CN117305367A - 一种表达全长耳畸蛋白的双aav载体系统及其应用 - Google Patents
一种表达全长耳畸蛋白的双aav载体系统及其应用 Download PDFInfo
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Abstract
本发明公开了一种表达全长耳畸蛋白的双AAV载体系统及其应用。所述双AAV载体系统包括:第一AAV载体,其包含在5’和3’ITR之间的第一核酸序列,所述第一核酸序列包含:依次连接的启动子、耳畸蛋白N端编码序列、剪接供体信号序列、重组序列;第二AAV载体,其包含在5’和3’ITR之间的第二核酸序列,所述第二核酸序列包含:依次连接的重组序列、剪接受体信号序列、耳畸蛋白C端编码序列。所述第一AAV载体和第二AAV载体能够在内毛细胞中发生高效的同源重组或AAV交联,继而高效的表达全长耳畸蛋白,最终恢复或改善OTOF基因缺失或突变患者的听力。
Description
技术领域
本发明涉及分子生物学领域、医药领域,具体涉及一种表达全长耳畸蛋白的双AAV载体系统及其应用。
背景技术
耳是人体的重要器官,由外耳、中耳和内耳构成,其主要功能是感知声音和维持身体平衡。当耳功能异常时会导致耳聋、耳鸣、眩晕等。耳聋是一种听觉功能异常的常见疾病,可分为先天性耳聋和后天性耳聋,与遗传因素和环境因素相关。每1000个新生婴儿中约有2位先天性耳聋患者,其中约60%的患者由基因突变引起,基因突变导致的耳聋可分为显性基因突变和隐性基因突变。对于这些由基因突变引发耳聋时,基因治疗可以作为耳聋根治的首选策略。
目前,大部分基因治疗都需要载体递送,腺相关病毒(Adeno-Associated Virus,AAV)是其中一种安全、高效的递送载体,其包装容量约为4.7kb。然而,在耳聋领域,许多基因编码区长度超过4kb,如BDP1、CDH23、COL11A2、LOXHD1、MET、MYO15A、MYO3A、MYO7A、OTOG、OTOF、OTOGL、PCDH15、PTPRQ、STRC、TECTA、TARA等,它们连同相关调控元件会超过AAV载体的包装限制。
在编码序列长于4kb的耳聋相关基因中,OTOF在听力中发挥了重要作用。OTOF蛋白(耳畸蛋白)主要表达在耳蜗的内耳毛细胞中,主要作用是结合钙离子(Ca2+),并启动下游神经递质的释放。OTOF基因的缺失或功能丧失性突变会引起DFNB9耳聋。现有技术为了解决OTOF基因的包装问题,目前主要有三种方法:(1)过载包装,将长度为7.5kb的基因表达元件包装到一个AAV病毒,并注射到小鼠耳蜗,其作用一段时间后能够观察到约30%的内耳毛细胞表达OTOF蛋白,部分小鼠听力平均恢复到58dB左右。但过载包装存在包装效率低、产品不易控制和转染效率低的问题,并不是最佳的解决方案;(2)缩短功能型OTOF所需要的编码序列长度,OTOF是一个C2结构域蛋白,由A、B、C、D、E、F六个C2结构域和一个TEM域组成,研究发现由其中几个结构域组成的迷你OTOF能够部分恢复OTOF电生理的功能,但是并不能恢复动物听力;(3)用双载体的方法进行DNA重组,产生全长完整成熟的OTOF mRNA并完成蛋白的翻译,但目前治疗方法和疗效皆有限,且临床获益完全未知。
因此,我们亟需寻找一种能高效治疗DFNB9耳聋的药物或治疗方法。
发明内容
本发明针对现有缺乏高效治疗DFNB9耳聋的药物和方法的问题,提供了一种表达全长耳畸蛋白的双AAV载体系统及其应用,所述的双AAV载体系统在体内重组效率高,能高效表达全长耳畸蛋白,改善DFNB9耳聋患者的听功能,同时对存在DFNB9耳聋风险的患者起到预防作用。
基于上述,本发明首先提供一种表达全长耳畸蛋白的双AAV载体系统,所述双AAV载体系统包括:
第一AAV载体,其包含在5’和3’ITR之间的第一核酸序列,所述第一核酸序列包含:依次连接的启动子、耳畸蛋白N端编码序列、剪接供体信号序列、重组序列;
第二AAV载体,其包含在5’和3’ITR之间的第二核酸序列,所述第二核酸序列包含:依次连接的重组序列、剪接受体信号序列、耳畸蛋白C端编码序列;
其中,所述耳畸蛋白N端编码序列如SEQ ID NO.5所示,所述耳畸蛋白C端编码序列如SEQ ID NO.7所示。
优选地,所述AAV选自AAV1、AAV2、AAV5、AAV6、AAV8、AAV9、AAV-PHP.B、AAV-PHP.eB、AAV-ie和Anc80L65中的任意一种或两种。
优选地,所述重组序列为F1噬菌体序列或碱性磷酸酶序列,所述F1噬菌体序列包含SEQ ID NO:10所示的序列,所述碱性磷酸酶序列包含SEQ ID NO:11所示的序列。
优选地,所述第一AAV载体还包含Kozak序列,所述Kozak序列定位于所述启动子和耳畸蛋白N端编码序列之间。
优选地,所述第二AAV载体还包含WPRE序列和PolyA序列,所述WPRE序列和PolyA序列依次连接在所述耳畸蛋白C端编码序列的3’端。
优选地,所述WPRE序列包含SEQ ID NO:12所示的序列。
优选地,所述启动子选自:巨细胞病毒(CMV)启动子、人β肌动蛋白/CMV杂合启动子、鸡β肌动蛋白/CMV杂合启动子、磷酸甘油激酶1(PGK)启动子、CMV-肌动蛋白-球蛋白(CAG)杂合启动子、延伸因子1α(EF-1α)启动子、泛素(Ubc)启动子、SV40启动子、Myo6启动子、Myo7a启动子、Myo15启动子、Math1启动子、VGLUT3启动子、OTOF启动子、STRC启动子、TMC1启动子、GJB2启动子或Prestin启动子中的任意一种。
本发明另一方面还提供了如前面任一项所述的表达全长耳畸蛋白的双AAV载体系统在制备预防和/或治疗听神经病的药物中的应用,所述药物用于治疗DFNB9听力损失的患者,或在具有DFNB9突变的患者中预防DFNB9听力损失。
本发明另一方面还提供了一种预防和/或治疗听神经病的药物组合物,所述的药物组合物包括:前面任一项所述的表达全长耳畸蛋白的双AAV载体系统,以及药学上和生理学上可接受的载体。
相对于现有技术,本发明的有益效果至少包括:
本发明通过耳畸蛋白N端和C端序列位点的筛选、DNA元件的组合和优化,最终提供了一种双AAV载体系统,其中,第一AAV载体和第二AAV载体能够有效地转导所靶向的内毛细胞,并在内毛细胞中发生高效的同源重组或AAV交联,继而高效的表达全长耳畸蛋白,最终恢复或改善OTOF基因缺失或突变患者的听力,能够在不开启人工耳蜗的情况下,满足日常生活所需的听力及交流。
附图说明
图1为双AAV载体系统构建示意图。
图2为Otof-/-小鼠的听功能结果及免疫组化结果。
图3为Otof-/-小鼠注射3种双AAV载体系统治疗后的听功能结果比较。
图4为Otof-/-新生鼠注射双AAV载体系统治疗后4周的ABR结果。
图5为Otof-/-新生鼠注射双AAV载体系统治疗后4周的免疫组化结果。
图6为Otof-/-成年鼠注射双AAV载体系统治疗后3个月的ABR结果。
图7为DFNB9患者给与双AAV载体系统治疗后4周的ABR结果。
具体实施方式
以下结合附图和实施例对本发明的技术方案做进一步的说明。
由背景技术可知,耳畸蛋白在听力中发挥了重要作用。耳畸蛋白由OTOF基因编码。人类OTOF的编码区由47个外显子组成,表达的全长耳畸蛋白由1997个氨基酸组成。耳畸蛋白主要表达在耳蜗的内耳内毛细胞的突触处,参与囊泡释放,主要作用是结合钙离子(Ca2 +),并启动下游神经递质的释放。耳畸蛋白功能异常导致内毛细胞不能将机械声觉振动转化为大脑皮层能感知的神经信号。目前,上百个与OTOF相关的病理突变均可扰乱耳畸蛋白功能的正常发挥,这严重影响2-8%的先天性遗传性耳聋患者的生活。耳畸蛋白的缺失或功能丧失性会引起非综合征常染色体隐性耳聋-9(DFNB9)。然而,目前缺乏能够高效治疗DFNB9耳聋的药物或方法。
为解决上述技术问题,本发明申请人经过大量试验和实验验证,最终提供了一种表达全长耳畸蛋白的双AAV载体系统,所述双AAV载体系统包括:
第一AAV载体,其包含在5’和3’ITR之间的第一核酸序列,所述第一核酸序列包含:依次连接的启动子、耳畸蛋白N端编码序列、剪接供体信号序列、重组序列;
第二AAV载体,其包含在5’和3’ITR之间的第二核酸序列,所述第二核酸序列包含:依次连接的重组序列、剪接受体信号序列、耳畸蛋白C端编码序列。
一些实施例中,所述第一AAV载体还包含Kozak序列,所述Kozak序列定位于所述启动子和耳畸蛋白N端编码序列之间。
一些实施例中,所述第二AAV载体还包含WPRE序列和PolyA序列,所述WPRE序列和PolyA序列依次连接在所述耳畸蛋白C端编码序列的3’端。
术语说明
AAV载体
所述第一AAV载体、第二AAV载体均由衣壳及其内部DNA组成,第一AAV载体和第二AAV载体选自AAV1、AAV2、AAV5、AAV6、AAV8、AAV9、AAV-PHP.B、AAV-PHP.eB、AAV-ie或Anc80L65中的任意一种或两种;优选地,所述第一AAV载体和第二AAV载体选自AAV-PHP.eB、Anc80L65或AAV1中的任意一种或两种。
ITR序列
两侧反向末端重复(ITR)序列各自包含145个碱基。该名称来源于其对称性,所述对称性是AAV基因组所必需的。这些序列的另一个性质是其形成发卡结构的能力,所述发卡结构有助于所谓自启效应(self-priming)。所述ITR是AAV衣壳化以及形成完全组装且脱氧核酸酶耐受性AAV颗粒所必需的。
启动子
启动子是启动DNA转录序列,分为通用型启动子和特异性启动子。其中,通用型启动子包括CMV、CAG、PGK、EF-1α、Ubc、SV40等。而特异性在耳蜗表达的启动子包括MYO6、MYO15、MYO7A、MATH1、VGLUT3,OTOF、STRC、TMC1、GJB2及PRESTIN等。上述所述启动子的基因均可来源于各类动物。优选地,来源脊椎动物;更优选地,来源人或鼠的同源基因。
耳畸蛋白N端编码序列和C端编码序列
如本发明所用,术语“耳畸蛋白”表示耳畸蛋白多肽,在此处缩写“OTOF”。该多肽也称为DFNB9。在SEQ ID NO.1中显示野生型人耳畸形蛋白多肽的氨基酸序列全长,SEQ IDNO.2(对应于NM_001287489所示的编码序列)为编码SEQ ID NO.1的核苷酸序列。OTOF基因CDs区全长为5994bp,而AAV病毒包装限度大约为4.7kb。
因此,可行的,所述耳畸蛋白N端编码序列的长度为1-4700bp,耳畸蛋白C端编码序列的长度为1-4700bp。本发明经过筛选,提供了所述耳畸蛋白N端编码序列如SEQ ID NO.5所示,即所述第一AAV载体编码OTOF基因序列SEQ ID NO:1中氨基酸残基1-841,长度为2523bp,氨基酸序列如SEQ ID NO.4所示;所述耳畸蛋白C端编码序列如SEQ ID NO.7所示,即所述第二AAV载体编码OTOF基因序列SEQ ID NO:1中氨基酸残基842-1997,长度为3471bp,氨基酸序列如SEQ ID NO.6所示。其中:
SEQ ID NO:4:
MALLIHLKTVSELRGRGDRIAKVTFRGQSFYSRVLENCEDVADFDETFRW
PVASSIDRNEMLEIQVFNYSKVFSNKLIGTFRMVLQKVVEESHVEVTDTLI
DDNNAIIKTSLCVEVRYQATDGTVGSWDDGDFLGDESLQEEEKDSQETDG
LLPGSRPSSRPPGEKSFRRAGRSVFSAMKLGKNRSHKEEPQRPDEPAVLEM
EDLDHLAIRLGDGLDPDSVSLASVTALTTNVSNKRSKPDIKMEPSAGRPM
DYQVSITVIEARQLVGLNMDPVVCVEVGDDKKYTSMKESTNCPYYNEYF
VFDFHVSPDVMFDKIIKISVIHSKNLLRSGTLVGSFKMDVGTVYSQPEHQF
HHKWAILSDPDDISSGLKGYVKCDVAVVGKGDNIKTPHKANETDEDDIEG
NLLLPEGVPPERQWARFYVKIYRAEGLPRMNTSLMANVKKAFIGENKDL
VDPYVQVFFAGQKGKTSVQKSSYEPLWNEQVVFTDLFPPLCKRMKVQIR
DSDKVNDVAIGTHFIDLRKISNDGDKGFLPTLGPAWVNMYGSTRNYTLLD
EHQDLNEGLGEGVSFRARLLLGLAVEIVDTSNPELTSSTEVQVEQATPISES
CAGKMEEFFLFGAFLEASMIDRRNGDKPITFEVTIGNYGNEVDGLSRPQRP
RPRKEPGDEEEVDLIQNASDDEAGDAGDLASVSSTPPMRPQVTDRNYFHL
PYLERKPCIYIKSWWPDQRRRLYNANIMDHIADKLEEGLNDIQEMIKTEKS
YPERRLRGVLEELSCGCCRFLSLADKDQGHSSRTRLDRERLKSCMRELENMGQQARMLRAQVKRHTVRDKLRLCQNFLQKLRFLADE。
SEQ ID NO:6:
PQHSIPDIFIWMMSNNKRVAYARVPSKDLLFSIVEEETGKDCAKVKTLFLK
LPGKRGFGSAGWTVQAKVELYLWLGLSKQRKEFLCGLPCGFQEVKAAQ
GLGLHAFPPVSLVYTKKQAFQLRAHMYQARSLFAADSSGLSDPFARVFFI
NQSQCTEVLNETLCPTWDQMLVFDNLELYGEAHELRDDPPIIVIEIYDQDS
MGKADFMGRTFAKPLVKMADEAYCPPRFPPQLEYYQIYRGNATAGDLLA
AFELLQIGPAGKADLPPINGPVDVDRGPIMPVPMGIRPVLSKYRVEVLFWG
LRDLKRVNLAQVDRPRVDIECAGKGVQSSLIHNYKKNPNFNTLVKWFEV
DLPENELLHPPLNIRVVDCRAFGRYTLVGSHAVSSLRRFIYRPPDRSAPSW
NTTVRLLRRCRVLCNGGSSSHSTGEVVVTMEPEVPIKKLETMVKLDATSE
AVVKVDVAEEEKEKKKKKKGTAEEPEEEEPDESMLDWWSKYFASIDTMK
EQLRQQEPSGIDLEEKEEVDNTEGLKGSMKGKEKARAAKEEKKKKTQSS
GSGQGSEAPEKKKPKIDELKVYPKELESEFDNFEDWLHTFNLLRGKTGDD
EDGSTEEERIVGRFKGSLCVYKVPLPEDVSREAGYDSTYGMFQGIPSNDPI
NVLVRVYVVRATDLHPADINGKADPYIAIRLGKTDIRDKENYISKQLNPVF
GKSFDIEASFPMESMLTVAVYDWDLVGTDDLIGETKIDLENRFYSKHRATC
GIAQTYSTHGYNIWRDPMKPSQILTRLCKDGKVDGPHFGPPGRVKVANRV
FTGPSEIEDENGQRKPTDEHVALLALRHWEDIPRAGCRLVPEHVETRPLLN
PDKPGIEQGRLELWVDMFPMDMPAPGTPLDISPRKPKKYELRVIIWNTDE
VVLEDDDFFTGEKSSDIFVRGWLKGQQEDKQDTDVHYHSLTGEGNFNWR
YLFPFDYLAAEEKIVISKKESMFSWDETEYKIPARLTLQIWDADHFSADDF
LGAIELDLNRFPRGAKTAKQCTMEMATGEVDVPLVSIFKQKRVKGWWPL
LARNENDEFELTGKVEAELHLLTAEEAEKNPVGLARNEPDPLEKPNRPDT
AFVWFLNPLKSIKYLICTRYKWLIIKIVLALLGLLMLGLFLYSLPGYMVKKLLGA*。
剪接供体信号序列和剪接受体信号序列
剪接供体和受体是RNA在剪切体剪切过程中识别的序列,包括GU-AG和AU-AC等类型供体和受体,优选地,供体序列为GU(A/G)AGU,受体序列为高嘧啶序列-AG。本发明一实施例中,所述剪接供体信号序列(SD序列)为:GUAAGUAUCAAGGUUACAAGACAGGUUUAAGGAGACCAAUAGAAACUGGGCUUGUCGAGACAGAGAAGACUCUUGCGUUUCU(SEQ ID NO.8),所述剪接受体信号序列(SK序列)为:GAUAGGCACCUAUUGGU CUUACUGACAUCCACUUUGCCUUUCUCUCCACAG(SEQ ID NO.9)。
重组序列
重组序列为双载体AAV DNA中互补的区域,实现双载体DNA的重组。其重组序列可以选自OTOF中互补的序列、其他基因的序列(如碱性磷酸酶AP、噬菌体序列AK)、或AAV的ITR序列等。本发明一实施例中,所述重组序列为AK序列:GGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAAT GAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAAT(SEQ ID NO.10);本发明另一实施例中,所述重组序列为AP序列:GGAATGGCTGGCGAAGCGCCAGGGTGCCCGGTATGTGTGGAACCGCACTGAGCTCATGCAGGCTTCCCTGGACCCGTCTGTGACCCATCTCATGGGTCTCTTTGAGCCTGGAGACATGAAATACGAGATCCACCGAGACTCCACACTGGACCCCTCCCTGATGGAGATGACAGAGGCTGCCCTGCGCCTGCTGAGCAGGAACCCCCGCGGCTTCTTCCTCTTCGTGGAGGGTGGTCGCATCGACCATGGTCATCATGAAAGCAGGGCTTACCGGGCAC(SEQ ID NO.11)。
PolyA序列
PolyA序列为稳定mRNA不被降解的信号序列,可选自于各种基因,优选于牛生长激素基因、SV40病毒序列等。本发明一实施例中,所述PolyA序列选自牛生长激素的PolyA序列。
WPRE序列
WPRE是一段顺式作用的RNA元件,为转录后调控序列,放在多聚腺苷酸化PolyA信号之前可显著增加mRNA的表达水平和翻译效率,进而增强基因的表达,并且WPRE插入病毒载体中还可以大大提升病毒包装的滴度。本发明一实施例中,所述WPRE序列如SEQ IDNO.12所示。
通过以上技术方案,本发明申请人使用双AAV载体方法将耳畸蛋白基因分成两部分提供至内毛细胞,在内毛细胞中发生同源重组或AAV交联并表达全长蛋白。经过实验验证,本发明所述的第一AAV载体和第二AAV载体能够有效地转导所靶向的内毛细胞,并在内毛细胞中发生高效的同源重组或AAV交联并表达全长耳畸蛋白,恢复或改善OTOF基因缺失或先天性突变患者的听力。
本发明还提供了所述的表达全长耳畸蛋白的双AAV载体系统在制备预防和/或治疗听神经病的药物中的应用,所述药物用于治疗DFNB9听力损失的患者,或在具有DFNB9突变的患者中预防DFNB9听力损失。
本发明还提供了一种预防和/或治疗听神经病的药物组合物,所述的药物组合物包括:本发明所述的所述表达全长耳畸蛋白的双AAV载体系统,以及药学上和生理学上可接受的载体。
合适的药学上可接受的载体是本领域普通技术人员所熟知的。在Remington’sPharmaceutical Sciences中可找到关于药学上可接受的载体的充分说明。在组合物中药学上可接受的载体可含有液体,如水、磷酸盐缓冲液、ringer溶液、生理盐水、平衡盐溶液、甘油或山梨醇等。另外,这些载体中还可能存在辅助性的物质,如润滑剂、助流剂、润湿剂或乳化剂、pH缓冲物质和稳定剂,如白蛋白等。
本发明的药物制剂可以是任何剂型,包括但不限于注射剂型和软膏剂型,腺相关病毒为主要活性成分;优选地,制剂或配方或药物可以包含常用溶剂、缓冲液,如常用的药物载体和辅剂,包括中性盐缓冲液、酸性盐缓冲液、碱性盐缓冲液、葡萄糖、甘露糖、甘露糖醇、蛋白、多肽及氨基酸、抗生素、螯合剂、辅剂或防腐剂中的一种或多种;更优选地,所述制剂的配方为人工耳淋巴液或PF-68含量为0.001%-0.1%的PBS或HEPES溶液。
本发明所述的药物组合物可通过多种途径施用于DFNB9患者,例如局部施用于内耳(诸如通过注射装置或导管经圆窗膜、半规管、小管造口术、镫骨底板等将药物施用于内耳毛细胞)、静脉、皮下、口服、肌肉、腹腔、动脉、吸入、灌注施用。药物组合物在一定条件下需要使用一次或一次以上。在一些实施例中,双耳同时给药;在另一些实施例中,双耳间隔给药。在一些实施例中,药物组合物中双AAV载体同时施用;在另一些实施例中,双AAV载体间隔实施。双AAV载体可具有相同或不同的血清型。
本发明所述治疗的受试者是患有OTOF突变相关的感音神经性耳聋或具有所述疾病风险的受试者。受试者经本领域技术人员已知的标准方法(听力学评估、基因诊断)筛选。听力学评估使用临床已知的标准方法(诸如ABR、OAE、ASSR、PTA、MAIS、CAP等)评价。基因诊断筛选使用已知的常规方法(外显子测序、Sanger测序)检测。
药物组合物在临床给药上,所使用的给药量、给药装置、给药路径、给药术式都在本领域临床领域的技术人员的操作技能范围内。给药量包括一次或一次以上施用单位剂量的有预期疗效的药物组合物。本文所述的AAV载体药物可具有以下滴度:约1×1010VG/mL至1×1017VG/mL;给药体积可包含0.001mL至1mL;给药剂量可包含约1×107VG/mL/耳至约1×1017VG/mL/耳。
本发明所述的药物组合物递送至体内后,可重组成编码全长OTOF的核酸序列,并表达出完整的耳畸蛋白,增加野生型耳畸蛋白的表达量,从而改善受试者的听功能。经标准的听力测试(ABR、OAE、ASSR、PTA、MAIS、CAP等)检查发现,受试者经本发明药物治疗后,测定的听力阈值可降低约5%至80%或至正常水平,听功能与言语识别能力会显著改善和提高。
下面对本发明的实验过程及实验结果进行详细说明。
下述实施例中所使用的实验方法,如无特殊说明,均按照常规或者制造厂商所建议的条件进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
(一)设计并构建双AAV载体系统
按照图1所示构建第一AAV载体和第二AAV载体,所述第一AAV载体和第二AAV载体均选用AAV1,其中,在所述第一AAV载体中的5’和3’ITR之间包含:依次连接的启动子(选用Myo15或CMV启动子)、OTOF N端编码序列(2523bp,SEQ ID NO.5)、剪接供体信号序列SD(SEQID NO.8)和重组序列(AK序列SEQ ID NO.10或AP序列SEQ ID NO.11)。
在所述第二AAV载体中的5’和3’ITR之间包含:依次连接的重组序列(AK序列SEQID NO.10或AP序列SEQ ID NO.11)、剪接受体信号序列SA(SEQ ID NO.9)、OTOF C端编码序列(3741bp,SEQ ID NO.7)、WPRE序列(SEQ ID NO.12)和PolyA序列。同时纳入无重组序列的双AAV载体系统(即第一AAV载体和第二AAV载体均无重组序列)作为对照。
需要说明的是,本发明未提供具体序列的,如ITR序列、PolyA序列等均为本领域技术人员根据公知和常规技术能容易得到或简单修改、替换的,在此不做赘述。
(二)使用双AAV载体系统进行Otof-/-小鼠的体内治疗实验
1.实验方法
(1)Otof-/-耳聋小鼠模型的构建
通过基因编辑技术构建129品系的Otof-/-耳聋小鼠模型,在Otof基因组外显子21的一段序列(TCAGGTGAAGCGGCACACTG)前插入一个腺嘌呤(A);纯合基因型的小鼠表现出极重度听力损失。本研究方案获复旦大学伦理委员会批准。所有动物均在复旦大学实验动物科学系饲养,所有实验程序均按照动物研究的政策和伦理进行。
(2)新生Otof-/-小鼠内耳给药及检测
将第一AAV载体与第二AAV载体在体外充分混合后经圆窗膜(RWM)递送至P0-P7新生Otof-/-小鼠内耳中。用低温麻醉的方法麻醉小鼠,显微操作定位圆窗龛后,控制显微注射系统向圆窗膜内注射不同滴度的双AAV载体混合物(7.5×109VG、1.5×1010VG、3×1010VG、6×1010VG),注射体积为2μL,注射速度为5nL/s,确保手术切口无活动性出血后将其送回母亲笼盒内。同时纳入新生P0-P7野生型小鼠和未给药P0-P7新生Otof-/-小鼠作为对照。注射4周后,进行听性脑干反应(ABR)测试,具体地:
使用RZ6声学系统(Tucker-Davis Technologies,Alachua,FL,USA)在隔音室中记录听觉脑干反应(ABRs)。将甲苯噻嗪(20mg/kg)和氯胺酮(100mg/kg)混合液,通过腹腔注射将小鼠麻醉;麻醉状态良好的小鼠放入TDT工作站隔音箱中;采集电极插入小鼠颅顶中线皮下;刺激电极插入小鼠耳后乳突皮下;接地电极插入小鼠后背皮下;选择声音刺激频率(4kHz、8kHz、16kHz、24kHz和32kHz),声强范围从90dB~20dB以5dB依次递减,每个频率的声强从90dB开始逐渐减弱至20dB,对小鼠在不同频率下听力的阈值进行检测;同时记录小鼠I波潜伏期及振幅。
完成ABR测试后处死小鼠,取出耳蜗后将其置于预冷的1×PBS溶液中,显微镜下在蜗壳顶部打一孔;将耳蜗放到4% PFA溶液中进行固定,常温下摇床过夜,或4℃冰箱存放1天;然后放入预先装有10% EDTA的1.5mL EP管中进行脱钙;脱钙后的耳蜗分为顶、中、底回染色;1×PBS溶液中,常温5min,浸洗三次;1%PBST(1% Triton X-100)10%血清封闭,常温下孵育1小时;然后用耳畸蛋白Otoferlin一抗(1:200,Abcam)、Myo7a一抗(1:500,Proteus BioSciences)在4℃下孵育过夜,再用1×PBS冲洗三次。在室温下使用对应二抗孵育2小时。细胞核用DAPI(Sigma-Aldrich)染色。用Leica TCS SP8激光扫描共聚焦显微镜(40×物镜)采集图像。图像由Image J软件处理和分析。
(3)成年Otof-/-小鼠内耳给药及检测
将第一AAV载体与第二AAV载体在体外充分混合,P30-P35的成年Otof-/-小鼠麻醉、备皮、消毒后,在右侧耳后作一切口,显微操作定位圆窗龛后,然后将不同滴度的双AAV载体混合药物(1.5×1010VG、3×1010VG、6×1010VG)注射到耳蜗中,注射体积为2μL,注射速度为5nL/s,确保手术切口无活动性出血后将其送回笼盒内。注射3个月后,进行ABR检测和免疫组织化学染色评估小鼠的听功能。
2.实验结果
(1)Otof-/-小鼠的听功能完全丧失,成功模拟DFNB9重度耳聋患者
为了评估所构建Otof-/-小鼠的听功能,利用ABR和免疫组化手段分别评估小鼠的听力及耳畸蛋白的表达情况。
结果如图2所示,在无药物干预的情况下,Otof-/-小鼠的听力完全丧失(图2中的A)。另外,单独注射第一AAV载体或第二AAV载体皆不能恢复小鼠的听力(图2中的A)。免疫组化染色结果表明,Otof-/-小鼠的内耳毛细胞(红色Myo7a标记)中不表达耳畸蛋白(白色Otoferlin标记,图2中的B)。
(2)三种双AAV载体系统改善新生Otof-/-小鼠的听功能,其中AK型效果较好
由于双AAV载体系统在基因治疗中的挑战性,为了筛选出疗效更好的双AAV载体治疗体系,本发明设计并比较了三种双AAV载体系统对Otof-/-小鼠的听功能改善情况。三种双AAV载体系统的区别在于重组序列分别为AK序列、AP序列或无重组序列(TS)。
结果如图3所示,由ABR结果可知,三种双AAV载体系统均能明显改善Otof-/-小鼠的听功能,其中AK型双AAV载体系统(DualAAV1-hyb(AK))的疗效更优。
(3)本发明双AAV载体系统改善新生Otof-/-小鼠的听功能
为了研究双AAV载体系统(AK型)对新生Otof-/-小鼠听力功能的影响,记录了ABR,评估治疗组小鼠4kHz至32kHz频率范围内的听力恢复情况。
首先通过实验证实了双AAV载体系统不影响野生型小鼠的听力功能。结果如图4所示,注射4周后,与未治疗组(Otof-/-组)相比,经过双AAV载体系统治疗的小鼠,其ABR波中出现明显的I、II、III、IV、V波形(图4中的A),且所有测试频率的ABR阈值均显著降低(图4中的B)。并且,随着双AAV载体系统注射量的增加,治疗效果越明显,其中,注射滴度达6×1010VG时,小鼠的ABR阈值与野生型(WT组)小鼠最接近。
以上结果表明,本发明实施例构建的双AAV载体系统可显著改善新生Otof-/-小鼠的听功能,且一定范围内呈浓度依赖性。
(4)本发明双AAV载体系统恢复新生Otof-/-小鼠耳蜗中耳畸蛋白的表达
为了评估双AAV载体系统(AK型)在新生Otof-/-小鼠耳蜗中耳畸蛋白的表达,我们在完成ABR测试后处死小鼠,进行了相关分析。
结果如图5所示,蓝色DAPI标记细胞核,红色Myo7a抗体特异性标记内耳毛细胞,白色Otoferlin抗体表示耳畸蛋白在内耳内毛细胞中出现明显的表达,说明双AAV载体系统成功恢复了新生Otof-/-小鼠耳蜗中耳畸蛋白的表达。
(5)本发明双AAV载体系统改善成年Otof-/-小鼠的听功能
成年鼠的耳蜗发育状态更接近人的耳蜗发育情况,为了研究双AAV载体系统(AK型)对成年Otof-/-小鼠听力功能的影响,记录了ABR,评估治疗组小鼠4kHz至32kHz频率范围内的听力恢复情况。
首先通过实验证实了双AAV载体系统不影响野生型小鼠的听力功能。结果如图6所示,注射4周后,与未治疗组(Otof-/-组)相比,经过双AAV载体系统治疗的耳蜗中所有测试频率的ABR阈值均显著降低。并且,随着双AAV载体系统注射量的增加,治疗效果越明显,其中,注射滴度达6×1010VG时,小鼠的ABR阈值与野生型(WT组)小鼠最接近。
以上结果表明,本发明实施例构建的双AAV载体系统可显著改善成年Otof-/-小鼠的听功能,且一定范围内呈浓度依赖性。
(三)使用双AAV载体系统进行DFNB9患者的体内治疗实验
基于前述研究,本发明还进行了临床试验,以验证本发明提出的双AAV载体系统的体内治疗效果。该临床试验经伦理委员会审批,并在中国临床试验中心注册和审核(ChiCTR2200063181),严格按照临床方案开展。具体地:
在临床医生的专业操作下将双AAV载体系统混合后注射至经确认和筛选后的DFNB患者内耳中。手术在全麻下进行(麻醉医生负责麻醉安全)。手术暴露圆窗,采取经圆窗注射。注射参数:注入药物体积:30μL;输注速率:10.0mL/min;输入治疗药物量:9×1011vg。
注射4周后通过ABR等测试评估患者的听力改善情况。结果如图7所示,相较于治疗前患者的ABR阈值,治疗后该患者的ABR阈值显著降低20dB以上,其中在刺激频率为1和2kHz的情况时,ABR阈值至50dB以下。
实施例2
本发明申请还对耳畸蛋白的核苷酸序列进行了密码子优化,优化后耳畸蛋白的核苷酸序列如SEQ ID NO.3所示,氨基酸序列仍是SEQ ID NO.1。依据实施例1双载体构建方法,构建重组成SEQ ID NO.3序列并表达耳畸蛋白的双载体AAV系统,并进行听力检测。
结果发现,本实施例构建的双AAV载体系统具有更优异重组和表达率,改善听力的效果更好。
综上所述,本发明提供了一种双AAV载体系统,其包括第一AAV载体和第二AAV载体,所述第一AAV载体和第二AAV载体能够有效地转导所靶向的内毛细胞,并在内毛细胞中发生高效的同源重组或AAV交联,继而高效的表达全长耳畸蛋白,最终恢复或改善OTOF基因缺失或突变患者的听力,能够在不开启人工耳蜗的情况下,满足日常生活所需的听力及交流。
尽管本发明的内容已经通过上述优选实施例作了详细介绍,但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后,对于本发明的多种修改和替代都将是显而易见的。因此,本发明的保护范围应由所附的权利要求来限定。
Claims (9)
1.一种表达全长耳畸蛋白的双AAV载体系统,其特征在于,所述双AAV载体系统包括:
第一AAV载体,其包含在5’和3’ITR之间的第一核酸序列,所述第一核酸序列包含:依次连接的启动子、耳畸蛋白N端编码序列、剪接供体信号序列、重组序列;
第二AAV载体,其包含在5’和3’ITR之间的第二核酸序列,所述第二核酸序列包含:依次连接的重组序列、剪接受体信号序列、耳畸蛋白C端编码序列;
其中,所述耳畸蛋白N端编码序列如SEQ ID NO.5所示,所述耳畸蛋白C端编码序列如SEQ ID NO.7所示。
2.如权利要求1所述的表达全长耳畸蛋白的双AAV载体系统,其特征在于,所述AAV选自AAV1、AAV2、AAV5、AAV6、AAV8、AAV9、AAV-PHP.B、AAV-PHP.eB、AAV-ie和Anc80L65中的任意一种或两种。
3.如权利要求1所述的表达全长耳畸蛋白的双AAV载体系统,其特征在于,所述重组序列为F1噬菌体序列或碱性磷酸酶序列,所述F1噬菌体序列包含SEQ ID NO:10所示的序列,所述碱性磷酸酶序列包含SEQ ID NO:11所示的序列。
4.如权利要求1所述的表达全长耳畸蛋白的双AAV载体系统,其特征在于,所述第一AAV载体还包含Kozak序列,所述Kozak序列定位于所述启动子和耳畸蛋白N端编码序列之间。
5.如权利要求1所述的表达全长耳畸蛋白的双AAV载体系统,其特征在于,所述第二AAV载体还包含WPRE序列和PolyA序列,所述WPRE序列和PolyA序列依次连接在所述耳畸蛋白C端编码序列的3’端。
6.如权利要求5所述的表达全长耳畸蛋白的双AAV载体系统,其特征在于,所述WPRE序列包含SEQ ID NO:12所示的序列。
7.如权利要求1所述的表达全长耳畸蛋白的双AAV载体系统,其特征在于,所述启动子选自:巨细胞病毒启动子、人β肌动蛋白/CMV杂合启动子、鸡β肌动蛋白/CMV杂合启动子、磷酸甘油激酶1启动子、CMV-肌动蛋白-球蛋白杂合启动子、延伸因子1α启动子、泛素启动子、SV40启动子、Myo6启动子、Myo7a启动子、Myo15启动子、Math1启动子、VGLUT3启动子、OTOF启动子、STRC启动子、TMC1启动子、GJB2启动子或Prestin启动子中的任意一种。
8.如权利要求1-7中任一项所述表达全长耳畸蛋白的双AAV载体系统在制备预防和/或治疗听神经病的药物中的应用,其特征在于,所述药物用于治疗DFNB9听力损失的患者,或在具有DFNB9突变的患者中预防DFNB9听力损失。
9.一种预防和/或治疗听神经病的药物组合物,其特征在于,所述的药物组合物包括:权利要求1-7中任一项所述表达全长耳畸蛋白的双AAV载体系统,以及药学上和生理学上可接受的载体。
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