物的受神经支配的接头数量增加(图14A和图14B);以及NMJ结构改善,其中,值得注意的是具有正常终板的NMJ的数量较高,碎片化终板和未成熟终板的数量减少(图14C)。
最后,处理对MuSK和nAchR二者的表达水平的影响均比预防性处理轻(图15),但如在腓肠肌中那样有所改善(图15D)。
总之,这些数据强烈支持CYP46A1作为ALS中的相关靶标。然后,本发明人决定评价增加的AAV-CYP46A1剂量是否仍能改善有益效果,并已用治疗性处理以5.1011vg对SOD1G93A动物同生群进行注射。行为的初步结果表明强的效果,以及由抱握得分测量的行为的明显改善(图16)。小鼠处于12周,并将进行生存评价。
最后,本发明人声称CYP46A1可能是ALS的家族形式和散发形式二者的相关靶标,因此不仅靶向SOD1突变的患者,而且还靶向TARDP患者的C9ORF72突变患者。为了该目的,本发明人决定在ALS的其它模型上测试他们的治疗策略,第一个模型是具有500次重复的GGGGCC的C9ORF72小鼠模型。
在C9ORF72模型中,本发明人在第4周给予5.1011vg AAV-CYP46A1-HA作为预防性处理,并且目前已经进行了直至16周的跟踪。未观察到生长曲线方面的差异(图17A)。基于缺口棒测试,已经证明了运动表现的明显改善(图17B)。正在进行小鼠追踪,并将对生存进行评估。
总体而言,这些数据支持CYP46A1的过表达具有治疗性能,促进神经元生理学的改善,尤其是在ALS的体外模型中的自噬(图18),并且在症状已经出现后,处理的有症状后递送的数据对于ALS治疗而言令人鼓舞。
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Claims (13)
1.一种用于治疗肌萎缩性侧索硬化(ALS)的载体,所述载体包含编码胆固醇24-羟化酶的核酸。
2.如权利要求1所述的用于治疗ALS的载体,其中,所述ALS与至少一种运动神经元相关紊乱有关联。
3.如权利要求2所述的用于治疗ALS的载体,其中,所述ALS与额颞痴呆有关联。
4.如权利要求1-3中任一项所述的用于治疗ALS的载体,所述载体包含编码氨基酸序列SEQ ID NO 2的核酸序列。
5.如权利要求1-4中任一项所述的用于治疗ALS的载体,所述载体包含核酸序列SEQ IDNO 1。
6.如权利要求1-5中任一项所述的用于治疗ALS的载体,所述载体选自于由腺病毒载体、慢病毒载体、逆转录病毒载体、疱疹病毒载体和腺相关病毒(AAV)载体所组成的组。
7.如权利要求1-6中任一项所述的用于治疗ALS的载体,所述载体为AAV载体。
8.如权利要求7所述的用于治疗ALS的载体,所述载体为AAV9、AAV10载体,例如AAVrh.10或AAVPHP.eB,优选AAVPHP.eB。
9.如权利要求1-8中任一项所述的用于治疗ALS的载体,所述载体将被直接静脉内给予或给予至患者的脑中。
10.如权利要求9所述的用于治疗ALS的载体,所述载体将被给予至脊髓和/或运动皮层。
11.如权利要求10所述的用于治疗ALS的载体,所述载体将被给予至运动神经元。
12.如权利要求1-11中任一项所述的用于治疗ALS的载体,所述载体将通过血管内、静脉内、鼻内、脑室内或鞘内注射进行给予。
13.一种用于治疗肌萎缩性侧索硬化的药物组合物,所述组合物包含治疗有效量的如权利要求1-12中任一项所定义的载体。
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US20110034540A1 (en) * | 2007-09-12 | 2011-02-10 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Use of the CYP46A1 Gene for the Treatment of Alzheimer's Disease |
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CA3118163A1 (en) | 2020-05-07 |
EP3873511A1 (en) | 2021-09-08 |
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ES2936254T3 (es) | 2023-03-15 |
EP4205757A1 (en) | 2023-07-05 |
WO2020089154A1 (en) | 2020-05-07 |
EP3873511B1 (en) | 2022-12-07 |
US20220054597A1 (en) | 2022-02-24 |
AU2019373340A1 (en) | 2021-06-03 |
HUE060949T2 (hu) | 2023-04-28 |
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