CN113543814A - 用于治疗肾病综合征的aav基因疗法 - Google Patents
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Abstract
本发明提供了一种腺相关病毒(AAV)载体基因疗法,其用于治疗单基因形式的肾病综合征,其中AAV载体包含NS相关转基因和最小nephrin启动子NPHS1或podocin启动子NPHS2。
Description
发明领域
本发明涉及基因疗法,其用于治疗单基因形式的肾病综合征。
发明背景
肾病综合征(NS)是一种慢性肾脏疾病,其特征在于显著的蛋白尿、低白蛋白血症、水肿和高脂血症,并且是儿童中最常见的原发性肾小球疾病,在欧洲和美国影响了2/100,000的16岁以下儿童。NS与不同的发病年龄有关,从诊断时不到3个月大到成年早期,并根据其对皮质激素的敏感性分为不同的患者群体:约80%的患NS儿童被归类为患有激素敏感型肾病综合征(SSNS),并且可以用皮质类固醇疗法成功治疗。原本被归类为SSNS的一部分患者复发,并需要进一步的类固醇治疗,并且另外10-15%的NS患者在使用皮质类固醇治疗数周后没有达到缓解,并被归类为激素耐药型肾病综合征(SRNS)。高达50%的这些SRNS患者在10年内发展为终末期肾病,并且在肾移植后普遍面临着升高的复发风险,这突出表明对这些患者缺乏合适和有效的治疗。
足细胞功能障碍和导致的肾小球滤过屏障破坏是NS的发病机制的核心。足细胞分支出细胞突起以覆盖肾小球毛细血管的外部,称为足突,并且它们与相邻足突的交错接合形成肾小球裂孔膜,对肾小球滤过屏障的效率和蛋白质在血流中的保留至关重要。在NS的遗传形式中,编码关键足细胞过程,如足细胞的发育、迁移、基底膜相互作用或再生的基因突变导致肾小球裂孔膜的完整性丧失和肾病综合征表型。大约30%的儿童中SRNS病例是遗传性的,其中在儿童期最常见的突变是在NPHS2编码podocin,占散发性遗传病例的10-30%。
podocin是一种42kDa发夹样膜相关的足细胞特异性蛋白,是裂孔膜处蛋白复合物的关键组分;相邻足细胞足突之间的细胞间连接。其定位于脂筏并与其他重要的裂孔膜蛋白如nephrin、CD2AP和TRPC6相互作用。其在维持裂孔膜,及因而维持肾小球滤过屏障的完整性中至关重要。迄今为止报告有126个突变,但最常见的突变是R138Q,其导致podocin错误定位于内质网。
由于目前不存在对患有单基因形式的NS患者的有效治疗方法,使用基因疗法在患病的足细胞中转移功能性基因拷贝可能构成一个有希望的新策略,以解决单基因形式的NS,逆转NS表型并纠正肾功能障碍。事实上,US2003/0152954通常建议使用病毒载体来递送编码具有podocin活性的多肽的核酸,但未能公开或测试任何特定的基因疗法构建体。这可能是因为肾脏具有复杂的解剖学,有由肾小球、肾小管、脉管系统和小间隙组成的专门隔室,这使得它成为基因疗法载体的一个困难的靶标。迄今为止,肾脏靶向基因疗法在很大程度上是不成功的,因为肾脏高度分化的亚结构很难用病毒载体的方式进行靶向和特异性转导(van der Wouden et al.,2004)。
最近的一项研究试图使用rAAV载体结合CMV启动子和GFP或萤光素酶基因(经由尾静脉注射或肾静脉注射施用)来靶向肾脏(Rocca et al 2014)。然而,尾静脉注射证明不适合用于肾脏转导,并且尽管在足细胞中观察到低水平的基因表达,但在肝脏中也观察到广泛的表达,即使使用了一个据说的肾脏特异性启动子。该研究另外未能证明NS相关的转基因,如podocin的成功转导,也未能证明此类基因的长期功能表达。该研究也没有探索适合人肾细胞转导的AAV血清型。
本发明的目的是通过施用在足细胞特异性启动子的控制下表达NS相关转基因的AAV基因疗法,逆转NS表型,并纠正患有单基因形式的NS患者的足细胞相关的肾功能障碍。
发明概述
本发明提供了一种腺相关病毒(AAV)载体基因疗法,用于治疗单基因形式的肾病综合征,其中AAV载体包含:NS相关转基因;以及最小nephrin启动子NPHS1或podocin启动子NPHS2。该基因疗法载体可以逆转NS表型,并纠正患有单基因形式的NS患者的足细胞相关的肾功能障碍。
适合用于该载体中的AAV血清型包括2/9、LK03和3B。
AAV 2/9血清型已显示出对新生和成年小鼠肾脏的明显趋性,定位于肾小球和肾小管(Luo et al.,2011;Picconi et al.,2014;Schievenbusch et al.,2010),并且AAV2/9载体结合肾静脉注射已显示适用于肾脏靶向基因递送(Rocca at al.,2014)。因此,AAV2/9是一种用于本发明的基因疗法的合适载体。
合成的AAV衣壳如LK03也可以是用于本发明的基因疗法的合适载体。该载体已显示在体外和体内以高效率转导人原代肝细胞。然而,到目前为止,它还没有被用于肾脏靶向的基因递送。本发明人在本文中证明,AAV-LK03载体可以在体外人足细胞中实现接近100%的高转导,并可用于在体外特异性地转导足细胞。
AAV-LK03 cap序列由来自七种不同的野生型血清型(AAV1,2,3B,4,6,8,9)的片段组成,尽管AAV-3B代表了97.7%的cap基因序列和98.9%的氨基酸序列。AAV-3B也因其对人肝细胞的趋性而闻名,其是另一种用于本发明基因疗法的合适载体。到目前为止,它还没有被用于肾脏靶向的基因递送。
基因疗法中使用的NS相关转基因是与NS的单基因形式相关的基因,并在足细胞中表达,其编码一种约833个氨基酸或更少的蛋白质。这种尺寸限制使NS相关转基因适合本发明的基因疗法载体。
适合的NS相关转基因包括NPHS2;ADCK4;ALG1;ARHGAP24;ARGHDIA;CD151;CD2AP;COQ2;COQ6;DGKE;E2F3;EMP2;KANK2;LAGE3;LMNA;LMX1B;MAFB;NUP85;NUP93;NXF5;OSGEP;PAX2;PDSS2;PMM2;PODXL;SCARB2;SGPL1;Smad7;TP53RK;TPRKB;VDR;WDR73;WT1;ZMPSTE24;或APOL1。
在本发明的实施方案中,NS相关转基因可以是SRNS相关的转基因,如ADCK4;CD2AP;DGKE;EMP2;NPHS2;NUP86;NUP93;SGPL1;WDR73;或WT1。
在本发明的优选实施方案中,NS相关转基因是NPHS2,其编码podocin。合适的人NPHS2转基因cDNA序列的一个示例显示于图6中。
转基因物种优选与患者物种相匹配。例如,当治疗人类患者时,通常使用人类转基因。转基因可以是天然存在的,例如野生型,或者其可以是重组的。转基因通常作为一个cDNA序列包含在基因疗法载体中。
使用最小nephrin启动子如NPHS1或podocin启动子NPHS2允许基因疗法载体特异性地靶向至足细胞(Moeller et al.,2002;Picconi et al.,2014)。这使得转基因表达能够特异性地靶向在肾脏的肾小球基底膜中的足细胞,并最小化脱靶表达。由于足细胞是终末分化和非分裂的细胞,可以靶向它们以稳定表达转基因,并减少或避免任何载体稀释效应的风险。在本发明的优选实施方案中,启动子是NPHS1。NPHS1启动子的合适的DNA序列的一个示例显示于图5中。与转基因一样,启动子物种优选与患者物种相匹配。例如,在治疗人类患者时通常使用人NHPS1或人NPHS2。
AAV载体可以另外包含土拨鼠肝炎转录后调节元件(WPRE)。WPRE是DNA序列,当转录时,其产生三级结构增强表达。包含WPRE可以提高由载体递送的转基因的表达。可以突变WPRE序列以在不显著损失RNA增强活性的情况下减少致癌性(Schambach et al.,2005,通过引用并入本文)。合适的WPRE序列的一个示例显示于图7中。
NS相关转基因可以包含血凝素(HA)标签。HA可作为表位标签使用,并已显示不干扰所加入的蛋白质的生物活性或生物分布。HA标签可以促进转基因的检测、分离和纯化。
AAV载体可以另外包含启动子和podocin转基因之间的Kozak序列。已知Kozak序列在翻译过程的启动中起着重要作用,因此可以增强podocin转基因的表达。
AAV载体可以另外包含多腺苷酸化信号,如牛生长激素(bGH)多腺苷酸化信号,例如,如图8中所示。多腺苷酸化是在信使RNA上增加一个poly(A)尾。poly(A)尾由多个一磷酸腺苷组成;换句话说,它是一段仅具有腺嘌呤碱基的RNA。poly(A)尾对mRNA的核输出、翻译和稳定性非常重要。因此,包含多腺苷酸化信号可以增强podocin转基因的表达。
AAV载体基因疗法另外通常包括在载体的任一末端的末端反向重复(ITR)序列。例如,载体结构可以按顺序为:ITR-启动子-转基因(具有任选的HA标签)-任选的WRPE-多腺苷酸化信号-ITR。
因此,本发明的基因疗法载体可用于治疗或管理患者中的单基因形式的NS。如本文所用,术语“患者”可包括任何哺乳动物,包括人类。患者可以是成人或儿科患者,如新生儿或婴儿。在本发明的实施方案中,患者可以是年龄在约1岁和约16岁之间的儿科患者。
患者患有单基因形式的NS。换句话说,NS是由一个基因的突变引起的。优选地,突变在足细胞中表达的基因中。例如,NS可以是NPHS2(其编码podocin)中的基因突变引起的SRNS。或者,NS的单基因形式可以由ADCK4;ALG1;ARHGAP24;ARGHDIA;CD151;CD2AP;COQ2;COQ6;DGKE;E2F3;EMP2;KANK2;LAGE3;LMNA;LMX1B;MAFB;NUP85;NUP93;NXF5;OSGEP;PAX2;PDSS2;PMM2;PODXL;SCARB2;SGPL1;Smad7;TP53RK;TPRKB;VDR;WDR73;WT1;ZMPSTE24;或APOL1的任何一个中的一个或多个突变引起。在本发明的实施方案中,NS的单基因形式可以是由ADCK4;CD2AP;DGKE;EMP2;NPHS2;NUP86;NUP93;SGPL1;WDR73;或WT1的任何一个中的一个或多个突变引起的SRNS的单基因形式。
引起SRNS的基因突变可以是影响podocin表达的NPHS2突变,如下方表A中所列出的那些的一种或多种。
表A:podocin(NPHS2)突变的非详尽列表。
在本发明的优选实施方案中,基因突变可以是p.Arg138Gln,也称为R138Q。R138Q是高加索人群中患SRNS儿童中最常见的podocin突变。该突变导致podocin的内质网滞留,阻止其到达裂孔膜并与其他重要的裂孔膜蛋白相互作用以形成功能性滤过屏障。
由于NPHS2突变都影响同一基因,这些突变的任何组合都可以通过本发明中包含NPHS2转基因的AAV基因疗法载体来治疗。换句话说,患者可以具有p.Arg138Gln突变,并且可以具有以上表A中鉴定其他突变的一个或多个。
单基因形式的NS的存在或不存在可以通过实验室测试来确定,如英国布里斯托遗传学实验室提供的测试。通常情况下,基因测试可以通过分析从患者获得的血液样品来进行。
AAV载体基因疗法可以系统性地施用,如通过静脉内注射。在本发明的实施方案中,AAV载体基因疗法可以通过注射至肾动脉来施用。在本发明的另一个实施方案中,AAV载体基因疗法可以通过逆行施用来施用,例如使用导尿管经由输尿管。
基因疗法可以以单剂量施用,换句话说,可以不需要后续剂量的载体。在需要重复剂量的情况下,可以在载体中使用不同的AAV血清型。例如,用于首次剂量中的载体可以包含AAV-LK03或AAV-3B,而用于后续剂量中的载体可以包含AAV 2/9。
任选地,可以施用基因疗法结合患者的暂时性免疫抑制,例如通过在口服类固醇治疗的同时或之后施用基因疗法。在基因疗法治疗前和/或期间可以期望免疫抑制以抑制患者对载体的免疫反应。然而,AAV衣壳在转导的细胞中仅短暂地存在,因为它不是由载体编码的。因此,衣壳逐渐降解和清除,这意味着阻断对衣壳的免疫反应直到衣壳序列从转导细胞中清除的短期的免疫调节方案可以允许转基因的长期表达。因此,在施用基因疗法后约六周的时段内,免疫抑制可以是期望的。
AAV载体基因疗法可以以药物组合物的形式施用。换句话说,AAV载体基因疗法可以与一种或多种药学上可接受的载体或赋形剂相结合。合适的药物组合物优选是无菌的。
附图简述
现在将参照附图,仅以示例的方式对本发明进行详细描述。
图1显示了通过尾静脉注射施用的AAV 2/9转导肾脏,并在足细胞中表达带HA标签的podocin。A)用来表达小鼠或人podocin或GFP的AAV载体。所有载体均含有启动子和转基因之间的Kozak序列,以及WPRE(土拨鼠肝炎转录后调节元件)和牛生长激素(bGH)多腺苷酸化信号。B)在处于8周龄的iPod NPHS2fl/fl小鼠中经由尾静脉注射载体或盐水,并在10-14天后开始用多西环素诱导。C)qPCR显示在注射病毒载体的小鼠中的小鼠肾皮质中存在AAVITR。D)代表性免疫荧光显示在注射AAV 2/9的iPod NPHS2fl/fl小鼠中,带HA标签的podocin与足细胞特异性蛋白nephrin和podocin的表达。对照(盐水)图像是未注射完整iPodNPHS2fl/fl基因型的小鼠的图像,因此未出现蛋白尿或病变肾小球,因为具有病变肾小球的小鼠显示出足细胞标志物的缺失。
图2显示尾静脉注射在足细胞特异性启动子下表达野生型podocin的AAV 2/9改善条件性podocin敲除小鼠模型(iPod NPHS2fl/fl)中的蛋白尿。A)注射AAV 2/9mNPHS1.mpod相对于AAV 2/9hNPHS1.mpod相对于盐水的小鼠的尿白蛋白:肌酐比率(每组n=9,**p<0.01***p<0.001)。B)考马斯染色显示来自各实验组的一只小鼠中白蛋白尿程度的代表性图像。盐水组从第14天开始显示蛋白尿,并显示大量的白蛋白,而载体处理组显示白蛋白尿发作较晚且白蛋白尿较轻。C)生存曲线显示注射AAV 2/9hNPHS1.mpod或AAV 2/9mNPHS1.mpod的小鼠中改进的生存(对数秩(Mantel-Cox)检验p=0.049,每个病毒组中n=3,且盐水组中n=4)。D)每50ng总DNA中病毒DNA的拷贝数与第42天的尿白蛋白:肌酐比率呈负相关(斯皮尔曼r=-0.4596,p=0.0477)。E)多西环素后6周的血液结果,包括胆固醇、白蛋白、尿素和肌酐。(每组中n=至少3只小鼠,除胆固醇外,其每组中至少n=2)。F)组织学显示光镜下来自各组的代表性图像。注射盐水组显示肾小球肥大,胶原沉积和节段性硬化增加,伴有肾小管扩张,这与FSGS一致。注射表达小鼠podocin的AAV 2/9的那些表现出一系列的组织学发现,这些发现与它们死亡时的尿白蛋白:肌酐比率大致相关。一些小鼠具有健康正常的肾小球,而其他则显示轻微的疾病迹象,如注射AAV 2/9mNPHS1.mpodHA的小鼠中所见的假性新月形形成(箭头)。G)注射生理盐水的iPod NPHS2fl/fl小鼠显示podocin缺失,而nephrin表达显示出由主要的膜性染色向弥漫性模式变化。
图3.AAV LK03显示体外用最小人nephrin启动子对人足细胞的有效率的转导。A,C,E)免疫荧光表明AAV LK03 CMV GFP对人足细胞(Pod)、肾小球内皮细胞(GEnC)和近端小管上皮细胞(PTEC)的转导,当使用最小nephrin启动子AAV LK03 hNPHS1 GFP时,仅在足细胞中表达GFP。B)仅当使用利用AAV LK03的最小人nephrin启动子时,Western印迹表明足细胞中的GFP表达。D)流式细胞术表明使用AAV LK03 CMV GFP对足细胞的高效率的转导,并证实使用最小nephrin启动子的GFP表达仅见于足细胞中。相比之下,AAV 2/9CMV GFP显示在足细胞中的低转导效率(n=3)。F)条形图显示用AAV LK03转导的足细胞中的中位数荧光强度,且直方图显示用AAV LK03 CMV GFP(右侧峰)、AAV LK03 hNPHS1 GFP(中间峰)转导的足细胞和未转导的细胞(左侧峰)中的绿色荧光的程度。
图4.表达野生型人podocin的AAV LK03显示突变体podocinR138Q足细胞细胞系中的功能挽救。A)Western印迹显示AAV LK03.CMV.hpodocinHA和AAVLK03.hNPHS1.hpodocinHA转导R138Q足细胞并表达带HA标签的podocin。B)免疫荧光表明突变体podocin R138Q足细胞中带HA标签的野生型podocin的表达。C)粘附测定显示突变体podocinR138Q足细胞的粘附的降低,及用AAV LK03.hNPHS1.hpodHA.WPRE.bGH处理的R138Q足细胞中粘附的挽救。D)共聚焦显微术显示带HA标签的podocin不与钙连蛋白(一种内质网标志物)共定位。E)TIRF显微术表明100nm的质膜内带HA标签的podocin的表达,与小窝蛋白(一种脂筏标志物)有一些共定位。
图5显示最小人nephrin启动子(NPHS1)的示例DNA序列。
图6显示人nephrin转基因的示例cDNA序列。
图7显示WPRE序列的示例DNA序列。
图8显示bGH poly(A)信号序列的示例DNA序列。
图9显示使用具有最小人nephrin启动子的AAV LK03用HAVDR(A)或HASmad7(B)转导的人足细胞。
实施例
方法
载体产生
我们在我们的实验室中使用人(图6)和小鼠(序列未显示)podocin cDNA(Origene,Herford,Germany)以及人VDR和Smad7 cDNA,从CMV eGFP L22Y pUC-AV2构建体(Amit Nathwani的友好礼物)制备了pAV.hNPHS1.mpodHA.WPRE.bGH、pAV.mNPHS1.mpodHA.WPRE.bGH和pAV.hNPHS1.hpodHA.WPRE.bGH(图1A)pAV.mNPHS1.hHAVDR.WPRE.bGH和pAV.mNPHS1.hHASmad7.WPRE.bGH。用衣壳质粒(pAAV9来自Penn Vector Core,pAAV LK03是Mark Kay的友好礼物),具有腺病毒基因的辅助质粒和使用聚乙烯亚胺的转基因质粒转染人胚胎肾脏293T细胞。在转染后72小时收获细胞和上清液。细胞经历5个冻融循环,而上清液经历PEG沉淀(8%PEG 0.5N NaCl)。将这些合并,并与0.25%脱氧胆酸钠和70单位/ml Benzonase在37℃下温育30分钟。通过碘克沙醇梯度超速离心纯化载体,随后在PBS中浓缩。采用标准曲线法通过qPCR对载体进行滴定,使用如下引物:
ITR F GGAACCCCTAGTGATGGAGTT,
ITR R CGGCCTCAGTGAGCGA,
ITR探针FAM-5'-CACTCCCTCTGCGCTCG-3'-TAMRA。
动物
所有的动物实验和规程均由英国内政部根据1986年动物(科学规程)法案批准,并且在实验过程中遵循《实验动物护理和使用指南》(Guide for the Care and Use ofLaboratory Animals)。NPHS2flox/flox小鼠(Corinne Antignac的友好礼物,INSERM U983,Paris)与NPHS2-rtTA/Tet-On Cre小鼠交配以产生具有NPHS2-rtTA/Tet-On Cre/NPHS2flox /flox的后代。在暴露于多西环素时,这些小鼠出现足细胞特异性的podocin敲除。从这里开始,这些将称为iPod NPHS2fl/fl。小鼠处于混合背景且使用相等数量的每种性别。在8周龄时经由尾静脉注射对小鼠施用AAV。(图1B)10至14天后,向小鼠提供补充有多西环素2mg/ml和5%蔗糖的饮用水3周。每周获取尿。在多西环素开始后的6周,通过时间表1的方法处死小鼠。少数小鼠保留超过6周以测试对生存的影响。所有的小鼠均根据死亡时取得的组织中重新基因分型。
细胞培养
条件性永生化人足细胞(Pod)在具有L-谷氨酰胺和NaHCO3以及10%胎牛血清(Sigma Aldrich,Gillingham,UK)的RPMI中培养。条件性永生化人肾小球内皮细胞(GEnC)在补充有EGMTM-2内皮细胞生长培养基-2BulletKitTM(Lonza,Basel,Switzerland)的EBMTM-2内皮细胞生长基础培养基-2中培养。永生化近端小管上皮细胞(ATCC,Teddington,UK)(PTEC)在补充有胰岛素、转铁蛋白和硒、氢化可的松和10%FBS的DMEM/F12中培养。
用AAV以5x 105的MOI转导细胞。对于GFP表达,在转导后5-7天使用细胞,以允许在不同的细胞系之间进行比较。对于podocin、VDR和Smad7表达,在转导后的10-14天(足细胞最大程度地分化时)使用细胞。
定量PCR
使用DNeasy血液和组织试剂盒(Qiagen,Manchester,UK)从小鼠肾皮质提取DNA。使用上述引物检测AAV DNA,用于病毒滴定,并针对小鼠beta-肌动蛋白标准化。
使用RNeasy Mini Kit与RNase-Free DNase套装(Qiagen,Manchester,UK)提取RNA。
免疫荧光
使用4%PFA固定5μm的切片,并用3%BSA 0.3%Triton X-100和5%山羊或驴血清进行封闭。一抗是来自大鼠IgG1抗HA高亲和力(Roche,Basel,Switzerland),豚鼠抗nephrin(1243-1256)抗体(Origene,Herford,Germany)和兔抗NPHS2抗体(Proteintech,Manchester,UK)。
用4%PFA和/或冰冷的甲醇固定细胞,与0.03M甘氨酸温育5分钟,用0.3%Triton透化,然后用3%BSA进行封闭。一抗是小鼠HA.11表位标签抗体(Biolegend,San Diego,USA),小鼠抗GFP(Roche,Basel,Switzerland),兔抗钙连蛋白(Merck Millipore,Darmstadt,Germany)和兔抗小窝蛋白1(Cell Signaling,Danvers,USA)。
二抗是AlexaFluor 488驴抗小鼠、AlexaFluor 488驴抗兔、AlexaFluor 488山羊抗豚鼠、AlexaFluor 555山羊抗兔和AlexaFluor 633山羊抗大鼠,以及AlexaFluor 633鬼笔环肽(Invitrogen,Thermo Fisher Scientific,Waltham,USA)。切片用DAPI复染,并用Mowiol封装。使用LAS(Leica应用套件)X软件在附接到Leica DMi8倒置落射荧光显微镜的Leica SPE单通道共聚焦激光扫描显微镜,或附接到Leica DMI 6000倒置落射荧光显微镜的Leica SP5-II共聚焦激光扫描显微镜,或附接到Leica DMI 6000倒置落射荧光显微镜的Leica AM TIRF MC(多色)系统上拍摄图像。
Western印迹
在SDS裂解缓冲液中提取细胞。样品在12.5%凝胶上运行并转移到PVDF膜上。在TBST 0.1%中的5%乳中对膜进行封闭。使用的一抗是小鼠HA.11表位标签抗体(Biolegend,San Diego,USA),在TBST 0.1%中3%BSA中的小鼠抗GFP(Roche,Basel,Switzerland),或兔抗NPHS2抗体(Proteintech,Manchester,UK)。二抗是在TBST 0.1%中3%BSA中的抗兔或抗小鼠IgG过氧化物酶(Sigma Aldrich,Gillingham,UK)。膜在AmershamImager 600上成像。
流式细胞术
活细胞用碘化丙啶染色,且只有活的单细胞纳入分析中。在NovoCyte流式细胞仪上进行流式细胞术。
粘附测定
细胞经胰蛋白酶处理并以105/ml重悬,并允许恢复10分钟,然后将50μl的用PBS以1比2稀释的细胞铺板于96孔板中。使用技术上一式三份。在37℃下让细胞粘附约1小时。用PBS洗涤细胞以洗去非粘附的细胞,然后用4%PFA固定20分钟。用蒸馏水洗涤细胞,然后用2%乙醇中0.1%结晶紫在室温下染色60分钟。洗涤细胞并与10%乙酸在摇床上温育5分钟。在570nm处测量吸光度,并针对用AAV LK03 CMV GFP转导的野生型细胞系对结果标准化。
尿
使用小鼠白蛋白ELISA试剂盒(Bethyl Laboratories Inc,Montgomery,USA)测量白蛋白水平,并在Konelab Prime 60i分析仪上测量肌酐水平。
血液测试
以制造商供应的试剂和方案使用Konelab Prime 60i分析仪或Roche Cobas系统处理小鼠血浆。
统计学分析
除非另有说明,所有数据均以平均值±SEM表示。统计学分析在GraphPad Prism(Graphpad softward,La Jolla,USA)中进行。使用的统计学检验包括双尾t检验,单因素ANOVA与Tukey多重比较事后分析,双因素ANOVA与Tukey多重比较事后分析,以及对数秩(Mantel-Cox)检验用于生存分析。
结果
尾静脉注射AAV血清型9证明了肾细胞的转导和在足细胞中的表达
在8周龄时,经由尾静脉对小鼠施用1.5×1012vg的AAV2/9hNPHS1.mpod或AAV2/9mNPHS1.mpod,或盐水。6周后,在注射AAV的小鼠肾皮质中检测到AAV ITR(AAV 2/9hNPHS1.mpod=39,067±13,285拷贝ssDNA,AAV 2/9mNPHS1mpod=76,533.33±32047拷贝ssDNA,n=5-6/组)(图1C)。带HA标签的podocin显示与足细胞标志物nephrin和podocin共定位(图1D)
表达野生型podocin的AAV2/9减少了iPod NPHS2fl/fl小鼠中的白蛋白尿
载体处理组显示尿白蛋白:肌酐比率(ACR)的减少(图2A,2B)。尾静脉注射表达podocin的AAV 2/9对尿ACR的影响产生的F比率为F(2,24)=9.61,P<0.001(n=9/组)。在多西环素后14天,盐水组中的尿ACR高于任何一个载体处理组,尽管这并不显著(AAV 2/9hNPHS1.mpod=758.1±488.1mg/mmol,AAV 2/9mNPHS1.mpod=59.8±28.0mg/mmol,盐水=3,770.1±1337.6mg/mmol,AAV 2/9hNPHS1.mpod vs盐水p=0.40,AAV 2/9mNPHS1.mpodvs盐水p=0.25)。载体处理组中尿ACR在第28天(AAV 2/9hNPHS1.mpod=3,083.0±932.8mg/mmol,AAV 2/9mNPHS1.mpod=2,195.1±778.9mg/mmol,盐水=10,198±3,189.5mg/mmol,AAV 2/9hNPHS1.mpod vs盐水p=0.008,AAV 2/9mNPHS1.mpod vs盐水p=0.002)和第42天(AAV2/9hNPHS1.mpod=3,266.8±1,212.2mg/mmol,AAV 2/9mNPHS1.mpod=3,553.3±1,477.87mg/mmol,盐水=13,488.8±3,877.3mg/mmol,AAV 2/9hNPHS1.mpodvs盐水p<0.001,AAV 2/9mNPHS1.mpod vs盐水p<0.001)明显减少。在载体处理组中,AAV 2/9hNPHS1.mpod组中9只小鼠中的2只和AAV 2/9mNPHS1.mpod组中9只小鼠中的1只在第42天尿ACR小于30mg/mmol。
尽管载体处理组中的小鼠表现出改善,但各组内有很大程度的差异,我们假设这可能归因于系统性注射后到达肾脏的载体的量。肾皮质中检测到的病毒DNA的量与第42天的白蛋白尿的程度呈负相关(斯皮尔曼r=-0.4596,p=0.0477)(图2D)。
表达野生型podocin的AAV2/9部分挽救了iPod NPHS2fl/fl小鼠的表型
载体处理的小鼠显示出肌酐减少(盐水=39.0±8.5μmol/L,AAV 2/9hNPHS1.mpod=27.3±7.9μmol/L,AAV 2/9mNPHS1.mpod=18.6±4.4mmol/L,p=0.1622),尿素减少(盐水=39.4±17.6mmol/L,AAV 2/9hNPHS1.mpod=12.0±2.0mmol/L,AAV 2/9mNPHS1.mpod=11.6±1.6mmol/L,p=0.058),白蛋白增加(盐水=10.5±5.4g/L,AAV 2/9hNPHS1.mpod17.1=4.8±g/L,AAV 2/9mNPHS1.mpod=17.1±3.6g/L,p=0.5602)和胆固醇的显著减少(盐水=15.76±1.75mmol/L,AAV 2/9hNPHS1.mpod=2.64±0.60mmol/L,AAV 2/9mNPHS2.mpod=4.86±0.76mmol/L,p=0009)(图2E)。
盐水处理的小鼠在6周前出现FSGS的组织学特征。载体处理的小鼠在光镜下未显示FSGS的组织学特征,但表现出一系列的组织学发现,从完全正常的肾小球到假性新月形或肾小球系膜细胞过多(mesangial hypercellularity)。(图2F)
这些小鼠还表现出生存延长(n=3-4/组),其中盐水组中的中位数生存期为75.5天(范围为38至111天),相比于AAV 2/9hNPHS1.mpod中的中位数生存期为192天(范围为74至206天仍活着)和AAV 2/9mNPHS1.mpod中的中位数生存期为192天(范围为131至206天仍活着)(p=0.049)。
未经处理的小鼠显示出podocin的表达缺失,nephrin的表达模式变为弥漫性模式(图2G)。这与载体处理的小鼠中nephrin和podocin的主要膜性表达模式形成了鲜明的对比(图1D)。
AAV LK03在体外用最小人nephrin启动子有效率地转导人足细胞
使用具有CMV GFP的AAV LK03和AAV LK03 hNPHS1 GFP以5×105的MOI转导人足细胞、肾小球内皮细胞和近端小管上皮细胞。流式细胞术(n=3)显示,AAV LK03 CMV GFP对足细胞有高效率的转导(%GFP表达=98.83±0.84),AAV LK03 hNPHS1 GFP有良好的转导(%GFP表达=71.3±3.39),且未转导细胞的表达不明显(%GFP表达=0.89±0.36)(图3D)。这反映在免疫荧光(图3A、3C、3E)和Western印迹(图3B)上。虽然在用AAV LK03 hNPHS1 GFP转导的足细胞中,GFP表达阳性的细胞比例很高,但这些细胞的荧光强度低于用AAV LK03 CMVGFP转导的细胞(图3F)。
有趣的是,AAV LK03 CMV GFP在肾小球内皮细胞中显示更低的转导(%GFP表达=7.35±0.19)。AAV LK03 hNPHS1 GFP在肾小球内皮细胞中显示出最小的转导(%GFP表达=0.59±0.10),与未转导的肾小球内皮细胞(%GFP表达=0.23±0.02)水平相似。由于AAV2/9是啮齿动物肾脏中体内肾细胞中转导最好的血清型,我们测试了AAV 2/9CMV GFP在人肾细胞系上的表达。AAV 2/9CMV GFP在足细胞(%GFP表达=13.9±1.98)和肾小球内皮细胞(%GFP表达=21.99±4.35)两者中都显示低的转导效率(图3D)。使用AAV LK03与AAVLK03 hNPHS1 HAVDR和AAV LK03 hNPHS1 hSmad7转导人足细胞,显示两种蛋白的良好表达(图9)。
在最小nephrin启动子下表达人podocin的AAV LK03在突变体podocin R138Q足细胞细胞系中显示出功能挽救
R138Q podocin突变体导致podocin从质膜到内质网的错误定位。从患者肾脏中获得突变体podocin R138Q足细胞细胞系,并使用温度敏感的SV40T抗原进行条件性永生化。AAV LK03 hNPHS1 hpod转导R138Q足细胞并表达带HA标签的podocin(图4A,4B)。在共聚焦显微镜下见到带HA标签的podocin在质膜上,并如TIRF显微镜下所见,与小窝蛋白-1(一种脂筏蛋白)共定位(图4B,4E)。未转导的R138Q足细胞在质膜处未显示任何podocin表达(图4B)。带HA标签的podocin不与钙连蛋白(一种内质网标志物)共定位(图4D)。
足细胞在疾病状态下显示出粘附的减少或增加。我们实验室中以前的工作表明,R138Q突变导致足细胞粘附下降。AAV转导导致足细胞粘附下降,但与野生型足细胞相比,R138Q足细胞仍显示粘附降低,且用AAV LK03 hNPHS1 hpod转导导致R138Q足细胞的粘附功能得到挽救(图4C)。
讨论
此处,我们用使用最小nephrin启动子的AAV 2/9成功地靶向小鼠足细胞,以在条件性小鼠基因敲除模型中表达小鼠podocin,在载体处理的小鼠中看到部分表型的挽救和白蛋白尿的改善。作为首次原理证明研究,我们选择在多西环素诱导前注射载体,使得当podocin敲除时,载体的有效挽救就位。多西环素诱导的效果迅速,且进展至严重的肾病(8-14天)和FSGS相对快速的(约6周)。我们在此表明,在体外,将野生型人podocin引入R138Q足细胞能使podocin的表达达到质膜,并挽救足细胞粘附。
尽管我们已经证明该载体改善了这些小鼠中的白蛋白尿和生存,但在经处理和未经处理的两种小鼠中,白蛋白尿的程度存在很大程度的变化性。经处理小鼠内的变化性至少可以部分地用肾脏中的病毒转导的量来解释(图2D)。
AAV LK03在体外人足细胞中显示出接近100%的高转导,当使用最小人nephrin启动子时其降低至72.3%。我们已经证明,我们可以使用该血清型在体外特异性地转导足细胞,并且野生型podocin在R138Q突变体足细胞中的表达显示出功能性挽救。使用AAV LK03对转化有潜在影响,因为人足细胞的此类有效转导可以使人中的有效剂量显著减少。最近英国的一项研究显示23%的低抗AAV LK03中和抗体血清阳性率,并在儿童晚期达到最低点(Perocheau,D.P.et al.),这使得这一特殊血清型成为转化研究的有希望的候选者。
我们描述了首次原理证明研究,证明具有足细胞特异性启动子的AAV转导足细胞改善iPod NPHS2fl/fl小鼠模型中的白蛋白尿。我们还表明一种合成的衣壳AAV LK03,显示出对人足细胞的高效率的转导。综上所述,这项工作是朝向靶向足细胞的单基因疾病的AAV基因疗法的转化的第一步。
参考文献
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序列列表自由文本
[SEQ ID NO:1]显示ITR正向引物。
[SEQ ID NO:2]显示ITR反向引物。
[SEQ ID NO:3]显示ITR探针FAM-5'-CACTCCCTCTGCGCTCG-3'-TAMRA的DNA序列。
[SEQ ID NO:4]显示图5中所示的最小人nephrin启动子(NPHS1)的示例DNA序列。
[SEQ ID NO:5]显示图6所示的人podocin转基因的示例cDNA序列。
[SEQ ID NO:6]显示图7所示的WPRE序列的示例DNA序列。
[SEQ ID NO:7]显示图8所示的bGH poly(A)信号序列的示例DNA序列。
序列表
<110> 布里斯托尔大学
<120> 疗法
<130> P122719PCT
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> ITR正向引物
<400> 1
ggaaccccta gtgatggagt t 21
<210> 2
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> ITR反向引物
<400> 2
cggcctcagt gagcga 16
<210> 3
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> ITR探针
<400> 3
cactccctct ctgcgcgctc g 21
<210> 4
<211> 1192
<212> DNA
<213> 智人
<400> 4
cacctgaggt caggagttcg agaccagcgt ggccaacatg atgaaacccc gtctctagta 60
aaaatacaaa aattagccag gcatggtgct atatacctgt agcaccagct acttgggaga 120
cagaggtggg agaattactt gaacctggga ggttcaagcc atgggaggtg gaagttgcag 180
tgagccgaga tgccactgca ctccagcctg agcaacagag caagactatc tcaagaaaag 240
aaagaaagaa agaaagagac ttgccaaggt catgtatcag ggcaaggaag agctgggggc 300
ccagctggct gctcccctgc tgagctggga gaccaccttg atctgacttc tcccatcttc 360
ccagcctaag ccaggccctg gggtcacgga ggctggggag gcaccgagga acgcgcctgg 420
catgtgctga caggggattt tatgctccag ctgggccagc tgggaggagc ctgctgggca 480
gaggccagag ctgggggctc tggaaggtac ctgggggagg ttgcactgtg agaatgagct 540
caagctgggt cagagagcag ggctgactct gccagtgcct gcatcagcct catcgctctc 600
ctaggctcct ggcctgctgg actctgggct gcaggtcctt cttgaaaggc tgtgagtagt 660
gagacaagga gcaggagtga ggggtggcag gagagaagat agagattgag agagagagag 720
agagagagac agagagagag gaagagacag agacaaaagg agagagaacg gcttagacaa 780
ggagagaaag atggaaagat aaagagactg ggcgcagtgg ctcacgcctg taatcccaac 840
acttggggag gccaaggtgg gaggatggct tgaaggaaag agtctgagat caacctggcc 900
aacatagtga gaccccgtct ctaaaaaaaa aagaaaaaaa aaagaaaaaa gaaaaaaaag 960
tttttttaaa gagacagaga aagagactca gagattgaga ctgagagcaa gacagagaga 1020
gatactcaca gggaagaggg gaagaggaaa acgagaaagg gaggagagta acggaaagag 1080
ataaaaaaga aaagcaggtg gcagagacac acagagaggg acccagagaa agccagacag 1140
acgcaggtgg ctggcagcgg gcgctgtggg ggtcacagta gggggacctg tg 1192
<210> 5
<211> 1149
<212> DNA
<213> 智人
<400> 5
atggagagga gggcgcggag ctcctccagg gagtcccgcg ggcgaggcgg caggactccg 60
cacaaggaga acaagagggc aaaggccgag aggagcggcg ggggccgcgg gcgccaggag 120
gctgggcccg agccgtcggg ctccggacgg gcggggaccc cgggggagcc ccgagcgccc 180
gccgccacgg tggtggacgt ggatgaggtc cgaggctccg gcgaggaggg caccgaggtg 240
gtggcgctgt tggagagcga gcggcccgag gaaggtacca aatcctccgg cttaggggcc 300
tgtgagtggc ttcttgtcct catttccctg ctcttcatca tcatgacctt ccctttttcc 360
atctggttct gcgtaaaggt tgtacaagag tatgaaagag taattatatt ccgactggga 420
catctgcttc ctggaagagc caaaggccct ggtcttttct tttttttgcc ctgcctggat 480
acctaccaca aggttgacct tcgtctccaa actctggaga taccttttca tgagatcgtg 540
accaaagaca tgtttataat ggagatagat gccatttgct actaccgaat ggaaaatgcc 600
tctcttctcc taagcagtct tgctcatgta tctaaagctg tgcaattcct tgtgcaaacc 660
actatgaagc gtctcctagc acatcgatcc ctcactgaaa ttcttctaga gaggaagagc 720
atcgcccaag atgcaaaggt tgccttggat tcagtgacct gtatttgggg aatcaaagtg 780
gagagaatag aaattaaaga tgtgaggttg ccagctgggc ttcagcactc actggctgtg 840
gaggctgaag cgcaaagaca agccaaagtg cggatgattg ctgcagaagc ggaaaaggct 900
gcttctgagt ccctgaggat ggcagctgag attctgtcag gcacccctgc tgctgttcag 960
cttcgatacc tccacaccct tcagtctctg tccacagaga agccttccac tgtggtttta 1020
cctttgccat ttgacctact gaattgcctg tcttctccca gcaacagaac tcagggaagc 1080
ctccccttcc caagtccttc caaacctgtt gagccactaa atcctaaaaa gaaagactct 1140
cccatgtta 1149
<210> 6
<211> 589
<212> DNA
<213> 土拨鼠肝炎病毒
<400> 6
aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60
ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120
atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180
tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240
ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300
attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360
ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420
gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480
aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540
cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgc 589
<210> 7
<211> 225
<212> DNA
<213> 人工序列
<220>
<223> bGH poly(A)
<400> 7
ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60
tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120
tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180
gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgg 225
Claims (13)
1.腺相关病毒(AAV)载体基因疗法,其用于治疗单基因形式的肾病综合征,其中所述AAV载体包含:
NS相关转基因;和
最小nephrin启动子NPHS1或podocin启动子NPHS2。
2.根据权利要求1使用的AAV载体基因疗法,其中所述AAV载体是AAV血清型2/9、LK03或3B。
3.根据权利要求1或2使用的AAV载体基因疗法,其中所述NS相关转基因是NPHS2;ADCK4;ALG1;ARHGAP24;ARGHDIA;CD151;CD2AP;COQ2;COQ6;DGKE;E2F3;EMP2;KANK2;LAGE3;LMNA;LMX1B;MAFB;NUP85;NUP93;NXF5;OSGEP;PAX2;PDSS2;PMM2;PODXL;SCARB2;SGPL1;Smad7;TP53RK;TPRKB;VDR;WDR73;WT1;ZMPSTE24;或APOL1。
4.根据权利要求1至3中任一项使用的AAV载体基因疗法,其中所述AAV载体另外包含土拨鼠肝炎转录后调节元件(WPRE)。
5.根据权利要求1至4中任一项使用的AAV载体基因疗法,其中所述NS相关转基因是人的,和/或包含一个血凝素(HA)标签。
6.根据权利要求1至5中任一项使用的AAV载体基因疗法,其中所述AAV载体另外包含启动子和podocin转基因之间的Kozak序列。
7.根据权利要求1至6中任一项使用的AAV载体基因疗法,其中所述AAV载体另外包含多腺苷酸化信号,如牛生长激素(bGH)多腺苷酸化信号。
8.根据权利要求1至7中任一项使用的AAV载体基因疗法,其中所述AAV载体基因疗法将施用于人患者。
9.根据权利要求8使用的AAV载体基因疗法,其中所述患者是儿科患者。
10.根据权利要求1至9中任一项使用的AAV载体基因疗法,其中所述单基因形式的NS是单基因形式的激素耐药型肾病综合征。
11.根据权利要求1至10中任一项使用的AAV载体基因疗法,其中所述AAV载体基因疗法将系统性地施用。
12.根据权利要求1至11中任一项使用的AAV载体基因疗法,其中所述AAV载体基因疗法将通过静脉内注射施用。
13.根据权利要求1至12中任一项使用的AAV载体基因疗法,其中所述AAV载体基因疗法将通过注射至肾动脉来施用。
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CN114592052A (zh) * | 2022-04-01 | 2022-06-07 | 中国人民解放军陆军军医大学第二附属医院 | 一种肾病综合征患者pdss2致病突变基因及其检测试剂 |
CN117871216A (zh) * | 2024-03-12 | 2024-04-12 | 中日友好医院(中日友好临床医学研究所) | 一种石蜡标本中肾小球裂隙膜的三维可视化方法 |
CN117871216B (zh) * | 2024-03-12 | 2024-06-04 | 中日友好医院(中日友好临床医学研究所) | 一种石蜡标本中肾小球裂隙膜的三维可视化方法 |
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GB202003109D0 (en) * | 2020-03-04 | 2020-04-15 | Univ Bristol | Gene therapy |
GB202103470D0 (en) * | 2021-03-12 | 2021-04-28 | Univ Bristol | Promoter |
EP4273243A1 (en) | 2022-05-02 | 2023-11-08 | Fundació Hospital Universitari Vall d'Hebron - Institut de Recerca | Nucleic acid constructs and vectors for podocyte specific expression |
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CN114592052A (zh) * | 2022-04-01 | 2022-06-07 | 中国人民解放军陆军军医大学第二附属医院 | 一种肾病综合征患者pdss2致病突变基因及其检测试剂 |
CN117871216A (zh) * | 2024-03-12 | 2024-04-12 | 中日友好医院(中日友好临床医学研究所) | 一种石蜡标本中肾小球裂隙膜的三维可视化方法 |
CN117871216B (zh) * | 2024-03-12 | 2024-06-04 | 中日友好医院(中日友好临床医学研究所) | 一种石蜡标本中肾小球裂隙膜的三维可视化方法 |
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US20220125950A1 (en) | 2022-04-28 |
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