CN114736924A - 异位联合过表达Atoh1和Ikzf2再生耳蜗外毛细胞及其应用 - Google Patents
异位联合过表达Atoh1和Ikzf2再生耳蜗外毛细胞及其应用 Download PDFInfo
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Abstract
本发明提供了异位联合过表达Atoh1和Ikzf2再生耳蜗外毛细胞及其应用。具体地,本发明提供了一种可用于再生耳蜗外毛细胞的活性成分组合及其用途,所述活性成分组合包含Ikzf2蛋白和Atoh1蛋白,将所述活性成分组合施用于具有与耳蜗外毛细胞(OHC)变性和/或损伤相关的听力受损的受试者后,可通过再生OHC样细胞缓解听力受损,为临床上治疗听力受损提供了新方法。
Description
技术领域
本发明涉及生物制剂和生物技术领域,尤其涉及Atoh1和Ikzf2联合过表达再生耳蜗外毛细胞及其应用。
背景技术
哺乳动物耳蜗外毛细胞(OHC)对听力至关重要,但OHC对各种耳毒性药物、噪音和衰老非常敏感。OHC变性或损伤导致严重的听力障碍。在以往的体内研究中,虽然也有通过OHC再生以治疗听力损伤的先例,但其效率较低,且再生的新OHC通常无功能。
因此,研究本领域亟待开发一种在体内OHC变性或损伤的情况下再生OHC以治疗听力损伤的方法。
发明内容
本发明的目的就是提供一种能够有效治疗听力受损的基因治疗方法和治疗药物。
本发明的第一方面,提供了一种活性成分组合的用途,所述活性成分用于制备一制剂或药物,所述的制剂或药物用于:(i)再生耳蜗外毛细胞;和/或(ii)治疗或预防听力受损;
其中,所述的活性成分组合包括:
(a)Ikzf2蛋白、其编码序列、或其促进剂、或其组合;和
(b)Atoh1蛋白、其编码序列、或其促进剂、或其组合。
在另一优选例中,所述Ikzf2蛋白具有如SEQ ID NO.:1所示的氨基酸序列。
在另一优选例中,所述Atoh1蛋白具有如SEQ ID NO.:2所示的氨基酸序列。
在另一优选例中,所述的听力受损为与耳蜗外毛细胞(OHC)变性和/或损伤相关的听力受损。
在另一优选例中,所述的耳蜗外毛细胞为人或非人哺乳动物的耳蜗外毛细胞。
本发明的第二方面,提供了一种可用于再生耳蜗外毛细胞的活性成分组合,包括:
(a)Ikzf2蛋白、其编码序列、或其促进剂、或其组合;和
(b)Atoh1蛋白、其编码序列、或其促进剂、或其组合。
在另一优选例中,所述Ikzf2蛋白具有如SEQ ID NO.:1所示的氨基酸序列。
在另一优选例中,所述Atoh1蛋白具有如SEQ ID NO.:2所示的氨基酸序列。
本发明的第三方面,提供了一种表达载体,所述的表达载体包括:
(Z1)第一表达载体,所述第一表达载体含有用于表达Ikzf2蛋白的第一表达盒;
(Z1)第二表达载体,所述第二表达载体含有用于表达Atoh1蛋白的第二表达盒;
其中,所述的第一表达载体和第二表达载体是同一载体或不同的载体。
在另一优选例中,所述的表达载体包括病毒载体。
在另一优选例中,所述的表达载体选自下组:质粒、病毒载体。
在另一优选例中,所述的表达载体选自下组:慢病毒载体、腺病毒载体、腺相关病毒载体(AAV)、或其组合。
在另一优选例中,所述的表达载体为腺相关病毒AAV载体。
在另一优选例中,所述的AAV载体为AAV7m8、AAV-anc80、或AAV-ie。
在另一优选例中,所述表达载体用于表达Ikzf2蛋白和/或Atoh1蛋白。
在另一优选例中,所述表达载体含有编码Ikzf2蛋白和/或Atoh1蛋白的核苷酸序列。
在另一优选例中,所述编码Ikzf2蛋白的核苷酸序列如SEQ ID NO.:3所示。
在另一优选例中,所述编码Atoh1蛋白的核苷酸序列如SEQ ID NO.:4所示。
在另一优选例中,所述的第一表达盒从从5’端-3’端具有式I结构:
Z0-Z1-Z2 (I)
式中,
各“-”独立地为化学键或核苷酸连接序列;
Z0为无、或5’UTR序列;
Z1为编码Ikzf2蛋白的核苷酸序列;和
Z2为无、或3’UTR序列。
在另一优选例中,所述的第二表达盒从从5’端-3’端具有式II结构:
Z0’-Z1’-Z2’ (II)
式中,
各“-”独立地为化学键或核苷酸连接序列;
Z0’为无、或5’UTR序列;
Z1’为编码Atoh1蛋白的核苷酸序列;和
Z2’为无、或3’UTR序列。
在另一优选例中,各个核苷酸连接序列的长度为1-30nt,较佳地1-15nt,更佳地3-6nt。
在另一优选例中,所述的核苷酸连接序列来源于限制性内切酶酶切形成的核苷酸接头序列。
本发明的第四方面,提供了一种宿主细胞,所述宿主细胞含有本发明第三方面所述的表达载体。
在另一优选例中,所述宿主细胞为哺乳动物细胞,所述哺乳动物包括人和非人哺乳动物。
在另一优选例中,所述宿主细胞选自下组:耳蜗支持细胞(SC),包括柱状细胞(PC)、Deiters细胞(DC)或其组合;较佳地,所述宿主细胞为柱状细胞(PC)。
本发明的第五方面,提供了一种药物制剂,所述的制剂含有(a)如本发明第二方面所述的活性成分组合,或本发明第三方面所述的载体,或如本发明第四方面所述的细胞,以及(b)药学上可接受的载体或赋形剂。
在另一优选例中,所述药物制剂的剂型选自下组:冻干制剂、液体制剂、或其组合。
在另一优选例中,所述的载体选自下组:慢病毒载体、腺病毒载体、腺相关病毒载体、或其组合;较佳地,所述载体为AAV载体;更佳地为AAV7m8、AAV-anc80、或AAV-ie。
在另一优选例中,所述药物制剂中载体的含量为1x1012-1x1014个病毒/毫升,较佳地1x1013个病毒/毫升。
在另一优选例中,所述药物制剂的治疗有效量为1x109-1x1011个病毒,较佳地1x1010个病毒。
在另一优选例中,所述药物制剂用于治疗或预防听力受损,较佳地治疗耳蜗外毛细胞变性和/或损伤。
本发明的第六方面,提供了一种如本发明第二方面所述的活性成分组合,本发明第三方面所述的表达载体,本发明第四方面所述的宿主细胞,或如本发明第五方面所述的药物制剂在治疗或预防听力受损中的用途。
在另一优选例中,所述的听力受损为与耳蜗外毛细胞(OHC)变性和/或损伤相关的听力受损。
本发明的第七方面,提供了一种治疗或预防听力受损的方法,所述方法包括将本发明第五方面所述的药物制剂施用于有需要的对象。
在另一优选例中,所述的听力受损为与耳蜗外毛细胞变性和/或损伤相关的听力受损。
在另一优选例中,所述方法为在患有或有风险形成听力受损的患者中减少耳蜗外毛细胞变性和/或损伤的方法。
在另一优选例中,所述有需要的对象包括人和非人哺乳动物。
在另一优选例中,所述有需要的对象患有与耳蜗外毛细胞变性和/或损伤相关的听力受损
在另一优选例中,所述方法包括直接将本发明所述的载体施用至需要的对象的耳部。
在另一优选例中,所述方法包括直接将本发明所述的宿主细胞植入需要的对象的耳蜗。
在另一优选例中,所述方法可以使得由于耳蜗外毛细胞变性和/或损伤相关的听力受损的需要对象的耳蜗外毛细胞再生。
在另一优选例中,所述方法使得在处理的耳中听觉功能基本得以恢复或维持。
本发明的第八方面,提供了一种再生耳蜗外毛细胞的方法,包括将本发明第二方面所述的活性成分组合,或本发明第三方面所述的表达载体,转导进入耳蜗支持细胞。
在另一优选例中,所述耳蜗支持细胞为柱状细胞(PC),包括Deiters细胞(DC)或其组合。
本发明的第九方面,提供了一种活性成分的用途,用于制备一制剂或药物,所述的制剂或药物用于:(i)促进再生耳蜗外毛细胞;和/或(ii)辅助治疗或预防听力受损;
其中,所述的活性成分包括:Ikzf2蛋白、其编码序列、或其促进剂、或其组合。
在另一优选例中,所述Ikzf2蛋白具有如SEQ ID NO.:1所示的氨基酸序列。
在另一优选例中,所述的听力受损为与耳蜗外毛细胞(OHC)变性和/或损伤相关的听力受损。
在另一优选例中,所述的耳蜗外毛细胞为人或非人哺乳动物的耳蜗外毛细胞。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
附图说明
图1:在内毛细胞异位过表达Ikzf2后,内毛细胞开始表达Prestin。(A)用图示解释如何在毛细胞中启动Ikzf2的异位表达。Tdtomato和Ikzf2(HA标签)关联表达。Atoh1-CreER+作为早期毛细胞的有效的启动工具。(B-C”')在P42的对照组Atoh1-CreER+(B-B”')和实验组Atoh1-CreER+;Rosa26-CAG-LSL-Ikzf2/+(C-C”')中同时染Prestin,vGlut3和Tdtomato。其中,Prestin只在野生型外毛细胞中表达(B-B”')。在(C-C”')中,箭头指出了同时表达Tdtomato/vGlut3/Prestin的内毛细胞,星号显示了实验组中不表达Prestin的内毛细胞。(D)对Prestin阳性内毛细胞的定量分析。**p<0.01。(E-F)在P42的对照组(E)和实验组(F)中共染HA/Tdtomato。其中标尺(Scale bars):20μm。
图2:同时在成年小鼠的PC和DC中过表达Atoh1和Ikzf2,可以产生少量OHC样细胞。在4种P30、P31给TMX,P60分析的小鼠中,同时染HA、Tdtomato和Prestin。(A-A”')在Fgfr3-iCreER+;Ai9/+(Fgfr3-Ai9)的小鼠中,所有Tdtomato阳性的细胞都是支持细胞SC:PCs和DCs。在(A')中嵌入的是支持细胞层面。箭头标记了一个Prestin+/Tdtomato-的外毛细胞。(B-B”')Fgfr3-iCreER+;CAG-LSL-Atoh1+(Fgfr3-Atoh1)的小鼠中没有Tdtomato信号,也没有Prestin+/HA+双阳的信号。在(B)中嵌入的图显示的是支持细胞层面。(C-D”')在Fgfr3-iCreER+;Rosa26-CAG-LSL-Ikzf2/+(Fgfr3-Ikzf2)的小鼠中,(C-C”')显示的是毛细胞层,(D-D”')显示的是支持细胞层面。箭头指的是两个层面中HA+/Tdtomato+,但是Pestin-的细胞,同时原位的Prestin+的外毛细胞是异常的。(E-F”')在Fgfr3-iCreER+;CAG-LSL-Atoh1+;Rosa26-CAG-LSL-Ikzf2/+(Fgfr3-Atoh1-Ikzf2)的小鼠的毛细胞(E-E”')和支持细胞层面(F-F”')。根据位置和形态,(E-E”')中的箭标出了由成年DCs中转变来的HA+/Tdtomato+/Prestin+的OHC样细胞,(F-F”')中的箭显示了由成年PC中转变来的HA+/Tdtomato+/Prestin+的OHC样细胞。箭头标记了Prestin+/Tdtomato-的原位外毛细胞落入了支持细胞层面。显然,OHC样的细胞中Prestin的信号比原位的外毛细胞弱。(G)对Fgfr3-Atoh1-Ikzf2小鼠中的OHC样的细胞的定量分析。数据采用Mean±SEM(n=3)来统计分析。(H)对3种模型重编程结果的总结。其中标尺(Scale bars):20μm。
图3:通过遗传学和药理学方法特异性损伤外毛细胞。(A-B')Prestin-P2A-DTR/+在P36给白喉毒素(DT)(A-A',对照无)或(B-B',实验组有),在P42分析。样品同时染Prestin和vGlut3,(A')、(B')中显示的分别是(A)、(B)中的白色方框区域。(B'中的箭头)显示出,在给药6天后,大部分外毛细胞迅速死亡,剩下大量绿色的残存碎片,只有少量残存的外毛细胞(B'中的箭头)。(C)Prestin-P2A-DTR/+小鼠的听觉脑干反应,对照(蓝色线,无DT),实验组(红色线,有DT)。(D)实验组(红色线)和对照组(蓝色线)中外毛细胞与内毛细胞的比率。其中Scale bars:200μm(B),20μm(B')。
图4:损伤原位外毛细胞后,可以促进在成年支持细胞中过表达Atoh1和Ikzf2的重编程效率。(A)对不同的细胞模型,P30、31给TMX,P36给DT,P60分析。(B)用卡通图解释了在细胞水平上,在成年PCs和DCs中过表达Atoh1和Ikzf2后,Tdtomato可以永久地标记支持细胞。(C-G”')在4种不同的:1)Prestin-DTR/+(C和D),2)Fgfr3-iCreER+;CAG-LSL-Atoh1+;Prestin-DTR/+(Fgfr3-Atoh1-DTR)(E),3)Fgfr3-iCreER+;Rosa26-CAG-LSL-Ikzf2/+;Prestin-DTR/+(Fgfr3-Ikzf2-DTR)(F),和4)Fgfr3-iCreER+;CAG-LSL-Atoh1+;Rosa26-CAG-LSL-Ikzf2/+;Prestin-DTR/+(Fgfr3-Atoh1-Ikzf2-DTR)(G-G””)小鼠模型中同时染HA、Tdtomato and Prestin。在P60,相比于没有DT处理的Prestin-DTR/+小鼠中完整的毛细胞(C),DT处理过的只残留了少量毛细胞(D中的箭头)。相比于P42(图2),死掉的外毛细胞的碎片消失。尽管在前三个小鼠模型中没有OHC样细胞的产生,但是在Fgfr3-Atoh1-Ikzf2-DTR小鼠模型中产生了Tdtomato+/HA+/Prestin+OHC样细胞(G-G”'中的箭头)(H)在Fgfr3-Atoh1-Ikzf2-DTR小鼠中,对OHC样细胞数的定量分析。(I)对Fgfr3-Atoh1-Ikzf2-DTR和Fgfr3-Atoh1-Ikzf2(没有破坏原位外毛细胞)小鼠模型中OHC样细胞数量的比较。其中Scale bars:20μm。
图5:OHC样细胞扫描电镜(SEM)分析结果。(A-A')在没有DT处理的Prestin-DTR/+的小鼠中,外毛细胞呈现出V或W型的纤毛束。(A')显示的是(A)中黑色框区域的放大图。(B)在P36给过DT的Prestin-DTR/+的小鼠中,黑色星号指出的是一个丢失内毛细胞。(C)在Fgfr3-iCreER+;CAG-LSL-Atoh1+;Rosa26-CAG-LSL-Ikzf2/+;Prestin-DTR/+(Fgfr3-Atoh1-Ikzf2-DTR)的模型中,观察到了不规则的纤毛束。他们应该是来自OHC样的细胞。(C')黑色框显示的是(C)中的放大图。C中嵌入的图中的纤毛束很少被观察到,黑色星号指出的是一个丢失的内毛细胞。其中Scale bars:5μm(A,B,C),1μm(C')和500nm(A')。
图6:OHC样的细胞与P1的野生型的外毛细胞相似。(A)手挑3种小鼠模型中的Tdtomato+的细胞,以及进行单细胞测序的卡通图。(B-C”')在P60的对照(B)和Fgfr3-Atoh1-Ikzf2-DTR(C)小鼠耳蜗样品中染Myo7a,Prestin和Tdtomato。箭指出了Tdtomato+/Myo7a+/Prestin+的OHC样细胞,箭头指出了Tdtomato+/Myo7a-/Prestin-的细胞。星号标出了Tdtomato+/Myo7a+/Prestin-的细胞(C”-C”')。(D)对5种细胞类型的UMAP分析,其中,用浅蓝色虚线框标出的42个Tdtomato+的细胞来自P60的Fgfr3-Atoh1-Ikzf2-DTR的小鼠模型,它们被分成了3种亚群。(E)对(D)中5种不同细胞类型的Pearson相关性分析。(F-G)对(D-E)中除了不能转变成毛细胞的支持细胞以外的细胞类型(野生型外毛细胞:E16、P1、P7、P30、以及P60的支持细胞,重编程的毛细胞)的UMAP分析。
具体实施方式
本发明人经过广泛而深入的研究,首次成功地将成人耳蜗SC(主要是柱状细胞和Deiters细胞)通过异位表达Atoh1和Ikzf2这两个OHC发育所必需的关键转录因子(TF)转化为Prestin+OHC样细胞。转化得到的OHC样细胞分别上调数百个OHC基因,下调其原始SC基因。通过单细胞转录组比较发现,OHC样细胞与野生型OHC极为相似。总而言之,本发明建立了一种新的、有效的方法来再生受损耳蜗内的OHC,该方法在临床上具有再生OHC的潜力。
在此基础上,完成了本发明。
术语
为了可以更容易地理解本公开,首先定义某些术语。如本申请中所使用的,除非本文另有明确规定,否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成,其将部分地取决于如何测量或测定值或组成。例如,如本文所用,表述“约100”包括99和101和之间的全部值(例如,99.1、99.2、99.3、99.4等)。
如本文所用,术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之,所述术语也包括“基本上由…构成”、或“由…构成”。
如本文使用的,术语“受试者”、“需要的对象”指任何哺乳动物或非哺乳动物。哺乳动物包括但不限于人类、脊椎动物诸如啮齿类、非人类灵长类、牛、马、狗、猫、猪、绵羊、山羊。
如本文所用,术语“听力受损”、“听力障碍”和“听力损伤”是指由于耳蜗外毛细胞变性和/或损伤导致的听力功能异常。
耳蜗外毛细胞(OHC)
如本文所用,术语“耳蜗外毛细胞”、“OHC”和“外毛细胞”具有相同的意义,在本文中可以互换使用。
哺乳动物的声音受体毛细胞(HC)分布于听觉上皮,称为Corti器官(OC)。接近HC的是几种支持细胞(SC)亚型,从内侧到外侧,分别是柱状细胞(PC)和Deiters细胞(DC)。听觉HC有两种亚型:内毛细胞(IHC)和外毛细胞(OHC)。OHC特异性表达一种由Slc26a5编码的独特的运动蛋白Prestin。Prestin介导的电运动使OHC成为声音放大器,这对声音检测至关重要。Prestin-/-小鼠遭受严重的听力损害。与OHC不同,IHC是原代感觉细胞,受耳蜗螺旋神经节神经元(SGNs)支配。IHC特异性由Slc17a8编码的vGlut3决定,从IHC到SGNs的声音信息转换需要vGlut3。vGlut3-/-小鼠完全失聪。IHC和OHCs共享相同的Atoh1+祖细胞。
Ikzf2蛋白
Ikzf2(IKAROS Family Zinc Finger 2),是锌指类蛋白,另外一个常用名称是Helios。具体信息参见:
https://www.ncbi.nlm.nih.gov/gene/22807(人类);
https://www.ncbi.nlm.nih.gov/gene/22779(小鼠)。
在本发明中,所述Ikzf2蛋白的氨基酸序列如SEQ ID NO.:1(人类)和SEQ ID NO.:5(小鼠)所示;核苷酸编码序列如SEQ ID NO.:3(人类)和SEQ ID NO.:7(小鼠)所示。
Ikzf2本质是分布在细胞核内的转录因子,主要功能是启动特定基因的表达。在小鼠耳蜗中,Ikzf2出生后开始表达,并且特异性表达在耳蜗外毛细胞(OHC),耳蜗其他细胞不表达或者很低量地表达。小鼠Ikzf2突变型小鼠,表现出耳蜗外毛细胞发育异常、听力障碍等。
Atoh1蛋白
Atoh1(atonal bHLH transcription factor 1)是bHLH家族的核转录因子,另外一个常用的名称是math1。具体信息参见:
https://www.ncbi.nlm.nih.gov/gene/474(人类);
https://www.ncbi.nlm.nih.gov/gene/11921(小鼠)。
在本发明中,所述Atoh1蛋白的氨基酸序列如SEQ ID NO.:2(人类)和SEQ ID NO.:6(小鼠)所示;核苷酸编码序列如SEQ ID NO.:4(人类)和SEQ ID NO.:8(小鼠)所示。
Atoh1本质也是分布在细胞核内的转录因子,主要功能是启动特定基因的表达。在小鼠耳蜗中,Atoh1在胚胎中期的耳蜗开始表达,其主要功能是让耳蜗听觉上皮的祖细胞发育为毛细胞。Atoh1缺失的小鼠不能产生毛细胞。
腺相关病毒
腺相关病毒(adeno-associated virus,AAV),也称腺伴随病毒,属于微小病毒科依赖病毒属,是目前发现的一类结构最简单的单链DNA缺陷型病毒,需要辅助病毒(通常为腺病毒)参与复制。它编码两个末端的反向重复序列(ITR)中的cap和rep基因。ITR对于病毒的复制和包装具有决定性作用。cap基因编码病毒衣壳蛋白,rep基因参与病毒的复制和整合。AAV能感染多种细胞。
重组腺相关病毒载体(rAAV)源于非致病的野生型腺相关病毒,由于其安全性好、宿主细胞范围广(分裂和非分裂细胞)、免疫源性低,在体内表达外源基因时间长等特点,被视为最有前途的基因转移载体之一,在世界范围内的基因治疗和疫苗研究中得到广泛应用。经过10余年的研究,重组腺相关病毒的生物学特性己被深入了解,尤其是其在各种细胞、组织和体内实验中的应用效果方面已经积累了许多资料。在医学研究中,rAAV被用于多种疾病的基因治疗的研究(包括体内、体外实验);同时作为一种有特点的基因转移载体,还广泛用于基因功能研究、构建疾病模型、制备基因敲除鼠等方面。
表达载体和宿主细胞
本发明提供了一种用于Ikzf2蛋白和Atoh1蛋白表达的表达载体,它含有本发明的Ikzf2蛋白和Atoh1蛋白的编码序列。
通过提供的序列信息,熟练的技术人员可以使用可用的克隆技术以产生适于转导进入细胞的核酸序列或载体。
优选地,包含编码Ikzf2蛋白和Atoh1蛋白的核酸序列作为载体,优选地作为表达载体被提供。优选地,其可作为优选地适用于在靶细胞(例如耳蜗支持细胞)中转导和表达的基因治疗载体被提供。载体可以是病毒的或非病毒的(例如质粒)。病毒载体包括源自以下的那些病毒载体:腺病毒、包括突变的形式的腺相关病毒(AAV)、逆转录病毒、慢病毒、疱疹病毒、牛痘病毒、MMLV、GaLV、猿猴免疫缺陷病毒(SIV)、HIV、痘病毒和SV40。优选地,病毒载体是复制缺陷的(replication defective),或者可以是复制缺乏的(replicationdeficient)、能够复制或条件性复制的。病毒载体通常可以保持染色体外状态而不整合进入靶细胞的基因组。用于向靶细胞引入编码Ikzf2蛋白和Atoh1蛋白的核酸序列的优选的病毒载体是AAV载体。使用特定的AAV血清型(AAV血清型2到AAV血清型12)或这些血清型中的任何一个的修饰的版本可以实现选择性靶向。在本发明的优选例中,所述AAV载体优选为AAV7m8、AAV-anc80、或AAV-ie。
病毒载体可被修饰以缺失任何非必需的序列。例如,AAV中,病毒可被修饰以缺失全部或部分的IX基因、Ela和/或Elb基因。对于野生型AAV,没有辅助病毒诸如腺病毒的存在,复制是非常低效率的。对于重组的腺相关病毒,优选地,复制基因和衣壳基因以反式被提供(在pRep/Cap质粒中),并且仅AAV基因组的2ITR被保留并且包装进入病毒体,同时需要的腺病毒基因被腺病毒或另一个质粒提供。也可对慢病毒载体做出类似的修饰。
病毒载体具有进入细胞的能力。然而,非病毒载体诸如质粒可与试剂复合以有利于病毒载体被靶细胞的摄取。此类试剂包括聚阳离子剂。可选地,递送系统诸如基于脂质体的递送系统可被使用。用于在本发明中使用的载体优选地适于在体内或体外使用,并且优选地适于在人类中使用。
载体将优选地包含一个或多个调节序列以指导核酸序列在靶细胞中的表达。调节序列可以包括与核酸序列可操作地连接的启动子、增强子、转录终止信号、多腺苷酸化序列、复制起点、核酸限制性位点、和同源重组位点。载体还可包括选择性标记,例如来确定载体在生长系统(例如细菌细胞)中或在靶细胞中的表达。
“可操作地连接”意指,核酸序列在功能上与其可操作地连接的序列相关,以使得它们以使得它们影响彼此的表达或功能的方式连接。例如,与启动子可操作地连接的核酸序列将具有被启动子影响的表达模式。
启动子介导与其连接的核酸序列的表达。启动子可以是组成型的或可以是诱导型的。启动子可以指导基因在耳蜗细胞中普遍的表达,或耳蜗细胞特异的表达。在后一种情况中,启动子可以指导细胞类型特异的表达,例如耳蜗支持细胞(SC)(包括柱状细胞(PC)、Deiters细胞(DC))。合适的启动子将是本领域技术人员己知的。例如,合适的启动子可以选自由以下组成的组:L7、thy-1、恢复蛋白、钙结合蛋白、人类CMV、GAD-67、鸡肌动蛋白、CAG和CBA等。此外,使用细胞特异的启动子可以实现靶向特定细胞的基因表达。
合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列为能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而,也可使用其他组成型启动子序列,包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子,诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地,本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关,其能够在期望表达时,打开可操作地连接诱导型启动子的多核苷酸序列的表达,或当期望关闭表达时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
许多表达载体可应用于Ikzf2蛋白和Atoh1蛋白在哺乳动物细胞(较佳地为人,更佳地为人耳蜗支持细胞)表达。本发明优选用腺相关病毒AAV载体作为表达载体。
本发明还提供了一种宿主细胞,用于表达Ikzf2蛋白和Atoh1蛋白。优选地,所述宿主细胞为哺乳动物细胞(较佳地为人,更佳地为人耳蜗支持细胞),提高Ikzf2蛋白和Atoh1蛋白的表达量。
药物制剂和组合物
本发明提供一种药物制剂或组合物,所述药物制剂或组合物含有(a)本发明第二方面所述的活性成分组合、或本发明第三方面所述的载体、或本发明第四方面所述的宿主细胞,以及(b)药学上可接受的载体或赋形剂。
在另一优选例中,所述药物制剂用于治疗听力受损。
在另一优选例中,所述药物制剂用于治疗耳蜗外毛细胞变性和/或损伤。
本发明所述药物制剂中的“活性成分”是指本发明所述的载体(vector),例如病毒载体(包括腺相关病毒载体)。本发明所述的“活性成分”、制剂和/或组合物可用于治疗听力受损。“安全有效量”指的是:活性成分的量足以明显改善病情或症状,而不至于产生严重的副作用。“药学上可接受的载体或赋形剂(excipient)”指的是:一种或多种相容性固体或液体填料或凝胶物质,它们适合于人使用,而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的活性成分以及它们之间相互掺和,而不明显降低活性成分的药效。
组合物可以是液体或固体,例如粉末、凝胶或糊剂。优选地,组合物是液体,优选地可注射液体。合适的赋形剂将是本领域技术人员己知的。
在本发明中,所述载体可通过直接向耳部或耳蜗向受试者施用。在任一种施用模式中,优选地,载体作为可注射液体被提供。优选地,可注射液体作为胶囊或注射器被提供。
在本发明中,可以将本发明所述的宿主细胞植入受试者的耳蜗施用。优选地,宿主细胞作为可注射液体被提供。
药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液,和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
治疗方法
本发明提供了再生耳蜗外毛细胞的方法,所述方法包括将包含编码Ikzf2蛋白和Atoh1蛋白的核苷酸序列引入到耳蜗内。所述方法可包括向耳蜗的支持细胞施用包含编码Ikzf2蛋白和Atoh1蛋白的核苷酸序列的载体。
本发明提供了用于通过再生耳蜗外毛细胞在治疗听力受损的方法中使用的药物制剂或组合物,所述药物制剂或组合物包含编码Ikzf2蛋白和Atoh1蛋白的核苷酸序列的载体或含有所述载体的宿主细胞,以及药学上可接受的载体或赋形剂。本发明的药物制剂或组合物可以单独给药,或者与其他治疗药物联合给药(如配制在同一药物组合物中)。
本发明还提供了一种治疗听力受损的方法,所述方法包括将本发明所述的药物制剂或组合物施用于受试者。所述的听力受损为与耳蜗外毛细胞变性和/或损伤相关的听力受损。所述的方法包括直接将本发明所述包含编码Ikzf2蛋白和Atoh1蛋白的核苷酸序列的载体施用至需要的对象的耳部(耳蜗)。所述方法还包括直接将本发明所述的宿主细胞植入需要的对象的耳蜗。
如本文所用,再生耳蜗外毛细胞意指,之前不具有耳蜗外毛细胞能力或其耳蜗外毛细胞能力已经完全地或部分地退化的细胞,在其中表达编码Ikzf2蛋白和Atoh1蛋白的外来核酸序列后,变成具备耳蜗外毛细胞表征和功能的细胞。此类细胞在本文中可被称作转化的细胞,或“耳蜗外毛细胞样细胞(OHC样细胞)”,因为其在其中包含非天然的核酸。优选地,转化的耳蜗外毛细胞展现天然的耳蜗外毛细胞的一些或全部的耳蜗外毛细胞能力。优选地,转化的细胞展现天然的耳蜗外毛细胞的至少相同或大体上相同的听觉能力。优选地,转化的细胞展现比患病的或正在退化的天然的耳蜗外毛细胞高的听觉能力。因此,转化的细胞相比于来自同一来源、保持在同一条件下、未经处理的退化的或患病的细胞,将优选地具有增加的耳蜗外毛细胞。转化的细胞通过其中的外源核酸的存在可与天然的细胞区分。
相较于现有技术,本发明的主要优点在于:
(1)本发明人将Ikzf2蛋白和Atoh1蛋白异位联合共表达于耳蜗支持细胞中,使耳蜗支持细胞转化为OHC样细胞。转化得到的OHC样细胞具有与天然OHC相似的组织形态和基因特征,并具有相同的OHC功能。
(2)在发生OHC损伤后,本发明的方法可以快速且高效地产生OHC样细胞。
(3)本发明的方法为听力障碍和听力受损提供了一种基因治疗的新思路。
下面结合具体实施,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor LaboratoryPress,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数按重量计算。
方法和材料:
(1)通过CRISPR/Cas9方法构建Rosa26-CAG-LSL-Ikzf2/+和Prestin-DTR/+定点敲入的小鼠品系
同时通过在一细胞期的受精卵里同时注射Rosa26sgRNA(5'-actccagtctttctagaaga-3',SEQ ID No:5),供体DNA和Cas9 mRNA来构建Rosa26-CAG-LSL-Ikzf2/+定点敲入的小鼠。用同样的方法构建了Prestin-P2A-DTR/+(Prestin-DTR/+)定点敲入的小鼠品系(Prestin(Slc26a5),其sgRNA为5'-CGAGGCATAAAGGCCCTGTA-3'(SEQ IDNo:6)。
通过junction PCR的方法来筛选F0中正确的小鼠,之后将潜在正确的F0小鼠与野生型的C57BL/6小鼠交配得到F1的小鼠。首先对F1小鼠的尾巴进行junction PCR鉴定筛选出潜在正确的小鼠,之后参照如(Li et al.,2018)所述的方法,通过Southern blotting实验,来排除F1中有随机插入的小鼠。所有的小鼠都在SPF级别的鼠房进行繁殖和养育。所有的操作都根据神经科学研究所,中国科学院脑科学与智能卓越技术创新中心的指导进行。
(2)样品处理,免疫组化和细胞计数
对成年的小鼠用1xPBS和4%PFA进行心脏灌流以便彻底清除内耳里的血液并进行预固定。然后小心地解剖出内耳并放入含有4%PFA的管子里4摄氏度过夜进行后固定。第二天,用1xPBS洗3次内耳,每次10分钟,接着用120mM的EDTA对内耳脱钙2天直到内耳变软。然后解剖出整个耳蜗上皮并进行免疫组化。
使用的一抗如下表所示:
抗体信息表
用溶在PBST里的Hoechst33342(1:1000,62249,Thermo Scientific)染耳蜗组织以便观察细胞核,并用Prolong gold antifade medium(P36930,Thermo Scientific)来封片。用Nikon C2,TiE-A1和NiE-A1 plus共聚焦显微镜来拍图。
解剖时,每个耳蜗被分成3段,在10x物镜下拍图,之后通过软件在内外毛之间画一条线进而测出耳蜗的长度,并将耳蜗平均分成basal,middle,apical 3段。为了看Prestin-DTR/+小鼠模型中外毛细胞的死亡情况,在60x物镜下对每段随机拍2个位置,并对两个位置的计数结果进行平均,并用内毛细胞作为参照,因为DT不杀伤内毛细胞。而对于Atoh1CreER+;Rosa26-CAG-LSL-Ikzf2/+小鼠中的内毛细胞中的Prestin,和Fgfr3-Atoh1-Ikzf2和Fgfr3-Atoh1-Ikzf2-DTR小鼠模型中的Tdtomato+/HA+细胞,OHC样细胞和新生初期的毛细胞信号的计数,整个耳蜗用60x拼图方式拍摄以便更加精确地计数。用GraphPad Prism 6.0来做统计图,数据用Student's t test和Mean±SEM来进行统计分析。
(3)单细胞测序和生信分析
Tdtomato+的细胞在3个小鼠模型分别被用到:1)Prestin-CreER/+;Ai9/+的小鼠在P20,P21给予Tamoxifen,P30挑细胞(Fang et al.,2012);2)Fgfr3-iCreER+;Ai9/+的小鼠在P30,P31给予Tamoxifen,P60挑支持细胞(primarily PCs和DCs),根据的是以前的报道(Liu et al.,2012a;Liu et al.,2012b);3)Fgfr3-Atoh1-Ikzf2-DTR的小鼠在P30,P31给予Tamoxifen,P36给予DT,P60挑细胞。
在体视显微镜下手挑出Tdomato+的细胞,17个P30的野生型外毛细胞,16个P60的野生型的支持细胞,以及42个Fgfr3-Atoh1-Ikzf2-DTR模型中的细胞(Fig.6D),立即进行反转并用smart-Seq HT kit(Cat#634437,Takara)进行cDNA的扩增。用TruePrep DNALibrary Prep Kit V2(Cat#TD503,Vazyme)和TruePrep Index Kit V2(Cat#TD202,Vazyme)来对cDNA(1ng)进行质检。最后用Illumina Novaseq platform对cDNA库进行配对测序,每个库的测序深度是4G。
FastQC(v0.11.9)和trimmomatic(v0.39)被用来对原始测序数据进行质检。大约70-80%的小鼠参考基因的reads被高质量地覆盖到,用的是Hisat2(v2.1.0)默认参数。通过HTSeq(v0.10.0)来对原始数据进行计算。用StringTie(v1.3.5)默认的参数来估算基因的表达水平。基因的丰富度通过每百万转录本的方式呈现出来(TPM)。用R package“DESeq2”(p.adj<0.05,absolute value of(log2 Fold Change)>2)来分析不同基因的表达。DEGs被用于生物分析的富集过程(p<0.05,用bonferroni corrections进行校准),同时对数据库进行注释,可视化和整合。本发明所有的单细胞测序的原始数据GEO(GeneExpression Omnibus)库,都可以用搜索码:GSE161156获得。
用Seurat(R package v3.0)软件包对(Kolla et al.,2020)文献中的E16,P1和P7的野生型的外毛细胞的数据进行了分析。为了更加精确地比较10x和samrt-seq测序方法分析的转录组学数据,首先用“FindIntegrationAnchors”(k.filter=30)的功能对他们进行了整合。然后用“RunPCA”的功能进行了组成分计算,并用“RunTSNE”和“RunUMAP”对前20的主成分进行了降维分析。用“FindClusters”(resolution=0.5)功能进行了无监督聚类分析。另外,为了更加精确地分析单细胞测序中的(E16,E17和P7)的毛细胞与(E14)的毛细胞的祖细胞,设置的标准比传统的更加严格。其中,在E16的野生型外毛细胞中,Insm1,Myo6和Atoh1的表达大于0,在P1的野生型外毛细胞中Bcl11b,Myo6,Myo7a,和Atoh1大于0;P7的野生型外毛细胞中,Prestin(Slc26a5),Myo6,Ocm,Ikzf2大于0。用Monocle(R package v2.0)包来进行轨迹分析。用“importCDS”功能将预处理的Seurat数据通过“importCDS”功能导入Monocle。在三个年纪的外毛细胞中,发生显著改变的基因通过不同的表达分析被识别出来。通过Monocle中的“orderCells”功能,将细胞沿着一个假定的轴呈现出来。
(4)听觉脑干反应(ABR)
对小鼠听觉脑干反应的测试,参照了之前的方法(Li et al.,2018),对Prestin-DTR/+小鼠在P42和P60(在P36有或没有DT处理)和Fgfr3-Atoh1-Ikzf2-DTR小鼠分别用4kHz,5.6k Hz,8k Hz,11.3k Hz,16k Hz,22.6k Hz和32k Hz的频率下进行听力测试。数据用Student's t test进行统计分析。
(5)他莫西芬(Tamoxifen)和白喉毒素(Diphtheria toxin)
Tamoxifen(Cat#T5648,Sigma)溶在玉米油(Cat#C8267,sigma)中便于后续实验。根据体重按照3mg/40g(P0,P1)或9mg/40g(P20,P21和P30,P31)的剂量通过腹腔进行注射。Diphtheria toxin(Cat#D0564,Sigma)溶在0.9%NaCl,同样根据体重按照20ng/g的剂量在P36进行腹腔注射。
(6)扫描电镜(SEM)
SEM实验参照以前的方法(Parker et al.,2016)。首先,我们用镊子在耳蜗顶端扎一个孔,然后缓慢地用注射器将0.9%NaCl(Cat#10019318,Sinopharm Chemical ReagentCo,Ltd)从圆窗注入冲洗耳蜗,之后再注2.5%glutaraldehyde(Cat#G5882,Sigma)预固定耳蜗,然后用镊子尽可能多地暴露出柯蒂式器官(organ of Corti)以便充分固定,最后将耳蜗放入2.5%glutaraldehyde中在4℃过夜。第二天用1xPBS冲洗3次内耳,并用10%EDTA(Cat#ST066,Beyotime)脱钙1天,之后剖出耳蜗,并用1%osmium tetroxide(OsO4,Cat#18451,Tedpella)后固定1小时,用ddH2O洗6次,用TCH(thiocarbohydyazide,Cat#88535,Sigma)固定30分钟,用ddH2O洗6次,再用1%osmium tetroxide固定1小时,用ddH2O洗6次,之后用不同浓度的乙醇(30%,50%,75%,80%,95%,Cat#10009259,Sinopharm ChemicalReagent Co,Ltd),每步30分钟在4摄氏度对耳蜗样品进行梯度脱水。接着在100%乙醇中脱水3次,每次30分钟。接下来对样品进行临界点干燥(model:EM CPD300,Leica)。然后把耳蜗正面朝上粘贴在导电胶上,并对样品进行喷金处理(model:Q150T ES,Quorum)。最后用扫描电镜对样品进行拍摄(model:GeminiSEM 300,ZEISS)。
实施例1
在内毛细胞异位过表达Ikzf2后,内毛细胞开始表达Prestin
在野生型小鼠内,Ikzf2和Prestin都只表达在耳蜗外毛细胞中,内毛细胞从不表达Ikzf2和Prestin。构建了Rosa26-CAG-LSL-Ikzf2/+基因定点小鼠以过表达Ikzf2,图1A显示了Ikzf2的异位表达原理,当Ikzf2表达时,Ikzf2融合的HA标签,以及紧跟在其后的Tdtomato也随之表达,并使表达Ikzf2的细胞发出红色荧光。在给小鼠注射他莫昔芬后,与对照组小鼠相比(图1B-B”’),当内毛细胞内强制性过表达Ikzf2之后,prestin也随之异位表达在内毛细胞(图1C-C”’)。对Prestin阳性的内毛细胞进行定量分析,发现Apical turn中Prestin阳性的内毛细胞比Middle和Basal turn多(图1D)。经验证,只有实验组(图F)表达HA/Tdtomato,对照组(图E)不表达,且所有Tdtomato阳性的细胞都表达HA(Ikzf2),反之亦然。
实施例2
同时在成年小鼠的PCs和DCs中过表达Aoh1和Ikzf2,可以产生少量OHC样细胞
比较了不同的处理方式对OHC样细胞产生的影响。即特定在成年耳蜗支持细胞内,做了3种不同的处理:单独过表达Atoh1,单独过表达Ikzf2,和同时过表达Atoh1和Ikzf2。与对照组相比(图2A-A”’),单独过表达Atoh1(图2B-B”’)或者单独过表达Ikzf2(图2C-C”’和图2D-D”’)均不能产生新的Prestin+的外毛细胞。但是,同时过表达Atoh1和Ikzf2(图2E-E”’和图2F-F”’)可以产生少量新的Prestin+的外毛细胞。对同时过表达Atoh1和Ikzf2(Fgfr3-Atoh1-Ikzf2)小鼠中的OHC样细胞进行定量分析(图2G),发现尽管整个耳蜗范围都可以看见新生的细胞,但是数量很少,且变化很大。对3中不同的重编程处理方式进行总结,发现只有同时激活Atoh1和Ikzf2,才能产生OHC样的细胞(图2H)。
实施例3
通过遗传学和药理学方法特异性损伤外毛细胞
为了模拟人类听力受损模型,构建了Prestin-P2A-DTR/+基因敲入小鼠。在成年(P36)小鼠给予白喉毒素(DT)之后,耳蜗外毛细胞被特异杀伤(图3A-A’和图3B-B’)。统计学分析显示大于90%的外毛细胞死亡。但值得注意的是内毛细胞是正常的。并且,在DT处理后,整个耳蜗的听觉阈值显著升高(图3C)。统计分析了对照组和实验组的OHC/IHC比率,发现实验组的比率显著低于对照组(图3D)。以上结果说明,听力受损模型构建成功。
实施例4
损伤原位外毛细胞后,可以促进在成年支持细胞中过表达Atoh1和Ikzf2的重编程效率
通过遗传学和药理学方法建立听力受损模型,实现了特异杀伤成年外毛细胞。然后,在听力受损模型小鼠的成年支持细胞中单独过表达Atoh1(图4E),或者单独过表达Ikzf2(图4F),或者同时过表达Atoh1和Ikzf2(图4G-G”’)。图4A和图4B显示了模型构建的过程。实验结果显示,只有同时过表达Atoh1和Ikzf2可以产生大量(400-600个)新的Prestin+的OHC样的细胞。同时,对在Fgfr3-Atoh1-Ikzf2-DTR小鼠中,对OHC样细胞数的定量分析(图4H),对Fgfr3-Atoh1-Ikzf2-DTR和Fgfr3-Atoh1-Ikzf2(没有破坏原位外毛细胞)小鼠模型中OHC样细胞数量的比较。发现Fgfr3-Atoh1-Ikzf2-DTR比Fgfr3-Atoh1-Ikzf2有更多的OHC样细胞。以上结果说明杀伤内源性外毛细胞能够显著提高Atoh1和Ikzf2转分化支持细胞为新的外毛细胞的效率。
实施例5
OHC样细胞呈现出不规则的纤毛束
利用超高扫描电镜对三种不同基因型的小鼠耳蜗听觉上皮进行细致的分析。野生型小鼠外毛细胞顶部有呈‘V’或者‘W’型的纤毛(图5A-A’)。纤毛这个超微结构对于外毛细胞感知声音十分重要。纤毛结构在外毛细胞杀伤模型中消失(图5B),但在成年支持细胞过表达Atoh1和Ikzf2的模型中,重新出现(图5C-C’)。这个结果显示,新生的外毛细胞(即OHC样细胞)具有纤毛结构。
实施例6
OHC样细胞与P1的野生型外毛细胞相似
利用遗传学的手段把野生型的耳蜗支持细胞和外毛细胞标记为红色,然后消化耳蜗组织,用手工分选的办法进行单细胞转录组分析(图6A)。同时,采用类似的办法,把过表达Atoh1和Ikzf2的支持细胞也挑选出来进行单细胞转录组分析(图6A)。利用Myo7a和Prestin免疫组化(图6B-C”’),发现表达Atoh1和Ikzf2的支持细胞最终有3种细胞命运:1)没有转变成毛细胞(三角形);2)转变为非常不成熟的毛细胞(星号);3)转变为新生的Prestin+的OHC样细胞(本发明最关注的类型)(箭头)。UMAP分析显示,新产生的细胞更靠近成年的外毛细胞,但是并没有完全重合(图6D和图6E)。为了进一步分析,通过比对最近发表在2020年Nature Communications的文章的不同时期的毛细胞转录组数据,发现Prestin+的新生外毛细胞与出生后第一天(P1)野生型外毛细胞最相似(图6F和图6G)。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 中国科学院脑科学与智能技术卓越创新中心
<120> 异位联合过表达Atoh1和Ikzf2再生耳蜗外毛细胞及其应用
<130> P2020-2480
<160> 8
<170> PatentIn version 3.5
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Pro Gly Gly Gly Pro Arg Pro Thr Pro Pro Gly Pro Cys Arg Thr Arg
260 265 270
Phe Ser Gly Pro Ala Ser Ser Gly Gly Tyr Ser Val Gln Leu Asp Ala
275 280 285
Leu His Phe Pro Ala Phe Glu Asp Arg Ala Leu Thr Ala Met Met Ala
290 295 300
Gln Lys Asp Leu Ser Pro Ser Leu Pro Gly Gly Ile Leu Gln Pro Val
305 310 315 320
Gln Glu Asp Asn Ser Lys Thr Ser Pro Arg Ser His Arg Ser Asp Gly
325 330 335
Glu Phe Ser Pro His Ser His Tyr Ser Asp Ser Asp Glu Ala Ser
340 345 350
<210> 7
<211> 1581
<212> DNA
<213> 小鼠(Mus musculus)
<400> 7
atggaaacag acgcaattga tggctatata acatgtgaca atgagctttc acccgaaggg 60
gaacacgcca atatggccat tgacctcacc tcaagcacac ccaatggaca gcacgcctcg 120
ccaagtcaca tgacaagcac aaattctgta aagctggaaa tgcagagtga tgaagagtgt 180
gacaggcagc ccctgagccg tgaggatgag atcaggggcc acgatgaggg gagcagccta 240
gaagaacccc taattgagag cagcgaggtg gccgacaaca ggaaagtcca ggaccttcaa 300
ggcgagggag gaatccggct tccgaatggt aaactgaaat gtgacgtctg tggcatggtt 360
tgcattgggc ccaatgtgct tatggtacat aaaaggagtc acactggtga gcggcccttc 420
cactgtaacc agtgcggagc ttcttttacc cagaagggca accttctgag acacataaag 480
ttacactctg gagagaagcc cttcaaatgt cctttctgta gctatgcttg tagaagaagg 540
gacgctctca caggacacct caggacccat tctgtgggta aacctcacaa gtgtaactac 600
tgtggccgaa gctacaagca gcgcagctca ctggaggaac acaaggaacg ctgtcacaac 660
tatctccaga atgtcagcat ggaggctgcc gggcaggtca tgagtcacca tgtaccgcct 720
atggaagatt gtaaggaaca agagcctatc atggacaaca atatttctct ggtgcctttt 780
gagagacctg ctgtcataga gaagctcacg gcaaatatgg gaaagcgcaa aagctccact 840
cctcagaagt ttgtggggga aaagcttatg cgattcagct acccagatat tcattttgat 900
atgaacttaa catatgagaa ggaggctgag ctgatgcagt ctcatatgat ggaccaagcc 960
atcaacaatg caatcaccta ccttggagct gaggcccttc accctctgat gcagcatgca 1020
ccaagcacaa tcgctgaggt ggccccagtt ataagctcag cttattctca ggtctatcat 1080
ccaaacagga tagaaagacc cattagcagg gaaacatctg atagtcacga aaacaacatg 1140
gatggcccca tctctctcat cagaccaaag agtcgacccc aggaaagaga ggcctcgccc 1200
agcaatagct gcctcgattc tactgactca gaaagtagcc atgatgaccg ccagtcctac 1260
caaggaaacc ctgccttaaa tcccaagagg aaacaaagcc cagcttacat gaaggaggat 1320
gtcaaggctt tggatgctac caaggccccc aagggctctc tgaaggacat ctataaggtt 1380
ttcaatggag aaggagaaca gataagggcc ttcaagtgtg agcactgccg agtccttttt 1440
ctagaccatg tcatgtacac cattcacatg ggttgccatg gctaccggga cccactggaa 1500
tgcaacatct gtggctacag aagccaggac cgctacgaat tttcatcaca cattgttcga 1560
ggggagcaca cattccacta g 1581
<210> 8
<211> 1056
<212> DNA
<213> 小鼠(Mus musculus)
<400> 8
atgtcccgcc tgctgcatgc agaagagtgg gctgaggtaa aagagttggg ggaccaccat 60
cgccatcccc agccgcacca cgtcccgccg ctgacgccac agccacctgc taccctgcag 120
gcgagagacc ttcccgtcta cccggcagaa ctgtccctcc tggatagcac cgacccacgc 180
gcctggctga ctcccacttt gcagggcctc tgcacggcac gcgccgccca gtatctgctg 240
cattctcccg agctgggtgc ctccgaggcc gcggcgcccc gggacgaggc tgacagccag 300
ggtgagctgg taaggagaag cggctgtggc ggcctcagca agagccccgg gcccgtcaaa 360
gtacgggaac agctgtgcaa gctgaagggt ggggttgtag tggacgagct tggctgcagc 420
cgccagcgag ccccttccag caaacaggtg aatggggtac agaagcaaag gaggctggca 480
gcaaacgcaa gggaacggcg caggatgcac gggctgaacc acgccttcga ccagctgcgc 540
aacgttatcc cgtccttcaa caacgacaag aagctgtcca aatatgagac cctacagatg 600
gcccagatct acatcaacgc tctgtcggag ttgctgcaga ctcccaatgt cggagagcaa 660
ccgccgccgc ccacagcttc ctgcaaaaat gaccaccatc accttcgcac cgcctcctcc 720
tatgaaggag gtgcgggcgc ctctgcggta gctggggctc agccagcccc gggagggggc 780
ccgagaccta ccccgcccgg gccttgccgg actcgcttct caggcccagc ttcctctggg 840
ggttactcgg tgcagctgga cgctttgcac ttcccagcct tcgaggacag ggccctaaca 900
gcgatgatgg cacagaagga cctgtcgcct tcgctgcccg ggggcatcct gcagcctgta 960
caggaggaca acagcaaaac atctcccaga tcccacagaa gtgacggaga gttttccccc 1020
cactctcatt acagtgactc tgatgaggcc agttag 1056
Claims (10)
1.一种活性成分组合的用途,其特征在于,用于制备一制剂或药物,所述的制剂或药物用于:(i)再生耳蜗外毛细胞;和/或(ii)治疗或预防听力受损;
其中,所述的活性成分组合包括:
(a)Ikzf2蛋白、其编码序列、或其促进剂、或其组合;和
(b)Atoh1蛋白、其编码序列、或其促进剂、或其组合。
2.如权利要求1所述的用途,其特征在于,所述的听力受损为与耳蜗外毛细胞(OHC)变性和/或损伤相关的听力受损。
3.一种可用于再生耳蜗外毛细胞的活性成分组合,其特征在于,包括:
(a)Ikzf2蛋白、其编码序列、或其促进剂、或其组合;和
(b)Atoh1蛋白、其编码序列、或其促进剂、或其组合。
4.一种表达载体,其特征在于,所述的表达载体包括:
(Z1)第一表达载体,所述第一表达载体含有用于表达Ikzf2蛋白的第一表达盒;
(Z1)第二表达载体,所述第二表达载体含有用于表达Atoh1蛋白的第二表达盒;
其中,所述的第一表达载体和第二表达载体是同一载体或不同的载体。
5.一种宿主细胞,其特征在于,所述宿主细胞含有权利要求4所述的表达载体。
6.如权利要求5所述的宿主细胞,其特征在于,所述宿主细胞选自下组:耳蜗支持细胞(SC),包括柱状细胞(PC)、Deiters细胞(DC)或其组合。
7.一种药物制剂,其特征在于,所述的制剂含有(a)如权利要求3所述的活性成分组合,或权利要求4所述的载体,或如权利要求5所述的细胞,以及(b)药学上可接受的载体或赋形剂。
8.一种如权利要求3所述的活性成分组合,如权利要求4所述的表达载体,如权利要求5所述的宿主细胞,或如权利要求7所述的药物制剂在治疗或预防听力受损中的用途。
9.一种再生耳蜗外毛细胞的方法,其特征在于,包括将权利要求3所述的活性成分组合,或权利要求4所述的表达载体,转导进入耳蜗支持细胞。
10.一种活性成分的用途,其特征在于,用于制备一制剂或药物,所述的制剂或药物用于:(i)促进再生耳蜗外毛细胞;和/或(ii)辅助治疗或预防听力受损;
其中,所述的活性成分包括:Ikzf2蛋白、其编码序列、或其促进剂、或其组合。
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US18/260,527 US20240075098A1 (en) | 2021-01-07 | 2021-02-22 | Cochlear outer hair cell regenerated by ectopic joint overexpression of atoh1 and ikzf2 and application thereof |
PCT/CN2021/077285 WO2022147891A1 (zh) | 2021-01-07 | 2021-02-22 | 异位联合过表达Atoh1和Ikzf2再生耳蜗外毛细胞及其应用 |
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