CN105755044A - 具有变异衣壳的腺相关病毒病毒体及其使用方法 - Google Patents
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Abstract
本公开提供了衣壳蛋白改变的腺相关病毒(AAV)病毒体,其中所述AAV病毒体在经玻璃体内注射施用时,与野生型AAV相比,表现出更强的视网膜细胞感染性。本公开进一步提供了将基因产物递送至个体的视网膜细胞的方法和治疗眼病的方法。
Description
本申请是申请日为2012年4月20日、发明名称为“具有变异衣壳的腺相关病毒病毒体及其使用方法”的中国发明专利申请201280024852.3的分案申请。
交叉引用
本申请要求2011年4月22日提交的美国临时专利申请No.61/478,355的权益,所述申请通过引用整体并入本文。
关于联邦资助研究的声明
本发明通过由国立卫生研究院的国立眼科研究所授予的批准号EY016994-02和EY1018241在政府支持下进行。政府具有本发明的某些权利。
背景
光感受器是视网膜中接收和处理视觉信息,通过光转导将可见电磁辐射转换为超极化反应的第一个神经元。绝大多数遗传视网膜疾病直接导致这些细胞丢失,例如影响视紫红质蛋白折叠的显性突变,或间接导致这些细胞丢失,例如视网膜色素上皮细胞(RPE)中影响视网膜再循环途径的隐性突变。
AAV属于细小病毒科(Parvoviridae)和依赖病毒属(Dependovirus),其成员需要与辅助病毒例如腺病毒一起协同感染以启动复制,并且AAV在没有辅助病毒时建立了潜伏性感染。病毒体由涵盖具有两个开放阅读框:rep和cap的4.9kb单链DNA基因组的25nm二十面体外壳构成。非结构rep基因编码病毒复制所必需的4种调节蛋白,而cap编码装配成60-mer衣壳外壳的3种结构蛋白(VP1-3)。这种病毒衣壳介导AAV载体克服病毒转导的许多生物屏障的能力—包括细胞表面受体结合、胞吞作用、胞内运输和细胞核中的解包装。
文献
美国专利公布No.2005/0053922;美国专利公布No.2009/0202490;Allocca等(2007)J.Virol.81:11372;Boucas等(2009)J.GeneMed.11:1103。
发明概述
本公开提供了衣壳蛋白改变的腺相关病毒(AAV)病毒体,其中所述AAV病毒体在经玻璃体内注射施用时,与野生型AAV相比,表现出更强的视网膜细胞感染性。本公开进一步提供了将基因产物递送至个体的视网膜细胞的方法和治疗眼病的方法。
附图简述
图1提供了在氨基酸587之后含有无规七聚物的AAV2的代表性三维模型。
图2描绘了相对于AAV2(左),AAV27M8变异体(右)的更高水平的玻璃体内转导。
图3提供了视网膜低温切片的代表性荧光图像,显示出由携带GFP基因的7M8,在普遍存在的CAG启动子(左)或光感受器特异性Rho启动子(右)的控制下产生的绿色荧光蛋白(GFP)表达。
图4描绘了在经7M8或载有4个酪氨酸突变(7m8.4YF)的7M8转导后,通过流式细胞术计数的每一百万个视网膜细胞的GFP+光感受器细胞。
图5提供了AAV2VP1的氨基酸序列(SEQIDNO:1)。
图6提供了与不同AAV血清型的AAV衣壳蛋白VP1的AAV2(图5)的氨基酸570-610相对应的氨基酸序列。
图7A-I描绘了基因转移后,Rs1h-/-小鼠视网膜中的结构改进。
图8A-D描绘了RS1基因递送后,视网膜电流图A和B波的功能性救援。
图9A-E描绘了7m8-rho-RS1治疗后10个月时测量的视网膜厚度持续增加。
图10提供了视网膜劈裂蛋白(retinoschisin)的氨基酸序列。
图11提供了脑源性神经营养因子的氨基酸序列。
图12提供了RPE65的氨基酸序列。
图13A-C提供了7m8-rho-RS1构建体的核苷酸序列。
图14提供了外周蛋白-2的氨基酸序列。
图15提供了外周蛋白的氨基酸序列。
图16提供了色素性视网膜炎GTP酶调节因子相互作用蛋白-1的氨基酸序列。
图17A-C提供了AAV衣壳蛋白环IV(GH环)区域的氨基酸序列的比对。***位点用粗体和下划线示出。
图18A-C提供了具有异源肽***的AAV衣壳蛋白GH环区域的氨基酸序列的比对。
图19提供了荧光眼底图像,显示在施用携带受连接蛋白36启动子控制的GFP的7m8后9周,灵长类动物中央视网膜中的GFP表达。
定义
术语“视网膜细胞”在本文中可指任何细胞类型,包括视网膜,例如视网膜神经节细胞、无长突细胞、水平细胞、双极细胞和光感受器细胞(包括杆状和锥状细胞)、缪勒胶质细胞和视网膜色素上皮细胞。
“AAV”是腺相关病毒的缩写,并且可用于指病毒本身或其衍生物。除非另有需要时,所述术语包括亚型及自然存在和重组形式。缩写“rAAV”指重组腺相关病毒,也称为重组AAV载体(或“rAAV载体”)。术语“AAV”包括1型AAV(AAV-1)、2型AAV(AAV-2)、3型AAV(AAV-3)、4型AAV(AAV-4)、5型AAV(AAV-5)、6型AAV(AAV-6)、7型AAV(AAV-7)、8型AAV(AAV-8)、禽AAV、牛AAV、犬AAV、马AAV、灵长类AAV、非灵长类AAV和羊AAV。“灵长类AAV”指感染灵长类动物的AAV,“非灵长类AAV”指感染非灵长类哺乳动物的AAV,“牛AAV”指感染牛哺乳动物的AAV等。
不同亚型AAV的基因组序列,以及天然末端重复序列(TR)、Rep蛋白和衣壳亚基的序列在本领域中已知。此类序列可在文献或公用数据库例如基因库中找到。见,例如,基因库登记号NC_002077(AAV-1)、AF063497(AAV-1)、NC_001401(AAV-2)、AF043303(AAV-2)、NC_001729(AAV-3)、NC_001829(AAV-4)、U89790(AAV-4)、NC_006152(AAV-5)、AF513851(AAV-7)、AF513852(AAV-8)和NC_006261(AAV-8);其公开内容通过引用并入本文,以讲授AAV核酸和氨基酸序列。同样见,例如,Srivistava等(1983)J.Virology45:555;Chiorini等(1998)J.Virology71:6823;Chiorini等(1999)J.Virology73:1309;Bantel-Schaal等(1999)J.Virology73:939;Xiao等(1999)J.Virology73:3994;Muramatsu等(1996)Virology221:208;Shade等,(1986)J.Virol.58:921;Gao等(2002)Proc.Nat.Acad.Sci.USA99:11854;Moris等(2004)Virology33:375-383;国际专利公布WO00/28061、WO99/61601、WO98/11244;和美国专利No.6,156,303。
如本文所使用,“rAAV载体”指包含非AAV起源的多核苷酸序列(即,与AAV异源的多核苷酸),通常是细胞遗传转化的目标序列的AAV载体。一般而言,异源多核苷酸两侧有至少一个,而通常有两个AAV反向末端重复序列(ITR)。术语rAAV载体涵盖rAAV载体颗粒和rAAV载体质粒。rAAV载体可为单链(ssAAV)或自身互补(scAAV)。
“AAV病毒”或“AAV病毒颗粒”或“rAAV载体颗粒”指由至少一种AAV衣壳蛋白(通常为野生型AAV的所有衣壳蛋白)和壳体化多核苷酸rAAV载体构成的病毒颗粒。如果所述颗粒包含异源多核苷酸(即除野生型AAV基因组外的多核苷酸,例如递送至哺乳动物细胞的转基因),通常将其称为“rAAV载体颗粒”或简称为“rAAV载体”。因此,rAAV颗粒的生成必定包括rAAV载体的生成,因为在rAAV颗粒中含有这种载体。
“包装”指导致AAV颗粒装配和壳体化的一系列胞内事件。
AAV“rep”和“cap”基因指编码腺相关病毒的复制和壳体化蛋白的多核苷酸序列。本文将AAVrep和cap称为AAV“包装基因”。
AAV的“辅助病毒”指允许哺乳动物细胞复制并包装AAV(例如野生型AAV)的病毒。在本领域中已知AAV的多种此类辅助病毒,包括腺病毒、疱疹病毒和痘病毒(例如牛痘)。虽然C亚类的5型腺病毒最常用,但是腺病毒涵盖许多不同亚类。已知人、非人类哺乳动物和禽类来源的许多腺病毒并且可从贮藏所例如ATCC获得。疱疹家族的病毒包括(例如)单纯疱疹病毒(HSV)和埃-巴二氏病毒(Epstein-Barrviruses)(EBV)以及巨细胞病毒(CMV)和假狂犬病病毒(PRV);也可从贮藏所例如ATCC获得。
“辅助病毒功能”指辅助病毒基因组中编码的允许AAV复制和包装(连同对本文所述复制和包装的其它要求)的功能。如本文所述,可以多种方式提供“辅助病毒功能”,包括通过提供辅助病毒或为生产细胞提供(例如)编码必需功能的反式多核苷酸序列。例如,将包含编码一种或多种腺病毒蛋白的核苷酸序列的质粒或其它表达载体连同rAAV载体一起转染至生产细胞中。
“传染性”病毒或病毒颗粒是包含适当装配的病毒衣壳并且能够将多核苷酸组分递送至对其而言病毒物种是向性的细胞中的病毒或病毒颗粒。所述术语不一定暗指病毒的任何复制能力。在本公开和本领域的其它地方描述了为传染性病毒颗粒计数的测定法。可用传染性病毒颗粒与总病毒颗粒之比表示病毒感染性。在本领域中已知测定传染性病毒颗粒与总病毒颗粒之比的方法。见,例如,Grainger等(2005)Mol.Ther.11:S337(描述了TCID50传染性滴度测定);和Zolotukhin等(1999)GeneTher.6:973。同样见实施例。
“有复制能力的”病毒(例如有复制能力的AAV)指有传染性,并且也能够在受感染细胞中(即,在辅助病毒或辅助病毒功能的存在下)复制的野生表现型病毒。在AAV的情况下,复制能力通常需要功能AAV包装基因的存在。一般而言,在哺乳动物细胞中(尤其在人类细胞中),由于缺乏一个或多个AAV包装基因,如本文所述的rAAV载体无复制能力。通常,为了将通过AAV包装基因和引入rAAV载体之间重组产生有复制能力的AAV的可能性降到最低,此类rAAV载体缺乏任何AAV包装基因。在许多实施方案中,如本文所述的rAAV载体制剂是含有很少(如果有)有复制能力的AAV(rcAAV,也称为RCA)的rAAV载体制剂(例如,每102个rAAV颗粒小于约1个rcAAV,每104个rAAV颗粒小于约1个rcAAV,每108个rAAV颗粒小于约1个rcAAV,每1012个rAAV颗粒小于约1个rcAAV或没有rcAAV)。
术语“多核苷酸”指任何长度的核苷酸聚合形式,包括脱氧核糖核苷酸或核糖核苷酸或其类似物。多核苷酸可包含经修饰的核苷酸,例如甲基化核苷酸和核苷酸类似物,并且可被非核苷酸组分中断。若存在,可在聚合物装配之前或之后给予对核苷酸结构的修饰。如本文所使用,术语多核苷酸可交替地指双链和单链分子。除非另有说明或要求,本文所述为多核苷酸的本发明任何实施方案涵盖双链形式和已知或预测构成双链形式的两种互补单链形式的每一种。
核酸杂交反应可在不同“严格性”的条件下进行。增强杂交反应的严格性的条件众所周知并且在本领域中公开。见,例如,通过引用并入本文的Sambrook等MolecularCloning,ALaboratoryManual,第2版,ColdSpringHarborLaboratoryPress,ColdSpringHarbor,N.Y.,1989。例如,见Sambrook等的第7.52页。相关条件的实例包括(按增强严格性的顺序):25℃、37℃、50℃和68℃的孵育温度;10×SSC、6×SSC、1×SSC、0.1×SSC的缓冲液浓度(其中1×SSC为0.15MNaCl和15mM柠檬酸盐缓冲液)及其使用其它缓冲***的等效物;0%、25%、50%和75%的甲酰胺浓度;从5min至24h的孵育时间;1个、2个或更多个洗涤步骤;1、2或15min的洗涤孵育时间;和6×SSC、1×SSC、0.1×SSC的洗液,或去离子水。严格杂交条件的实例为在50℃或更高温度和0.1×SSC(15mM氯化钠/1.5mM柠檬酸钠)下杂交。严格杂交条件的另一实例是在42℃下于溶液:50%甲酰胺、1×SSC(150mMNaCl、15mM柠檬酸钠)、50mM磷酸钠(pH7.6)、5×登哈特溶液(Denhardt'ssolution)、10%硫酸葡聚糖和20μg/ml变性、剪切鲑鱼精DNA中过夜孵育,接着在约65℃下于0.1XSSC中洗涤过滤器。再如,严格杂交条件包括:在65℃下于包含6X单一强度柠檬酸盐(SSC)(1XSSC为0.15MNaCl、0.015M柠檬酸钠;pH7.0)、5×登哈特溶液、0.05%焦磷酸钠和100μg/ml鲱鱼精DNA的溶液中预杂交8h至过夜;在65℃下于含有6XSSC、1X登哈特溶液、100μg/ml酵母tRNA和0.05%焦磷酸钠的溶液中杂交18-20h;并且在65℃下于含有0.2XSSC和0.1%SDS(十二烷基硫酸钠)的溶液中洗涤过滤器1h。
严格杂交条件是至少与以上代表性条件一样严格的杂交条件。其它严格杂交条件在本领域中已知并且也可用于鉴定本发明这个特定实施方案的核酸。
“Tm”是按摄氏度计,在实验条件下,由在反向平行方向通过沃森-克里克(Watson-Crick)碱基配对氢键结合的互补链构成的50%多核苷酸双链体解离成单链的温度。可根据标准公式预测Tm,例如:
Tm=81.5+16.6log[X+]+0.41(%G/C)-0.61(%F)-600/L。
其中[X+]是以mol/L计的阳离子浓度(通常为钠离子,Na+);(%G/C)是按双链体中总残基的百分比计,G和C残基的数量;(%F)是溶液中的甲酰胺百分比(wt/vol);并且L是双链体每条链中核苷酸的数量。
多核苷酸或多肽与另一多核苷酸或多肽具有一定百分比的“序列同一性”,意味着,比对时,碱基或氨基酸的百分比在比较两个序列时相同。可以许多不同方式测定序列相似性。为测定序列同一性,可使用所述方法和计算机程序,包括万维网上在ncbi.nlm.nih.gov/BLAST/可用的BLAST比对序列。另一比对算法为FASTA,在来自OxfordMolecularGroup,Inc.的全资子公司Madison,Wisconsin,USA的遗传学计算机组(GCG)包中可用。在Enzymology,第266卷:美国加利福尼亚圣地亚哥HarcourtBrace&Co.的分公司AcademicPress,Inc.,Doolittle编辑,ComputerMethodsforMacromolecularSequenceAnalysis(1996)中的Methods中描述了其它比对技术。特别感兴趣的是在序列中允许有空位的比对程序。Smith-Waterman是在序列比对中允许有空位的一类算法。见Meth.Mol.Biol.70:173-187(1997)。同样,可利用使用Needleman和Wunsch比对方法的GAP程序比对序列。见J.Mol.Biol.48:443-453(1970)。
感兴趣的是使用Smith和Waterman的局部同源性算法(AdvancesinAppliedMathematics2:482-489(1981)测定序列同一性的BestFit程序。空位生成罚分范围通常将从1到5,一般从2到4并且在许多实施方案中将为3。空位拓展罚分范围通常将从约0.01到0.20并且在许多情况下将为0.10。程序具有由输入以待比较的序列确定的缺省参数。优选地,使用由程序确定的缺省参数测定序列同一性。该程序也可从来自Madison,Wisconsin,USA的遗传学计算机组(GCG)包中获得。
另一个感兴趣的程序是FastDB算法。在SequenceComparisonandAnalysis、MacromoleculeSequencingandSynthesis、SelectedMethodsandApplications中的CurrentMethods中,第127-149页,1988,AlanR.Liss,Inc.描述了FastDB。基于下列参数通过FastDB计算序列同一性百分比:
错配罚分:1.00;
空位罚分:1.00;
空位大小罚分:0.33;和
连接罚分:30.0。
“基因”指含有至少一个能够在转录和翻译后编码特定蛋白的开放阅读框的多核苷酸。
“基因产物”是由特定基因表达产生的分子。基因产物包括(例如)多肽、适配体、干扰RNA、mRNA等。
“小干扰”或“短干扰RNA”或siRNA是靶向基因目标(“靶基因”)的核苷酸的RNA双链体。“RNA双链体”指通过RNA分子的两个区域之间互补配对形成的结构。siRNA“靶向”基因,因为siRNA的双链体部分的核苷酸序列与靶基因的核苷酸序列互补。在一些实施方案中,siRNA双链体的长度小于30个核苷酸。在一些实施方案中,双链体的长度可为29、28、27、26、25、24、23、22、21、20、19、18、17、16、15、14、13、12、11或10个核苷酸。在一些实施方案中,双链体的长度为19-25个核苷酸。siRNA的RNA双链体部分可为发夹结构的一部分。除双链体部分外,发夹结构可含有位于形成双链体的两个序列之间的环部分。所述环的长度可改变。在一些实施方案中,所述环的长度为5、6、7、8、9、10、11、12或13个核苷酸。发夹结构也可含有3'或5'突出部分。在一些实施方案中,所述突出的长度为3'或5'突出0、1、2、3、4或5个核苷酸。
“短发夹RNA”或shRNA是可使其表达干扰RNA例如siRNA的多核苷酸构建体。
如应用于多核苷酸,“重组”意指多核苷酸是克隆、限制或连接步骤的不同组合和产生不同于在自然界中发现的多核苷酸的构建体的其它程序的产物。重组病毒是包含重组多核苷酸的病毒颗粒。所述术语分别包括原多核苷酸构建体和原病毒构建体子代的复制品。
“控制元件”或“控制序列”是牵涉于分子相互作用,有助于多核苷酸的功能调控,包括多核苷酸的复制、重复、转录、剪接、翻译或降解的核苷酸序列。调控可影响所述过程的频率、速度或特异性,并且实际上可为增强性或抑制性。本领域已知的控制元件包括(例如)转录调控序列例如启动子和增强子。启动子是在某些条件下能够结合RNA聚合酶并且引发通常位于启动子下游(3'方向)的编码区域转录的DNA区域。
“操作性连接”或“可操作地连接”指遗传元件的并列,其中所述元件呈允许其以预期方式操作的关系。例如,如果启动子帮助引发编码序列的转录,则启动子与编码区操作性连接。只要维持这种功能关系,在启动子和编码区域之间可能有***残基。
“表达载体”是包含编码目标多肽的区域的载体,并且用于在预定靶细胞中实现蛋白质的表达。表达载体还包含与编码区域操作性连接以促进蛋白质在靶标中的表达的控制元件。控制元件和与之可操作地连接以便表达的基因的组合有时称为“表达盒”,许多表达盒在本领域中已知并且可用或可易于由在本领域中可用的组分构建体。
“异源”指源自和与之比较的其余实体基因型不同的实体。例如,通过基因工程技术引入源自不同物种的质粒或载体中的多核苷酸为异源多核苷酸。取自其天然编码序列并且与并非发现与之天然连接的编码序列操作性连接的启动子为异源启动子。因此,例如,包括编码异源基因产物的异源核酸的rAAV是包括在自然存在的野生型AAV中一般不包括的核酸的rAAV,并且编码的异源基因产物是一般并非自然存在的野生型AAV编码的基因产物。
术语“遗传改变”和“遗传修饰”(及其语法变型)在本文中可交换用于指其中除通过有丝***或减数***外,将遗传元件(例如,多核苷酸)引入细胞的过程。所述元件可与细胞异源,或可为细胞中已经存在的元件的另一拷贝或改良形式。例如,可通过本领域已知的任何过程,例如电穿孔、磷酸钙沉淀用重组质粒或其它多核苷酸转染细胞,或使细胞与多核苷酸-脂质体复合物接触,而实现遗传改变。例如,也可通过用DNA或RNA病毒或病毒载体转导或感染实现遗传改变。一般地,将遗传元件引入细胞内的染色体或微型染色体中;但是改变细胞及其子代的表现型和/或基因型的任何改变均包括在该术语中。
如果在细胞体外扩大培养期间,序列可用于执行其功能,则将细胞称为经基因序列“稳定”改变、转导、遗传修饰或转化。一般地,此类细胞经“可遗传性”改变(遗传修饰),因为引入了受改变细胞的子代也可遗传的遗传改变。
术语“多肽”、“肽”和“蛋白质”在本文中可交换用于指任何长度的氨基酸聚合物。所述术语还涵盖已经修饰的氨基酸聚合物;例如,二硫键形成、糖基化、脂化、磷酸化或与标记组分结合。多肽例如抗血管生成多肽、神经保护多肽等,当在将基因产物递送至哺乳动物受试者的上下文中讨论时,及其组合物,指各个完整多肽或其保留了完整蛋白质的所需生物化学功能的任何片段或基因工程衍生物。类似地,提到编码抗血管生成多肽的核酸、编码神经保护多肽的核酸和用于将基因产物递送至哺乳动物受试者的其它此类氨基酸(也可称为待递送至受体细胞的“转基因”),包括编码完整多肽或其具有所需生物化学功能的任何片段或基因工程衍生物的多核苷酸。
“分离的”质粒、核酸、载体、病毒、病毒体、宿主细胞或其它物质指没有在所述物质或类似物质自然存在或最初制备时也可能存在的至少一些其它组分的物质的制剂。因此,例如,可使用纯化计数制备分离物质以使其从源混合物中富集。可绝对地测量富集物,例如每体积溶液的重量,或可相对于源混合物中存在的第二潜在干扰物质测量。逐渐多次分离本公开实施方案越来越多的富集物。在一些实施方案中纯化分离的质粒、核酸、载体、病毒、宿主细胞或其它物质,例如从约80%至约90%纯度,至少约90%纯度,至少约95%纯度,至少约98%纯度,或至少约99%纯度或更高纯度。
如本文所使用,术语“治疗(treatment)”、“治疗(treating)”等指获得所需药理学和/或生理学作用。所述作用在完全或部分预防疾病或其症状方面可为预防性和/或在对疾病和/或可归因于所述疾病的副作用的部分或完全治愈方面可为治疗性。如本文所使用,“治疗”包括对哺乳动物(特别是人类)疾病的任何治疗,并且包括:(a)预防疾病在可能易患病或有患病风险但尚未诊断为患病的受试者中出现;(b)抑制疾病,即阻止其发展;和(c)缓解疾病,即引起疾病消退。
术语“个体”、“宿主”、“受试者”和“患者”在本文中可交换使用,并且指哺乳动物,包括但不限于人和非人灵长类动物,包括猿和人;哺乳类运动动物(例如,马);哺乳类家畜(例如,绵羊、山羊等);哺乳类宠物(狗、猫等);和啮齿动物(例如,小鼠、大鼠等)。
在进一步描述本发明之前,应理解本发明不限于描述的特定实施方案,当然,同样可能变化。还应理解,本文使用的术语仅仅是为了描述特定实施方案,并非旨在限制,因为本发明的范围将仅受所附权利要求的限制。
提供值的范围时,应理解,介于该范围的上下限之间的每个居间数值(至下限单位的十分之一,除非上下文另外明确指出)以及所述范围内的任意其它规定或居间的数值涵盖在本发明内。这些较小范围的上下限可独立地包括在较小范围内,并且也涵盖在本发明内,以规定范围内明确排除的任何限值为限。规定范围包括一个或两个限值时,将那些包括的限值的任一个或两个排除在外的范围也包括在本发明中。
除非另有定义,否则本文使用的所有技术和科学术语具有与本发明所属领域中普通技术人员通常所理解的相同含义。虽然在本发明的实践和教学中也可使用与本文所述类似或等效的任何方法和材料,但现在描述优选方法和材料。本文提及的所有出版物通过引用并入本文以公开和描述连同出版物一起引用的方法和/或材料。
必须指出的是,如本文和所附权利要求中所使用,除非上下文另外明确指出,否则单数形式“一种”、“一个”和“所述”包括多个指示物。因此,例如,提到“一种重组AAV病毒体”包括多个此类病毒体而提到“所述光感受器细胞”包括提到一个或多个光感受器细胞及其本领域技术人员已知的等效物,等等。应进一步指出,权利要求可起草为排除任何任选要素。同样,该声明旨在用作使用诸如“只是”、“仅仅”等专有术语连同对权利要求要素的叙述,或使用“消极”限制的前提基础。
应了解,为清楚起见,在独立实施方案的上下文中描述的本发明的某些特征也可在单个实施方案中组合提供。相反,为简洁起见,在单个实施方案的上下文中描述的本发明的各种特征,也可单独地或以任何适合的子组合提供。本发明明确包括关于本发明的实施方案的所有组合并且在本文中公开,正如单独明确地公开每个和每种组合一样。另外,本发明还明确包括各实施方案及其要素的所有子组合并且在本文中公开,正如在本文单独明确地公开每个和每种此类子组合一样。
本文讨论的出版物只是为了在本申请的提交日期之前公开而提供。不得将本文任何内容解释为承认无权凭借先前发明使本发明先于此出版物。进一步地,提供的出版日期可能与实际出版日期不同,可能需要单独确认。
发明详述
本公开提供了衣壳蛋白改变的腺相关病毒(AAV)病毒体,其中所述AAV病毒体在经玻璃体内注射施用时,与野生型AAV在经玻璃体内注射施用时相比,表现出更强的视网膜细胞感染性。本公开进一步提供了将基因产物递送至个体的视网膜细胞的方法和治疗眼病的方法。
视网膜细胞可为光感受器(例如,杆状细胞、锥状细胞)、视网膜神经节细胞(RGC)、缪勒细胞(Müllercell)(缪勒胶质细胞)、双极细胞、无长突细胞、水平细胞或视网膜色素上皮(RPE)细胞。
变异AAV衣壳多肽
本公开提供了一种变异AAV衣壳蛋白,其中相对于相应亲本AAV衣壳蛋白,所述变异AAV衣壳蛋白在衣壳蛋白GH环或IV环中的***位点中包含约5个氨基酸至约11个氨基酸的***,并且其中与包含所述相应亲本AAV衣壳蛋白的AAV病毒体对视网膜细胞的感染性相比,所述变异衣壳蛋白,当存在于AAV病毒体中时,赋予增强的视网膜细胞感染性。在一些情况下,视网膜细胞为光感受器细胞(例如,杆状细胞、锥状细胞)。在其它情况下,视网膜细胞为RGC。在其它情况下,视网膜细胞为RPE细胞。在其它情况下,视网膜细胞为缪勒细胞。其它视网膜细胞包括无长突细胞、双极细胞和水平细胞。“约5个氨基酸至约11个氨基酸的***”本文也称为“肽***”(例如异源肽***)。“相应亲本AAV衣壳蛋白”指无肽***的相同AAV血清型的AAV衣壳蛋白。
***位点在AAV衣壳蛋白的GH环或IV环中,例如在AAV衣壳蛋白的GH环或IV环的溶剂可及部分。对于AAV衣壳的GH环/IV环,见,例如,vanVliet等(2006)Mol.Ther.14:809;Padron等(2005)J.Virol.79:5047;和Shen等(2007)Mol.Ther.15:1955。例如,如图17A和17B所描绘,***位点可在AAV衣壳蛋白的氨基酸411-650内。例如,如图6所描绘,***位点可在AAV2的氨基酸570-611内,AAV1的氨基酸571-612内,AAV5的氨基酸560-601内,AAV6的氨基酸571-612内,AAV7的氨基酸572-613内,AAV8的氨基酸573-614内,AAV9的氨基酸571-612内,或AAV10的氨基酸573-614内。
在一些情况下,相对于相应亲本AAV衣壳蛋白,在所述衣壳蛋白的GH环或IV环中的***位点***约5个氨基酸至约11个氨基酸。例如,***位点可介于AAV2的氨基酸587和588之间,或另一AAV血清型的衣壳亚基的相应位置之间。应指出的是,***位点587/588基于AAV2衣壳蛋白。可在除AAV2外的AAV血清型(例如,AAV8、AAV9等)中的相应位点***约5个氨基酸至约11个氨基酸。本领域的技术人员将了解,基于对不同AAV血清型的衣壳蛋白的氨基酸序列的比较,其中“与AAV2的氨基酸587-588相对应的”***位点将在任何指定AAV血清型的衣壳蛋白中。图6中示出了不同AAV血清型中与AAV2(见图5)的衣壳蛋白VP1的氨基酸570-611相对应的序列。见,例如,基因库登记号NP_049542的AAV1;基因库登记号AAD13756的AAV5;基因库登记号AAB95459的AAV6;基因库登记号YP_077178的AAV7;基因库登记号YP_077180的AAV8;基因库登记号AAS99264的AAV9和基因库登记号AAT46337的AAV10。
在一些实施方案中,***位点是介于位于任何AAV血清型的VP1的氨基酸570-614之间的两个相邻氨基酸之间的单个***位点,例如,***位点介于位于任何AAV血清型或变异体的VP1的氨基酸570-610、氨基酸580-600、氨基酸570-575、氨基酸575-580、氨基酸580-585、氨基酸585-590、氨基酸590-600或氨基酸600-614中的两个相邻氨基酸之间。例如,***位点可介于氨基酸580和581、氨基酸581和582、氨基酸583和584、氨基酸584和585、氨基酸585和586、氨基酸586和587、氨基酸587和588、氨基酸588和589或氨基酸589和590之间。***位点可介于氨基酸575和576、氨基酸576和577、氨基酸577和578、氨基酸578和579或氨基酸579和580之间。***位点可介于氨基酸590和591、氨基酸591和592、氨基酸592和593、氨基酸593和594、氨基酸594和595、氨基酸595和596、氨基酸596和597、氨基酸597和598、氨基酸598和599或氨基酸599和600之间。
例如,***位点可介于AAV2的氨基酸587和588之间,介于AAV1的氨基酸590和591之间,介于AAV5的氨基酸575和576之间,介于AAV6的氨基酸590和591之间,介于AAV7的氨基酸589和590之间,介于AAV8的氨基酸590和591之间,介于AAV9的氨基酸588和589之间,或介于AAV10的氨基酸588和589之间。
再如,如图17A所示,***位点可介于AAV衣壳蛋白的氨基酸450和460之间。例如,如图17A所示,***位点可在AAV2的氨基酸453、AAV1的氨基酸454、AAV6的氨基酸454、AAV7的氨基酸456、AAV8的氨基酸456、AAV9的氨基酸454或AAV10的氨基酸456处(例如,直接在其N端)。
在一些实施方案中,主题衣壳蛋白包括包含与图18A-C所示的氨基酸有至少约85%、至少约90%、至少约95%、至少约98%、至少约99%或100%氨基酸序列同一性的氨基酸序列的GH环。
***肽
如上所述,长度约5个氨基酸至约11个氨基酸的肽***AAV衣壳的GH环中。***肽的长度为5个氨基酸、6个氨基酸、7个氨基酸、8个氨基酸、9个氨基酸、10个氨基酸或11个氨基酸。
***肽可包含以下提出的任一公式的氨基酸序列。
例如,***肽可为长度为5-11个氨基酸的肽,其中所述***肽是式I的***肽:
Y1Y2X1X2X3X4X5X6X7Y3Y4
其中:
Y1-Y4(若存在)的每一个独立选自Ala、Leu、Gly、Ser和Thr;
X1(若存在)选自Leu、Asn和Lys;
X2选自Gly、Glu、Ala和Asp;
X3选自Glu、Thr、Gly和Pro;
X4选自Thr、Ile、Gln和Lys;
X5选自Thr和Ala;
X6选自Arg、Asn和Thr;
X7(若存在)选自Pro和Asn。
再如,***肽可为长度为5-11个氨基酸的肽,其中所述***肽是式IIa的***肽:
Y1Y2X1X2X3X4X5X6X7Y3Y4
其中:
Y1-Y4(若存在)的每一个独立选自Ala、Leu、Gly、Ser和Thr;
X1-X4的每一个为任何氨基酸;
X5为Thr;
X6为Arg;并且
X7为Pro。
再如,***肽可为长度为5-11个氨基酸的肽,其中所述***肽是式IIb的***肽:
Y1Y2X1X2X3X4X5X6X7Y3Y4
其中:
Y1-Y4(若存在)的每一个独立选自Ala、Leu、Gly、Ser和Thr;
X1(若存在)选自Leu和Asn;
X2(若存在)选自Gly和Glu;
X3选自Glu和Thr;
X4选自Thr和Ile;
X5为Thr;
X6为Arg;并且
X7为Pro。
再如,***肽可为长度为5-11个氨基酸的肽,其中所述***肽是式III的***肽:
Y1Y2X1X2X3X4X5X6X7Y3Y4
其中:
Y1-Y4(若存在)的每一个独立选自Ala、Leu、Gly、Ser和Thr;
X1(若存在)为Lys;
X2选自Ala和Asp;
X3选自Gly和Pro;
X4选自Gln和Lys;
X5选自Thr和Ala;
X6选自Asn和Thr;
X7(若存在)为Asn。
再如,***肽可为长度为5-11个氨基酸的肽,其中所述***肽是式IV的***肽:
Y1Y2X1X2X3X4X5X6X7Y3Y4
其中:
Y1-Y4(若存在)的每一个独立选自Ala、Leu、Gly、Ser和Thr;
X1(若存在)为带正电的氨基酸或不带电的氨基酸;或选自Leu、Asn、Arg、Ala、Ser和Lys;
X2为带负电的氨基酸或不带电的氨基酸;或选自Gly、Glu、Ala、Val、Thr和Asp;
X3为带负电的氨基酸或不带电的氨基酸;或选自Glu、Thr、Gly、Asp或Pro;
X4选自Thr、Ile、Gly、Lys、Asp和Gln;
X5为极性氨基酸、醇(具有游离羟基的氨基酸)或疏水氨基酸;或选自Thr、Ser、Val和Ala;
X6为带正电的氨基酸或不带电的氨基酸;或选自Arg、Val、Lys、Pro、Thr和Asn;并且
X7(若存在)为带正电的氨基酸或不带电的氨基酸;或选自Pro、Gly、Phe、Asn和Arg。
作为非限制性实例,***肽可包含选自LGETTRP(SEQIDNO:13)、NETITRP(SEQIDNO:14)、KAGQANN(SEQIDNO:15)、KDPKTTN(SEQIDNO:16)、KDTDTTR(SEQIDNO:57)、RAGGSVG(SEQIDNO:58)、AVDTTKF(SEQIDNO:59)和STGKVPN(SEQIDNO:60)的氨基酸序列。
在一些情况下,***肽在LGETTRP(SEQIDNO:13)、NETITRP(SEQIDNO:14)、KAGQANN(SEQIDNO:15)、KDPKTTN(SEQIDNO:16)、KDTDTTR(SEQIDNO:57)、RAGGSVG(SEQIDNO:58)、AVDTTKF(SEQIDNO:59)和STGKVPN(SEQIDNO:60)的任一个的氨基末端和/或羧基末端具有1-4个间隔氨基酸(Y1-Y4)。适合的间隔氨基酸包括但不限于亮氨酸、丙氨酸、甘氨酸和丝氨酸。
例如,在一些情况下,***肽具有下列氨基酸序列之一:LALGETTRPA(SEQIDNO:45)、LANETITRPA(SEQIDNO:46)、LAKAGQANNA(SEQIDNO:47)、LAKDPKTTNA(SEQIDNO:48)、LAKDTDTTRA(SEQIDNO:61)、LARAGGSVGA(SEQIDNO:62)、LAAVDTTKFA(SEQIDNO:63)和LASTGKVPNA(SEQIDNO:64)。再如,在一些情况下,***肽具有下列氨基酸序列之一:AALGETTRPA(SEQIDNO:49)、AANETITRPA(SEQIDNO:50)、AAKAGQANNA(SEQIDNO:51)和AAKDPKTTNA(SEQIDNO:52)。又如,在一些情况下,***肽具有下列氨基酸序列之一:GLGETTRPA(SEQIDNO:53)、GNETITRPA(SEQIDNO:54)、GKAGQANNA(SEQIDNO:55)和GKDPKTTNA(SEQIDNO:56)。再如,在一些情况下,***肽包含在C端一侧为AA而在N端一侧为A的KDTDTTR(SEQIDNO:57)、RAGGSVG(SEQIDNO:58)、AVDTTKF(SEQIDNO:59)和STGKVPN(SEQIDNO:60)的其中一个;或包含在C端一侧为G而在N端一侧为A的KDTDTTR(SEQIDNO:57)、RAGGSVG(SEQIDNO:58)、AVDTTKF(SEQIDNO:59)和STGKVPN(SEQIDNO:60)的其中一个。
在一些实施方案中,除相对于相应亲本AAV衣壳蛋白,GH环或IV环中约5个氨基酸至约11个氨基酸的***外,主题变异AAV衣壳不包括任何其它氨基酸取代、***或缺失。在其它实施方案中,与亲本AAV衣壳蛋白相比,除相对于相应亲本AAV衣壳蛋白,GH环或IV环中约5个氨基酸至约11个氨基酸的***外,主题变异AAV衣壳包括1个至约25个氨基酸***、缺失或取代。例如,在一些实施方案中,与亲本AAV衣壳蛋白相比,除相对于相应亲本AAV衣壳蛋白,GH环或IV环中约5个氨基酸至约11个氨基酸的***外,主题变异AAV衣壳包括1个至约5个、约5个至约10个,约10个至约15个,约15个至约20个或约20个至约25个氨基酸***、缺失或取代。
在一些实施方案中,主题变异衣壳多肽不包括以下1、2、3或4个氨基酸取代:Y273F、Y444F、Y500F和Y730F。
在一些实施方案中,除如上所述的***肽外,主题变异衣壳多肽包含以下1、2、3或4个氨基酸取代:Y273F、Y444F、Y500F和Y730F。
在一些实施方案中,变异AAV衣壳多肽为嵌合衣壳,例如,衣壳包含第一AAV血清型的AAV衣壳的一部分和第二血清型的AAV衣壳的一部分;并且相对于相应亲本AAV衣壳蛋白,在GH环或IV环中包含约5个氨基酸至约11个氨基酸的***。
在一些实施方案中,主题变异衣壳蛋白包含与图5中提供的氨基酸序列有至少约85%、至少约90%、至少约95%、至少约98%或至少约99%氨基酸序列同一性的氨基酸序列;和相对于相应亲本AAV衣壳蛋白,在GH环或IV环中约5个氨基酸至约11个氨基酸的***。
在一些实施方案中,主题变异衣壳蛋白经分离,例如纯化。在一些情况下,在同样提供的AAV载体中包括主题变异衣壳蛋白。如以下所详细描述,在重组AAV病毒体中可包括主题变异衣壳蛋白。
重组AAV病毒体
本公开提供了一种重组腺相关病毒(rAAV)病毒体,包含:a)变异AAV衣壳蛋白,其中相对于相应亲本AAV衣壳蛋白,所述变异AAV衣壳蛋白在衣壳蛋白GH环或IV环中的***位点中包含约5个氨基酸至约11个氨基酸的***,并且其中与包含所述相应亲本AAV衣壳蛋白的AAV病毒体对视网膜细胞的感染性相比,所述变异衣壳蛋白赋予增强的视网膜细胞感染性;和b)包含编码基因产物的核苷酸序列的异源核酸。在一些情况下,视网膜细胞为光感受器细胞(例如,杆状和/或锥状细胞)。在其它情况下,视网膜细胞为RGC细胞。在其它情况下,视网膜细胞为RPE细胞。在其它情况下,视网膜细胞为缪勒细胞。在其它情况下,视网膜细胞可包括无长突细胞、双极细胞和水平细胞。“约5个氨基酸至约11个氨基酸的***”本文也称为“肽***”(例如,异源肽***)。“相应亲本AAV衣壳蛋白”指无肽***的相同AAV血清型的AAV衣壳蛋白。
***位点在AAV衣壳蛋白的GH环或IV环中,例如在AAV衣壳蛋白的GH环或IV环的溶剂可及部分。对于GH环,见,例如,vanVliet等(2006)Mol.Ther.14:809;Padron等(2005)J.Virol.79:5047;和Shen等(2007)Mol.Ther.15:1955。例如,***位点在AAV2的氨基酸570-611内,AAV1的氨基酸571-612内,AAV5氨基酸的560-601内,AAV6的氨基酸571-612内,AAV7的氨基酸572-613内,AAV8的氨基酸573-614内,AAV9的氨基酸571-612内,或AAV10的氨基酸573-614内。
相对于相应亲本AAV衣壳蛋白,在所述衣壳蛋白的GH环或IV环中的***位点***约5个氨基酸至约11个氨基酸。例如,***位点可介于AAV2的氨基酸587和588之间,或另一AAV血清型的衣壳亚基的相应位置之间。应指出的是,***位点587/588基于AAV2衣壳蛋白。可在除AAV2外的AAV血清型(例如,AAV8、AAV9等)中的相应位点***约5个氨基酸至约11个氨基酸。本领域的技术人员将了解,基于对不同AAV血清型的衣壳蛋白的氨基酸序列的比较,其中“与AAV2的氨基酸587-588相对应的”***位点将在任何指定AAV血清型的衣壳蛋白中。图6中示出了不同AAV血清型中与AAV2(见图5)的衣壳蛋白VP1的氨基酸570-611相对应的序列。
在一些实施方案中,***位点是介于位于任何AAV血清型的VP1的氨基酸570-614之间的两个相邻氨基酸之间的单个***位点,例如,***位点介于位于任何AAV血清型或变异体的VP1的氨基酸570-614、氨基酸580-600、氨基酸570-575、氨基酸575-580、氨基酸580-585、氨基酸585-590、氨基酸590-600或氨基酸600-610中的两个相邻氨基酸之间。例如,***位点可介于氨基酸580和581、氨基酸581和582、氨基酸583和584、氨基酸584和585、氨基酸585和586、氨基酸586和587、氨基酸587和588、氨基酸588和589或氨基酸589和590之间。***位点可介于氨基酸575和576、氨基酸576和577、氨基酸577和578、氨基酸578和579或氨基酸579和580之间。***位点可介于氨基酸590和591、氨基酸591和592、氨基酸592和593、氨基酸593和594、氨基酸594和595、氨基酸595和596、氨基酸596和597、氨基酸597和598、氨基酸598和599或氨基酸599和600之间。
例如,***位点可介于AAV2的氨基酸587和588之间,介于AAV1的氨基酸590和591之间,介于AAV5的氨基酸575和576之间,介于AAV6的氨基酸590和591之间,介于AAV7的氨基酸589和590之间,介于AAV8的氨基酸590和591之间,介于AAV9的氨基酸588和589之间,或介于AAV10的氨基酸589和590之间。
***肽
如上所述,主题rAAV病毒体包含***AAV衣壳的GH环中的长度约5个氨基酸至约11个氨基酸的肽。***肽的长度为5个氨基酸、6个氨基酸、7个氨基酸、8个氨基酸、9个氨基酸、10个氨基酸或11个氨基酸。
***肽可包含以下提出的任一公式的氨基酸序列。
例如,***肽可为长度为5-11个氨基酸的肽,其中所述***肽是式I的***肽:
Y1Y2X1X2X3X4X5X6X7Y3Y4
其中:
Y1-Y4(若存在)的每一个独立选自Ala、Leu、Gly、Ser和Thr;
X1(若存在)选自Leu、Asn和Lys;
X2选自Gly、Glu、Ala和Asp;
X3选自Glu、Thr、Gly和Pro;
X4选自Thr、Ile、Gln和Lys;
X5选自Thr和Ala;
X6选自Arg、Asn和Thr;
X7(若存在)选自Pro和Asn。
再如,***肽可为长度为5-11个氨基酸的肽,其中所述***肽是式IIa的***肽:
Y1Y2X1X2X3X4X5X6X7Y3Y4
其中:
Y1-Y4(若存在)的每一个独立选自Ala、Leu、Gly、Ser和Thr;
X1-X4的每一个为任何氨基酸;
X5为Thr;
X6为Arg;并且
X7为Pro。
再如,***肽可为长度为5-11个氨基酸的肽,其中所述***肽是式IIb的***肽:
Y1Y2X1X2X3X4X5X6X7Y3Y4
其中:
Y1-Y4(若存在)的每一个独立选自Ala、Leu、Gly、Ser和Thr;
X1(若存在)选自Leu和Asn;
X2(若存在)选自Gly和Glu;
X3选自Glu和Thr;
X4选自Thr和Ile;
X5为Thr;
X6为Arg;并且
X7为Pro。
再如,***肽可为长度为5-11个氨基酸的肽,其中所述***肽是式III的***肽:
Y1Y2X1X2X3X4X5X6X7Y3Y4
其中:
Y1-Y4(若存在)的每一个独立选自Ala、Leu、Gly、Ser和Thr;
X1(若存在)为Lys;
X2选自Ala和Asp;
X3选自Gly和Pro;
X4选自Gln和Lys;
X5选自Thr和Ala;
X6选自Asn和Thr;
若存在,X7为Asn。
再如,***肽可为长度为5-11个氨基酸的肽,其中所述***肽是式IV的***肽:
Y1Y2X1X2X3X4X5X6X7Y3Y4
其中:
Y1-Y4(若存在)的每一个独立选自Ala、Leu、Gly、Ser和Thr;
X1(若存在)为带正电的氨基酸或不带电的氨基酸;或选自Leu、Asn、Arg、Ala、Ser和Lys;
X2为带负电的氨基酸或不带电的氨基酸;或选自Gly、Glu、Ala、Val、Thr和Asp;
X3为带负电的氨基酸或不带电的氨基酸;或选自Glu、Thr、Gly、Asp或Pro;
X4选自Thr、Ile、Gly、Lys、Asp和Gln;
X5为极性氨基酸、醇(具有游离羟基的氨基酸)或疏水氨基酸;或选自Thr、Ser、Val和Ala;
X6为带正电的氨基酸或不带电的氨基酸;或选自Arg、Val、Lys、Pro、Thr和Asn;并且
X7(若存在)为带正电的氨基酸或不带电的氨基酸;或选自Pro、Gly、Phe、Asn和Arg。
作为非限制性实例,***肽可包含选自LGETTRP(SEQIDNO:13)、NETITRP(SEQIDNO:14)、KAGQANN(SEQIDNO:15)、KDPKTTN(SEQIDNO:16)、KDTDTTR(SEQIDNO:57)、RAGGSVG(SEQIDNO:58)、AVDTTKF(SEQIDNO:59)和STGKVPN(SEQIDNO:60)的氨基酸序列。
在一些情况下,***肽在LGETTRP(SEQIDNO:13)、NETITRP(SEQIDNO:14)、KAGQANN(SEQIDNO:15)、KDPKTTN(SEQIDNO:16)、KDTDTTR(SEQIDNO:57)、RAGGSVG(SEQIDNO:58)、AVDTTKF(SEQIDNO:59)和STGKVPN(SEQIDNO:60)的任一个的氨基末端和/或羧基末端具有1-4个间隔氨基酸(Y1-Y4)。适合的间隔氨基酸包括但不限于亮氨酸、丙氨酸、甘氨酸和丝氨酸。
例如,在一些情况下,***肽具有下列氨基酸序列之一:LALGETTRPA(SEQIDNO:45)、LANETITRPA(SEQIDNO:46)、LAKAGQANNA(SEQIDNO:47)、LAKDPKTTNA(SEQIDNO:48)、LAKDTDTTRA(SEQIDNO:61)、LARAGGSVGA(SEQIDNO:62)、LAAVDTTKFA(SEQIDNO:63)和LASTGKVPNA(SEQIDNO:64)。再如,在一些情况下,***肽具有下列氨基酸序列之一:AALGETTRPA(SEQIDNO:49)、AANETITRPA(SEQIDNO:50)、AAKAGQANNA(SEQIDNO:51)和AAKDPKTTNA(SEQIDNO:52)。又如,在一些情况下,***肽具有下列氨基酸序列之一:GLGETTRPA(SEQIDNO:53)、GNETITRPA(SEQIDNO:54)、GKAGQANNA(SEQIDNO:55)和GKDPKTTNA(SEQIDNO:56)。再如,在一些情况下,***肽包含在C端一侧为AA而在N端一侧为A的KDTDTTR(SEQIDNO:57)、RAGGSVG(SEQIDNO:58)、AVDTTKF(SEQIDNO:59)和STGKVPN(SEQIDNO:60)的其中一个;或包含在C端一侧为G而在N端一侧为A的KDTDTTR(SEQIDNO:57)、RAGGSVG(SEQIDNO:58)、AVDTTKF(SEQIDNO:59)和STGKVPN(SEQIDNO:60)的其中一个。
在一些实施方案中,除相对于相应亲本AAV衣壳蛋白,GH环或IV环中约7个氨基酸至约10个氨基酸的***外,主题rAAV病毒体衣壳不包括任何其它氨基酸取代、***或缺失。在其它实施方案中,与亲本AAV衣壳蛋白相比,除相对于相应亲本AAV衣壳蛋白,GH环或IV环中约7个氨基酸至约10个氨基酸的***外,主题rAAV病毒体衣壳包括1个至约25个氨基酸***、缺失或取代。例如,在一些实施方案中,与亲本AAV衣壳蛋白相比,除相对于相应亲本AAV衣壳蛋白,GH环或IV环中约7个氨基酸至约10个氨基酸的***外,主题rAAV病毒体衣壳包括1个至约5个、约5个至约10个,约10个至约15个,约15个至约20个或约20个至约25个氨基酸***、缺失或取代。
在一些实施方案中,主题rAAV病毒体衣壳不包括以下1、2、3或4个氨基酸取代:Y273F、Y444F、Y500F和Y730F。
在一些实施方案中,除如上所述的***肽外,主题变异衣壳多肽包含以下1、2、3或4个氨基酸取代:Y273F、Y444F、Y500F和Y730F。
在一些实施方案中,主题rAAV病毒体衣壳为嵌合衣壳,例如,衣壳包含第一AAV血清型的AAV衣壳的一部分和第二血清型的AAV衣壳的一部分;并且相对于相应亲本AAV衣壳蛋白,在GH环或IV环中包含约5个氨基酸至约11个氨基酸的***。
在一些实施方案中,主题rAAV病毒体包含含与图5中提供的氨基酸序列有至少约85%、至少约90%、至少约95%、至少约98%或至少约99%氨基酸序列同一性的氨基酸序列的衣壳蛋白;和相对于相应亲本AAV衣壳蛋白,在GH环或IV环中约5个氨基酸至约11个氨基酸的***。
在一些实施方案中,主题rAAV病毒体包含衣壳蛋白,所述衣壳蛋白包括包含与图18A-C中提出的氨基酸序列有至少约85%、至少约90%、至少约95%、至少约98%、至少约99%或100%氨基酸序列同一性的氨基酸序列的GH环。
与包含相应亲本AAV衣壳蛋白的AAV病毒体对视网膜细胞的感染性相比,主题rAAV病毒体表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的视网膜细胞感染性。
在一些情况下,与包含相应亲本AAV衣壳蛋白的AAV病毒体在经玻璃体内注射施用时对视网膜细胞的感染性相比,主题rAAV病毒体在经玻璃体内注射施用时表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的视网膜细胞感染性。
在一些实施方案中,与包含相应亲本AAV衣壳蛋白的AAV病毒体对光感受器细胞的感染性相比,主题rAAV病毒体表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的光感受器(杆状或锥状)细胞感染性。
在一些实施方案中,与包含相应亲本AAV衣壳蛋白的AAV病毒体在经玻璃体内注射施用时对光感受器细胞的感染性相比,主题rAAV病毒体在经玻璃体内注射施用时表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的光感受器(杆状或锥状)细胞感染性。
在一些实施方案中,与包含相应亲本AAV衣壳蛋白的AAV病毒体对RGC的感染性相比,主题rAAV病毒体表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的RGC感染性。
在一些实施方案中,与包含相应亲本AAV衣壳蛋白的AAV病毒体在经玻璃体内注射施用时对RGC的感染性相比,主题rAAV病毒体在经玻璃体内注射施用时表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的RGC感染性。
在一些实施方案中,与包含相应亲本AAV衣壳蛋白的AAV病毒体对RPE细胞的感染性相比,主题rAAV病毒体表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的RPE细胞感染性。
在一些实施方案中,与包含相应亲本AAV衣壳蛋白的AAV病毒体在经玻璃体内注射施用时对RPE细胞的感染性相比,主题rAAV病毒体在经玻璃体内注射施用时表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的RPE细胞感染性。
在一些实施方案中,与包含相应亲本AAV衣壳蛋白的AAV病毒体对缪勒细胞的感染性相比,主题rAAV病毒体表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的缪勒细胞感染性。
在一些实施方案中,与包含相应亲本AAV衣壳蛋白的AAV病毒体在经玻璃体内注射施用时对缪勒细胞的感染性相比,主题rAAV病毒体在经玻璃体内注射施用时表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的缪勒细胞感染性。
在一些实施方案中,与包含相应亲本AAV衣壳蛋白的AAV病毒体对双极细胞的感染性相比,主题rAAV病毒体表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的双极细胞感染性。
在一些实施方案中,与包含相应亲本AAV衣壳蛋白的AAV病毒体在经玻璃体内注射施用时对双极细胞的感染性相比,主题rAAV病毒体在经玻璃体内注射施用时表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的双极细胞感染性。
在一些实施方案中,与包含相应亲本AAV衣壳蛋白的AAV病毒体对无长突细胞的感染性相比,主题rAAV病毒体表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的无长突细胞感染性。
在一些实施方案中,与包含相应亲本AAV衣壳蛋白的AAV病毒体在经玻璃体内注射施用时对无长突细胞的感染性相比,主题rAAV病毒体在经玻璃体内注射施用时表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的无长突细胞感染性。
在一些实施方案中,与包含相应亲本AAV衣壳蛋白的AAV病毒体对水平细胞的感染性相比,主题rAAV病毒体表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的水平细胞感染性。
在一些实施方案中,与包含相应亲本AAV衣壳蛋白的AAV病毒体在经玻璃体内注射施用时对水平细胞的感染性相比,主题rAAV病毒体在经玻璃体内注射施用时表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的水平细胞感染性。
在一些情况下,与包含相应亲本AAV衣壳蛋白的AAV病毒体穿过ILM的能力相比,主题rAAV病毒体表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的穿过内界膜(ILM)的能力。
在一些情况下,与包含相应亲本AAV衣壳蛋白的AAV病毒体在经玻璃体内注射施用时穿过ILM的能力相比,主题rAAV病毒体在经玻璃体内注射施用时表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的穿过内界膜(ILM)的能力。
主题rAAV病毒体可穿过ILM,并且也可经过细胞层,包括缪勒细胞、无长突细胞等,以达到光感受器细胞或RPE细胞。例如,主题rAAV病毒体在经玻璃体内注射施用时可穿过ILM,并且也可经过细胞层,包括缪勒细胞、无长突细胞等,以达到光感受器细胞或RPE细胞。
在一些实施方案中,主题rAAV病毒体选择性感染视网膜细胞,例如,主题rAAV病毒体以比非视网膜细胞,例如眼睛外部的细胞强10倍、15倍、20倍、25倍、50倍或50倍以上的特异性感染视网膜细胞。例如,在一些实施方案中,主题rAAV病毒体选择性感染视网膜细胞,例如,主题rAAV病毒体以比非视网膜细胞,例如眼睛外部的细胞强10倍、15倍、20倍、25倍、50倍或50倍以上的特异性感染光感受器细胞。
在一些实施方案中,主题rAAV病毒体选择性感染光感受器细胞,例如,主题rAAV病毒体以比眼部存在的非光感受器细胞,例如视网膜神经节细胞、缪勒细胞等强10倍、15倍、20倍、25倍、50倍或50倍以上的特异性感染光感受器细胞。
在一些实施方案中,与包含相应亲本AAV衣壳蛋白的AAV病毒体在经玻璃体内注射施用时对光感受器细胞的感染性相比,主题rAAV病毒体在经玻璃体内注射施用时表现出增强至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的光感受器细胞感染性。
基因产物
主题rAAV病毒体包含含编码基因产物的核苷酸序列的异源核酸。在一些实施方案中,基因产物为干扰RNA。在一些实施方案中,基因产物为适配体。在一些实施方案中,基因产物为多肽。在一些实施方案中,基因产物为提供对基因功能的位点特异性敲除的位点特异性核酸酶。
干扰RNA
基因产物为干扰RNA(RNAi)时,适合的RNAi包括降低细胞内凋亡或血管生成因子的水平的RNAi。例如,RNAi可为降低细胞内诱导或促进凋亡的基因产物水平的shRNA或siRNA。本文将基因产物诱导或促进凋亡的基因称为“促凋亡基因”而将那些基因的产物(mRNA;蛋白质)称为“促凋亡基因产物”。促凋亡基因产物包括(例如)Bax、Bid、Bak和Bad基因产物。见,例如,美国专利No.7,846,730。
干扰RNA也可抗血管生成产物,例如VEGF(例如,Cand5;见,例如,美国专利公布No.2011/0143400;美国专利公布No.2008/0188437;和Reich等(2003)Mol.Vis.9:210)、VEGFR1(例如,Sirna-027;见,例如,Kaiser等(2010)Am.J.Ophthalmol.150:33;和Shen等(2006)GenrTher.13:225)或VEGFR2(Kou等(2005)Biochem.44:15064)。同样见,美国专利No.6,649,596、6,399,586、5,661,135、5,639,872和5,639,736;和美国专利No.7,947,659和7,919,473。
适配体
基因产物为适配体时,示例性目标适配体包括对血管内皮生长因子(VEGF)的适配体。见,例如,Ng等(2006)Nat.Rev.DrugDiscovery5:123;和Lee等(2005)Proc.Natl.Acad.Sci.USA102:18902。例如,VEGF适配体可包含核苷酸序列5'-cgcaaucagugaaugcuuauacauccg-3'(SEQIDNO:17)。PDGF特异性适配体也适合使用,例如E10030;见,例如,Ni和Hui(2009)Ophthalmologica223:401;和Akiyama等(2006)J.CellPhysiol.207:407)。
多肽
基因产物为多肽时,多肽通常为增强视网膜细胞功能,例如杆状或锥状光感受器细胞、视网膜神经节细胞、缪勒细胞、双极细胞、无长突细胞、水平细胞或视网膜色素上皮细胞的功能的多肽。示例性多肽包括神经保护多肽(例如,GDNF、CNTF、NT4、NGF和NTN);抗血管生成多肽(例如,可溶性血管内皮生长因子(VEGF)受体;VEGF结合抗体;VEGF结合抗体片段(例如,单链抗VEGF抗体);内皮抑素(endostatin);肿瘤抑素(tumstatin);血管抑素(angiostatin);可溶性Flt多肽(Lai等(2005)Mol.Ther.12:659);包含可溶性Flt多肽的Fc融合蛋白(见,例如,Pechan等(2009)GeneTher.16:10);色素上皮源性因子(PEDF);可溶性Tie-2受体等);金属蛋白酶-3的组织抑制剂(TIMP-3);光反应性视蛋白(opsin),例如视紫红质;抗凋亡多肽(例如,Bcl-2、Bcl-X1)等。适合多肽包括但不限于胶质源性神经营养因子(GDNF);成纤维细胞生长因子2;神经营养因子(neurturin)(NTN);睫状神经营养因子(CNTF);神经生长因子(NGF);神经营养蛋白-4(NT4);源性神经营养因子(BDNF;例如,包含与图11所示氨基酸序列(SEQIDNO:11)的约200个氨基酸至247个氨基酸的连续段有至少约90%、至少约95%、至少约98%、至少约99%或100%氨基酸序列同一性的氨基酸序列的多肽);表皮生长因子;视紫红质;X连锁凋亡抑制蛋白;和音猬因子(Sonichedgehog)。
适合的光反应性视蛋白包括(例如)如美国专利公布No.2007/0261127(例如,ChR2;Chop2);美国专利公布No.2001/0086421;美国专利公布No.2010/0015095;和Diester等(2011)Nat.Neurosci.14:387中描述的光反应性视蛋白。
适合多肽还包括视网膜劈裂蛋白(例如,包含与图10所示氨基酸序列(SEQIDNO:10)的约200个氨基酸至224个氨基酸的连续段有至少约90%、至少约95%、至少约98%、至少约99%或100%氨基酸序列同一性的氨基酸序列的多肽)。适合多肽包括(例如)色素性视网膜炎GTP酶调节因子(RGPR)-相互作用蛋白-1(见,例如,基因库登记号Q96KN7、Q9EPQ2和Q9GLM3)(例如,包含与图16所示氨基酸序列(SEQIDNO:21)的约1150个氨基酸至约1200个氨基酸或约1200个氨基酸至约1286个氨基酸的连续段有至少约90%、至少约95%、至少约98%、至少约99%或100%氨基酸序列同一性的氨基酸序列的多肽);外周蛋白-2(Prph2)(见,例如,基因库登记号NP_000313(例如,包含与图14所示氨基酸序列(SEQIDNO:19)的约300个氨基酸至346个氨基酸的连续段有至少约90%、至少约95%、至少约98%、至少约99%或100%氨基酸序列同一性的氨基酸序列的多肽);和Travis等(1991)Genomics10:733);外周蛋白(例如,包含与图15所示氨基酸序列(SEQIDNO:20)的约400个氨基酸至470个氨基酸的连续段有至少约90%、至少约95%、至少约98%、至少约99%或100%氨基酸序列同一性的氨基酸序列的多肽);视网膜色素上皮特异性蛋白(RPE65),(例如,包含与图12所示氨基酸序列(SEQIDNO:12)的约200个氨基酸至247个氨基酸的连续段有至少约90%、至少约95%、至少约98%、至少约99%或100%氨基酸序列同一性的氨基酸序列的多肽)(见,例如,基因库AAC39660;和Morimura等(1998)Proc.Natl.Acad.Sci.USA95:3088)等。
适合多肽还包括:CHM(脉络膜缺损(choroidermia)(Rab护送蛋白1)),当缺损或缺失时,引起无脉络膜的一种多肽(见,例如,Donnelly等(1994)Hum.Mol.Genet.3:1017;和vanBokhoven等(1994)Hum.Mol.Genet.3:1041);和碎屑同源物1(CRB1),当缺损或缺失时,引起利伯氏先天性黑内障(Lebercongenitalamaurosis)和色素性视网膜炎的一种多肽(见,例如,denHollander等(1999)Nat.Genet.23:217;和基因库登记号CAM23328)。
适合多肽还包括当缺损或缺失时,导致全色盲的多肽,其中此类多肽包括(例如)杆状光感受器cGMP-门控通道亚基α(CNGA3)(见,例如,基因库登记号NP_001289;和Booij等(2011)Ophthalmology118:160-167);杆状光感受器cGMP-门控通道β亚基(CNGB3)(见,例如,Kohl等(2005)EurJHumGenet.13(3):302);鸟嘌呤核苷酸结合蛋白(G蛋白)、α转导活性多肽2(GNAT2)(ACHM4);和ACHM5;和缺损或缺乏时,导致各种形式的色盲的多肽(例如,L-视蛋白、M-视蛋白和S-视蛋白)。见Mancuso等(2009)Nature461(7265):784-787。
位点特异性核酸内切酶
在一些情况下,目标基因产物为提供对基因功能的位点特异性敲除的位点特异性核酸内切酶,例如,其中核酸内切酶敲除与视网膜疾病相关的等位基因。例如,若显性等位基因编码,野生型时,为视网膜结构蛋白和/或提供正常的视网膜功能的缺陷基因拷贝,则位点特异性核酸内切酶可靶向缺陷等位基因并敲除缺陷等位基因。
除敲除缺陷等位基因外,位点特异性核酸酶也可用于刺激与编码缺陷等位基因编码的蛋白质的功能拷贝的供体DNA的同源重组。因此,例如,主题rAAV病毒体可用于递送敲除缺陷等位基因的位点特异性核酸内切酶,并且可用于递送缺陷等位基因的功能拷贝,引起功能拷贝修复,从而提供功能性视网膜蛋白(例如,功能性视网膜劈裂蛋白、功能性RPE65、功能性外周蛋白等)的生成。见,例如,Li等(2011)Nature475:217。在一些实施方案中,主题rAAV病毒体包含编码位点特异性核酸内切酶的异源核苷酸序列;和编码缺陷等位基因的功能拷贝的异源核苷酸序列,其中所述功能拷贝编码功能性视网膜蛋白。功能性视网膜蛋白包括(例如)视网膜劈裂蛋白、RPE65、色素性视网膜炎GTP酶调节因子(RGPR)-相互作用蛋白-1、外周蛋白、外周蛋白-2等。
适合使用的位点特异性核酸内切酶包括(例如)锌指核酸酶(ZFN);和转录激活因子样效应核酸酶(TALEN),其中此类位点特异性核酸内切酶并非自然存在并且经修饰为靶向特定基因。此类位点特异性核酸酶可经工程化以切割基因组内的特定位置,然后非同源末端连接可在***或消去几个核苷酸的同时修复断裂处。然后此类位点特异性核酸内切酶(也称为“INDEL”)将蛋白质抛出框外并有效敲除基因。见,例如,美国专利公布No.2011/0301073。
调控序列
在一些实施方案中,编码目标基因产物的核苷酸序列与组成型启动子可操作地连接。在其它实施方案中,编码目标基因产物的核苷酸序列与诱导型启动子可操作地连接。在一些情况下,编码目标基因产物的核苷酸序列与组织特异性或细胞类型特异性调控元件可操作地连接。例如,在一些情况下,编码目标基因产物的核苷酸序列与光感受器特异性调控元件(例如,光感受器特异性启动子)可操作地连接,例如赋予光感受器细胞内可操作地连接的基因选择性表达的调控元件。适合的光感受器特异性调控元件包括(例如)视紫红质启动子;视紫红质激酶启动子(Young等(2003)Ophthalmol.Vis.Sci.44:4076);β磷酸二酯酶基因启动子(Nicoud等(2007)J.GeneMed.9:1015);色素性视网膜炎基因启动子(Nicoud等(2007)同上);和光感受器间维甲酸结合蛋白(IRBP)基因增强子(Nicoud等(2007)同上);IRBP基因启动子(Yokoyama等(1992)ExpEyeRes.55:225)。
药物组合物
本公开提供了一种药物组合物,包含:a)如上所述的主题rAAV病毒体;和b)药学上可接受的载体、稀释剂、赋形剂或缓冲液。在一些实施方案中,药学上可接受的载体、稀释剂、赋形剂或缓冲液适合用于人类。
此类赋形剂、载体、稀释剂和缓冲液包括可施用而无异常毒性的任何药剂。药学上可接受的赋形剂包括但不限于液体,例如水、盐水、甘油和乙醇。其中可包括药学上可接受的盐,例如矿物酸盐例如盐酸盐、氢溴酸盐、磷酸盐、硫酸盐等;和有机酸的盐例如醋酸盐、丙酸盐、丙二酸盐、苯甲酸盐等。另外,在此类媒介物中可存在辅助物质,例如润湿剂或乳化剂、pH缓冲物质等。在本领域中已知多种多样的药学上可接受的赋形剂而不需要在本文中详细讨论。在多种出版物中已经详尽地描述了药学上可接受的赋形剂,包括(例如)A.Gennaro(2000)"Remington:TheScienceandPracticeofPharmacy,"第20版,Lippincott,Williams,&Wilkins;PharmaceuticalDosageFormsandDrugDeliverySystems(1999)H.C.Ansel等编辑,第7版,Lippincott,Williams,&Wilkins;和HandbookofPharmaceuticalExcipients(2000)A.H.Kibbe等编辑,第3版,Amer.PharmaceuticalAssoc。
将基因产物递送至视网膜细胞的方法和治疗方法
本公开提供了一种将基因产物递送至个体的视网膜细胞的方法,所述方法包括向所述个体施用如上所述的主题rAAV病毒体。基因产物可为如上所述的多肽或干扰RNA(例如,shRNA、siRNA等)、适配体、位点特异性核酸内切酶。将基因产物递送至视网膜细胞可提供对视网膜疾病的治疗。视网膜细胞可为光感受器、视网膜神经节细胞、缪勒细胞、双极细胞、无长突细胞、水平细胞或视网膜色素上皮细胞。在一些情况下,视网膜细胞为光感受器细胞,例如杆状或锥状细胞。
本公开提供了一种治疗视网膜疾病的方法,所述方法包括向有需要的个体施用有效量的如上所述的主题rAAV病毒体。主题rAAV病毒体可经眼内注射、通过玻璃体内注射或通过其它方便的施用模式或途径施用。其它方便的施用模式或途径包括(例如)静脉内、鼻内等。
“治疗有效量”将落在通过实验和/或临床试验确定的相对较宽的范围内。例如,对于体内注射,即直接注射至眼内而言,治疗有效剂量将相当于约106至约1015个rAAV病毒体,例如约108至约1012个rAAV病毒体。对于体外转导而言,待递送至细胞的rAAV病毒体的有效量将相当于约108至约1013个rAAV病毒体。其它有效剂量可易于由本领域的普通技术人员通过确定剂量反应曲线的常规试验确定。
在一些实施方案中,可在不同间隔期内,例如每天、每周、每月、每年采用一次以上施用(例如,2、3、4次或更多次施用),以达到所需水平的基因表达。
可施用主题方法治疗的眼病包括但不限于急性黄斑区神经视网膜病变;***(Behcet'sdisease);脉络膜新生血管;糖尿病性葡萄膜炎;组织胞浆菌病;黄斑变性,例如急性黄斑变性、非渗出性老年性黄斑变性和渗出性老年性黄斑变性;水肿,例如黄斑水肿、黄斑囊样水肿和糖尿病性黄斑水肿;多灶性脉络膜炎;影响后眼部或位置的眼外伤;眼部肿瘤;视网膜病症,例如视网膜中央静脉阻塞、糖尿病性视网膜病(包括增生性糖尿病性视网膜病)、增生性玻璃体视网膜病变(PVR)、视网膜动脉阻塞性疾病、视网膜脱离、色素层炎视网膜疾病;交感性眼炎;VogtKoyanagi-Harada(VKH)综合征;眼色素层弥散;由眼部激光治疗引起或影响的眼后部病状;光动力疗法引起或影响的眼后部病状;光凝固法、辐射性视网膜病变;视网膜前膜病症;视网膜静脉分枝阻塞;前部缺血性视神经病变;非视网膜病变糖尿病性视网膜功能障碍;视网膜劈裂症;色素性视网膜炎;青光眼;尤塞氏综合征(Ushersyndrome);视锥-视杆细胞营养不良;斯特格氏病(Stargardtdisease)(眼底黄色斑点症);遗传性黄斑变性;脉络膜视网膜变性;利伯氏先天性黑内障;先天性静止性夜盲;无脉络膜;巴比二氏综合征(Bardet-Biedlsyndrome);黄斑毛细管扩张;利伯氏遗传性视神经病;早产儿视网膜病;和色觉障碍,包括全色盲、红色盲、绿色盲和蓝色盲。
核酸和宿主细胞
本公开提供了包含编码如上所述的主题变异腺相关病毒(AAV)衣壳蛋白的核苷酸序列的分离核酸,其中相对于相应亲本AAV衣壳蛋白,所述变异AAV衣壳蛋白在GH环或IV环中包含约5个氨基酸至约11个氨基酸的***,并且其中当存在于AAV病毒体中时,与包含相应亲本AAV衣壳蛋白的AAV病毒体对视网膜细胞的感染性相比,所述变异衣壳蛋白提供增强的视网膜细胞感染性。主题分离核酸可为AAV载体,例如重组AAV载体。
***肽
主题核酸编码的变异AAV衣壳蛋白具有***AAV衣壳的GH环中,长度约5个氨基酸至约11个氨基酸的***肽。***肽的长度为5个氨基酸、6个氨基酸、7个氨基酸、8个氨基酸、9个氨基酸、10个氨基酸或11个氨基酸。
***肽可包含以下提出的任一公式的氨基酸序列。
例如,***肽可为长度为5-11个氨基酸的肽,其中所述***肽是式I的***肽:
Y1Y2X1X2X3X4X5X6X7Y3Y4
其中:
Y1-Y4(若存在)的每一个独立选自Ala、Leu、Gly、Ser和Thr;
X1(若存在)选自Leu、Asn和Lys;
X2选自Gly、Glu、Ala和Asp;
X3选自Glu、Thr、Gly和Pro;
X4选自Thr、Ile、Gln和Lys;
X5选自Thr和Ala;
X6选自Arg、Asn和Thr;
X7(若存在)选自Pro和Asn。
再如,***肽可为长度为5-11个氨基酸的肽,其中所述***肽是式IIa的***肽:
Y1Y2X1X2X3X4X5X6X7Y3Y4
其中:
Y1-Y4(若存在)的每一个独立选自Ala、Leu、Gly、Ser和Thr;
X1-X4的每一个为任何氨基酸;
X5为Thr;
X6为Arg;并且
X7为Pro。
再如,***肽可为长度为5-11个氨基酸的肽,其中所述***肽是式IIb的***肽:
Y1Y2X1X2X3X4X5X6X7Y3Y4
其中:
Y1-Y4(若存在)的每一个独立选自Ala、Leu、Gly、Ser和Thr;
X1(若存在)选自Leu和Asn;
X2(若存在)选自Gly和Glu;
X3选自Glu和Thr;
X4选自Thr和Ile;
X5为Thr;
X6为Arg;并且
X7为Pro。
再如,***肽可为长度为5-11个氨基酸的肽,其中所述***肽是式III的***肽:
Y1Y2X1X2X3X4X5X6X7Y3Y4
其中:
Y1-Y4(若存在)的每一个独立选自Ala、Leu、Gly、Ser和Thr;
X1(若存在)为Lys;
X2选自Ala和Asp;
X3选自Gly和Pro;
X4选自Gln和Lys;
X5选自Thr和Ala;
X6选自Asn和Thr;
X7(若存在)为Asn。
再如,***肽可为长度为5-11个氨基酸的肽,其中所述***肽是式IV的***肽:
Y1Y2X1X2X3X4X5X6X7Y3Y4
其中:
Y1-Y4(若存在)的每一个独立选自Ala、Leu、Gly、Ser和Thr;
X1(若存在)为带正电的氨基酸或不带电的氨基酸;或选自Leu、Asn、Arg、Ala、Ser和Lys;
X2为带负电的氨基酸或不带电的氨基酸;或选自Gly、Glu、Ala、Val、Thr和Asp;
X3为带负电的氨基酸或不带电的氨基酸;或选自Glu、Thr、Gly、Asp或Pro;
X4选自Thr、Ile、Gly、Lys、Asp和Gln;
X5为极性氨基酸、醇(具有游离羟基的氨基酸)或疏水氨基酸;或选自Thr、Ser、Val和Ala;
X6为带正电的氨基酸或不带电的氨基酸;或选自Arg、Val、Lys、Pro、Thr和Asn;并且
X7(若存在)为带正电的氨基酸或不带电的氨基酸;或选自Pro、Gly、Phe、Asn和Arg。
作为非限制性实例,***肽可包含选自LGETTRP(SEQDNO:13)、NETITRP(SEQIDNO:14)、KAGQANN(SEQIDNO:15)、KDPKTTN(SEQIDNO:16)、KDTDTTR(SEQIDNO:57)、RAGGSVG(SEQIDNO:58)、AVDTTKF(SEQIDNO:59)和STGKVPN(SEQIDNO:60)的氨基酸序列。
在一些情况下,***肽在LGETTRP(SEQIDNO:13)、NETITRP(SEQIDNO:14)、KAGQANN(SEQIDNO:15)、KDPKTTN(SEQIDNO:16)、KDTDTTR(SEQIDNO:57)、RAGGSVG(SEQIDNO:58)、AVDTTKF(SEQIDNO:59)和STGKVPN(SEQIDNO:60)的任一个的氨基末端和/或羧基末端具有1-4个间隔氨基酸(Y1-Y4)。适合的间隔氨基酸包括但不限于亮氨酸、丙氨酸、甘氨酸和丝氨酸。
例如,在一些情况下,***肽具有下列氨基酸序列之一:LALGETTRPA(SEQIDNO:45)、LANETITRPA(SEQIDNO:46)、LAKAGQANNA(SEQIDNO:47)、LAKDPKTTNA(SEQIDNO:48)、LAKDTDTTRA(SEQIDNO:61)、LARAGGSVGA(SEQIDNO:62)、LAAVDTTKFA(SEQIDNO:63)和LASTGKVPNA(SEQIDNO:64)。再如,在一些情况下,***肽具有下列氨基酸序列之一:AALGETTRPA(SEQIDNO:49)、AANETITRPA(SEQIDNO:50)、AAKAGQANNA(SEQIDNO:51)和AAKDPKTTNA(SEQIDNO:52)。又如,在一些情况下,***肽具有下列氨基酸序列之一:GLGETTRPA(SEQIDNO:53)、GNETITRPA(SEQIDNO:54)、GKAGQANNA(SEQIDNO:55)和GKDPKTTNA(SEQIDNO:56)。再如,在一些情况下,***肽包含在C端一侧为AA而在N端一侧为A的KDTDTTR(SEQIDNO:57)、RAGGSVG(SEQIDNO:58)、AVDTTKF(SEQIDNO:59)和STGKVPN(SEQIDNO:60)的其中一个;或包含在C端一侧为G而在N端一侧为A的KDTDTTR(SEQIDNO:57)、RAGGSVG(SEQIDNO:58)、AVDTTKF(SEQIDNO:59)和STGKVPN(SEQIDNO:60)的其中一个。
主题重组AAV载体可用于生成如上所述的主题重组AAV病毒体。因此,本公开提供了当引入适合细胞内,可提供主题重组AAV病毒体的生成的重组AAV载体。
本公开进一步提供了包含主题核酸的宿主细胞,例如经分离(经遗传修饰的)宿主细胞。主题宿主细胞可为分离细胞,例如体外培养的细胞。如下所述,主题宿主细胞可用于生成主题rAAV病毒体。主题宿主细胞用于生成主题rAAV病毒体时,将其称为“包装细胞”。在一些实施方案中,主题宿主细胞经主题核酸稳定地遗传修饰。在其它实施方案中,主题宿主细胞经主题核酸暂时地遗传修饰。
使用确定技术,包括但不限于电穿孔、磷酸钙沉淀、脂质体介导的转染等,稳定或暂时地将主题核酸引入宿主细胞中。为了稳定转化,主题核酸通常将进一步包括可选标记,例如几种众所周知的可选标记的任一种,例如新霉素抗性等。
通过将主题核酸引入多种细胞的任一种,例如哺乳动物细胞,包括(例如)鼠科动物细胞和灵长类动物细胞(例如,人类细胞)中生成主题宿主细胞。适合的哺乳动物细胞包括但不限于原代细胞和细胞系,其中适合的细胞系包括但不限于293细胞、COS细胞、HeLa细胞、Vero细胞、3T3小鼠成纤维细胞、C3H10T1/2成纤维细胞、CHO细胞等。适合宿主细胞的非限制性实例包括(例如)HeLa细胞(例如美国模式培养物保藏所(ATCC)No.CCL-2)、CHO细胞(例如,ATCCNo.CRL9618、CCL61、CRL9096)、293细胞(例如,ATCCNo.CRL-1573)、Vero细胞、NIH3T3细胞(例如,ATCCNo.CRL-1658)、Huh-7细胞、BHK细胞(例如,ATCCNo.CCL10)、PC12细胞(ATCCNo.CRL1721)、COS细胞、COS-7细胞(ATCCNo.CRL1651)、RAT1细胞、小鼠L细胞(ATCCNo.CCLI.3)、人胚肾(HEK)细胞(ATCCNo.CRL1573)、HLHepG2细胞等。也可使用杆状病毒感染产生AAV的昆虫细胞例如Sf9细胞制备主题宿主细胞(见,例如,美国专利No.7,271,002;美国专利公布12/297,958)。
在一些实施方案中,除包含如上所述编码变异AAV衣壳蛋白的核苷酸序列的核酸外,经遗传修饰的主题宿主细胞还包括包含编码一种或多种AAVrep蛋白的核苷酸序列的核酸。在其它实施方案中,主题宿主细胞进一步包含rAAV载体。可使用主题宿主细胞生成rAAV病毒体。例如,在美国专利公布No.2005/0053922和美国专利公布No.2009/0202490中描述了生成rAAV病毒体的方法。
实施例
提出下列实施例以便为本领域的普通技术人员提供如何制备和使用本发明的全面公开和描述,并非旨在限制发明人视为其发明的范围,也并非旨在表示以下实验是全部或唯一进行的实验。已经努力确保关于所用数字(例如,数量、温度等)的精确性,但是应考虑到一些实验误差和偏差。除非另外指出,份为重量份,分子量为重均分子量,温度按摄氏度计,而压力为或接近大气压。可使用标准偏差,例如bp,碱基对;kb,千碱基对;pl,皮升;s或sec,秒;min,分;h或hr,小时;aa,氨基酸;kb,千碱基对;bp,碱基对;nt,核苷酸;i.m.,肌肉内;i.p.,腹膜内;s.c.,皮下等等。
实施例1:视网膜细胞转导增强的AAV变异体
所用方法是通过聚合酶链反应(PCR)诱变将独特AvrII位点引入野生型AAV2基因组介于氨基酸587和588之间产生肽展示库。使用随机21核苷酸***,7merFor,连同反义引物7merRev一起合成dsDNA***。用NheI消化后将所得dsDNA***克隆至基因组的AvrII位点,生成不同的7mer展示库,然后包装7mer展示库(Perabo等,2003;Muller等,2003)。生成病毒,以致将每个病毒基因组包装或壳体化于该基因组编码的衣壳蛋白变异体中。在这点上,通过选择鉴定的功能改进可能与编码病毒衣壳中所含的这种改进功能的基因组序列有关联。
使这个库在rho-GFP小鼠体内经受阳性选择(Wensel等(2005)VisionRes.45:3445)。简言之,在第一轮选择中,为成年rho-GFP小鼠玻璃体内注射2μL经磷酸盐缓冲盐水(PBS)透析的碘克沙醇(iodixanol)纯化库,基因组滴度为约1×1012个病毒基因组(vg)/mL。超细301/2-规一次性针头穿过动物眼睛的巩膜,在中纬线处并且靠近边缘,进入玻璃体腔。通过直接观察在玻璃体腔中央的针头注射2μ1病毒。注射1周后,摘出眼睛并使用光、木瓜蛋白酶处理解离视网膜,接着荧光激活细胞分选仪(FACS)分离光感受器群体。然后由后续基因组萃取PCR扩增成功的病毒体并进一步克隆和重新包装用于注射。
进一步进行这种选择的重复,使变异体库缩小为具有大多数允许突变的亚类。重复3次后,进行一轮易错PCR以引起变异体的进一步生成以便选择。总计,重复这个过程两代。在这点上,与自然进化相似,这个定向进化过程通过应用阳性选择和诱变产生光感受器允许型AAV变异体。
随后,为50种变异体的空位基因测序以确定最显著和成功的变异体具有供玻璃体内光感受器转导的允许突变。50个克隆中,46个产生7mer***的可读序列。显著地,近三分之二的克隆含有同一个不同的7mer基序(~588LGETTRP~;SEQIDNO:13)。有趣的是,次显著变异体(~588NETITRP~;SEQIDNO:14)也含有由介于极性苏氨酸和非极性脯氨酸残基之间带正电的精氨酸残基组成的类似侧面基序(TRP)。
表1
表1来自定向进化的分离变异体的测序显示了病毒库中高度收敛。所有变异体源自AAV27mer库,大约64%的变异体含有同一7mer基序(~588LGETTRP~(SEQIDNO:13))。
在7mer***序列中,对特定位置有适度优先,例如位置1处带正电的氨基酸;位置2处带负电的氨基酸;位置5处的醇(例如,具有醇基(游离羟基)的氨基酸,例如Thr或Ser)。
如表2所示,7mer***两侧为间隔区:
克隆 | 频率 |
~588LALGETTRPA~(SEQ ID NO:45) | 31 |
~588LANETTRPA~(SEQ ID NO:46) | 5 |
~588LAKAGQANNA~(SEQ ID NO:47) | 3 |
~588LAKDPKTTNA~(SEQ ID NO:48) | 2 |
~588LAKDTDTTRA~(SEQ ID NO:61) | 2 |
~588LARAGGSVGA~(SEQ ID NO:62) | 1 |
~588LAAVDTTKFA~(SEQ ID NO:63) | 1 |
~588LASTGKVPNA~(SEQ ID NO:64) | 1 |
图1.在氨基酸587之后含有无规七聚物(橙色所示)的AAV2的代表性三维模型。AAV2衣壳的这个区域很可能参与细胞表面受体结合。
根据来自上述选择的高度库收敛,克隆重组形式的AAV2~588LGETTRP~(SEQIDNO:13;昵称7M8)并且包装具有scCAG-GFP转基因的载体以使其转导性质显现。成年小鼠玻璃体内注射3周后,观察到在许多细胞类型中稳健表达,包括视网膜神经节细胞(RGC)和缪勒细胞。重要的是,观察到正如通过外核层(ONL)细胞核(红色箭头)和外节(图2,蓝色箭头)中的GFP表达所见,注射了7M8的视网膜中光感受器转导,而AAV2未显示出可辩别的光感受器表达。
图2.相对于AAV2(左),AAV27M8变异体(右)展现出更高水平的玻璃体内光感受器转导。成年小鼠玻璃体内注射2μL的1×1012vg/mL的AAV27M8和AAV2scCAGGFP3周后视网膜横切面的共聚焦显微镜检查。红色箭头(上)指示光感受器细胞核而蓝色箭头(上)指示光感受器外节。
根据视网膜细胞中的这些增益,试图通过使用含有光感受器特异性视紫红质启动子的ssRho-eGFP转基因增加表达的特异性,以更好地解决特别是在光感受器中的转导效率(图3)。实际上,光感受器特异性Rho启动子的使用将GFP表达限于光感受器。试图通过使合理的设计方法与先前的定向进化方法结合提高7M8转导效率。因此,将7M8衣壳上4个表面暴露的酪氨酸残基诱变为苯丙氨酸(Y273F、Y444F、Y500F和Y730F),这先前已经证实提高了光感受器感染性(Petrs-Silva等,2009)。有趣的是,正如对来自受7m8或7m8-4YF感染的视网膜的GFP(+)光感受器的FAC分选(图4)所示,与原病毒相比,突变的增加使转导的光感受器数量减少。
图3.视网膜低温切片的代表性荧光图像,显示出由携带GFP基因的7m8,在普遍存在的CAG启动子(左)或光感受器特异性Rho启动子(右)的控制下产生的GFP表达。
图4.通过流式细胞术计数的每一百万个视网膜细胞的GFP(+)光感受器细胞。与携带4个酪氨酸突变(上)的7m8相比,7m8转导超过2×量的光感受器。
实施例2:视网膜劈裂症的治疗
使用表达构建体7m8-rho-RS1,将功能性网膜劈裂蛋白(RS1)递送至视网膜劈裂蛋白缺陷型小鼠(Rs1h缺陷型小鼠;Rs1h为人RS1的小鼠同源物)。载体包含在视紫红质启动子的转录控制下编码功能性视网膜劈裂蛋白的核苷酸序列。见图13A-C,其中粗体和加下划线的核苷酸序列(核苷酸4013-4851)为视紫红质启动子;并且核苷酸4866-5540(起始atg和终止tga序列以粗体显示)编码人RS1蛋白。
在P15时,经玻璃体内向Rs1h-/-小鼠施用7m8-rho-RS1构建体。如所述,通过靶向破坏Rs1h基因的外显子3产生Rs1h-/-小鼠(Weber等(2002)Proc.Natl.Acad.Sci.USA99:6222)。Rs1h-/-小鼠缺乏Rs1h蛋白产物,具有负电性ERG(例如,约化b波,而a波相对保存)和与在人类视网膜劈裂症患者中所见类似的视网膜层裂开。向Rs1h-/-注射7m8-rho-RS1载体导致视网膜内来自光感受器的高水平全视网膜RS1表达。RS1表达导致视网膜形态改进,如频域光学相干断层(SD-OCT)成像(图7A-I)中所见视网膜内腔的数量和尺寸减小,ERGb波救援(图8A-D)和视网膜的结构长期保存(图9A-E)。
图7A-I.注射了7m8-rho-GFP(左列)、7m8-rho-RS1(中间列)或未注射的野生型动物(右列)视网膜的代表性高分辨率SD-OCT图像。通过上视网膜的内核层得到眼底图像并且将其它层排除在外(a-c)。使用视神经***作为标志得到上视网膜(d-f)和下视网膜(g-i)的横向图像。
因为病理由于使内视网膜裂开的劈裂而进展,当从内界膜(ILM)到光感受器测量时,未经治疗的RS1视网膜的总厚度增加。这个过程与在不形成劈裂,但是表现出INL中进行性光感受器细胞死亡及伴随视网膜变薄和ERG振幅减小的大多数视网膜退行性疾病(RDD)中所观察到的过程不同。在RS1中,当光感受器因疾病而死时ONL变薄,但是这不同于视网膜总厚度变化。通常认为,对RS1的成功治疗将使视网膜总厚度恢复为野生型并且改善ONL中的光感受器损失。在除Rs1的大多数RDD中,以ONL变薄为特征的光感受器损失伴有通过ERG振幅测量的视网膜生理输出减少。RS1是病理使视网膜厚度增加,而伴随erg振幅减小的视网膜疾病的很少几个实例之一。总之,恢复RS1基因产物,细胞外视网膜“胶”使视网膜变薄回野生型厚度并且因为劈裂消退,erg振幅恢复至接近正常水平。
图8a示出了未经治疗的Rs1-/-眼与注射了AAV2-rho-RSl、7m8-rho-GFP和7m8-rho-RS1的眼在注射后1个月(左)和4个月(右)功能性救援的比较。注射后1个月,7m8-rho-RS1导致大量援救ERGb波振幅,而AAV2-rho.RSl与未经治疗的眼在统计上无区别。
4个月后,7m8-rho-RS1振幅增大至野生型振幅(右)。图8b示出了注射了7m8-rho-RS1的眼的代表性ERG轨迹,显示与注射了7m8-rho-GFP的眼相比,a波和b波振幅增大并且波形接近于野生型眼。图8c示出了从注射后1个月开始按月在每种情况的P15时记录的由高强度(1logcd×s/m2)刺激产生的全视野暗视b波的振幅。记录了3次反应并计算每只眼在每个时间点的平均值。
根据注射后的时间绘制平均ERGb波振幅。n=7用于两种情况。图8d示出了对暗视(上轨迹,刺激范围从-3到1logcd×s/m2)和明视(下轨迹,范围从-0.9到1.4logcd×s/m2)条件下ERG反应的分析,显示在一定刺激强度范围内杆状和锥状细胞功能改善。
图9A-E.7m8-rho-RS1治疗后10个月测量的视网膜厚度持续增加。集中在视神经***注射后10个月经a)7m8-rho-RS1或b)或7m8-rho-GFP治疗的视网膜的代表性横向SD-OCT图像。作为与视神经***的距离的函数绘制c)视网膜厚度、d)ONL厚度和e)内外节厚度的测量值。
实施例3:用于将蛋白质递送至猕猴视网膜细胞的AAV变异体
生成重组AAV2病毒体(携带受连接蛋白36启动子控制的GFP的7m8)。重组AAV2病毒体包括在AAV2衣壳的氨基酸587和588之间有LALGETTRPA肽***的AAV2衣壳变异体,和受连接蛋白36启动子的转录控制的在中间神经元中表达的GFP。将rAAV2病毒体经玻璃体内注射至猕猴眼内。图18中示出了数据。
图18提供了荧光眼底图像,显示在施用携带受连接蛋白36启动子控制的GFP的7m8后9周,在视网膜后部的GFP表达。与亲本AAV2血清型(Yin等,IOVS52(5);2775)相比,在视网膜中央凹环中看到更高水平的表达,并且在视网膜中央凹外的中央视网膜中看到可见荧光。
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虽然已经参考其特定实施方案描述了本发明,但是本领域的普通技术人员应理解,在不背离本发明的精神和范围的前提下,可做各种变化并代替等效方案。另外,可做许多修改以适应特殊情况、材料、物质组合物、工艺、一种或多种工序,对本发明的目的、精神和范围可做许多修改。旨在使所有此类修改均属于所附权利要求的范围之内。
Claims (27)
1.一种重组腺相关病毒(rAAV)病毒体,包含:
a)变异AAV衣壳蛋白,其中相对于相应亲本AAV衣壳蛋白,所述变异AAV衣壳蛋白在衣壳蛋白GH环中包含约5个氨基酸至约11个氨基酸的***,并且其中与包含所述相应亲本AAV衣壳蛋白的AAV病毒体对视网膜细胞的感染性相比,所述变异衣壳蛋白赋予增强的视网膜细胞感染性;和
b)包含编码基因产物的核苷酸序列的异源核酸。
2.根据权利要求1所述的rAAV病毒体,其中所述***为式I、式IIa、式III或式IV的肽。
3.根据权利要求2所述的rAAV病毒体,其中所述***包含选自LGETTRP(SEQIDNO:13)、NETITRP(SEQIDNO:14)、KAGQANN(SEQIDNO:15)、KDPKTTN(SEQIDNO:16)、KDTDTTR(SEQIDNO:57)、RAGGSVG(SEQIDNO:58)、AVDTTKF(SEQIDNO:59)和STGKVPN(SEQIDNO:60)的氨基酸序列。
4.根据权利要求1所述的rAAV病毒体,其中所述视网膜细胞为光感受器、视网膜神经节细胞、缪勒细胞、双极细胞、无长突细胞、水平细胞或视网膜色素上皮细胞。
5.根据权利要求1所述的rAAV病毒体,其中所述***位点介于AAV2的氨基酸587和588之间,介于AAV1的氨基酸590和591之间,介于AAV5的氨基酸575和576之间,介于AAV6的氨基酸590和591之间,介于AAV7的氨基酸589和590之间,介于AAV8的氨基酸590和591之间,介于AAV9的氨基酸588和589之间,或介于AAV10的氨基酸588和589之间。
6.根据权利要求1所述的rAAV病毒体,其中与包含所述相应亲本AAV衣壳蛋白的AAV病毒体对视网膜细胞的感染性相比,所述rAAV病毒体表现出增强至少10倍的视网膜感染性。
7.根据权利要求1所述的rAAV病毒体,其中与包含所述相应亲本AAV衣壳蛋白的AAV病毒体对视网膜细胞的感染性相比,所述rAAV病毒体表现出增强至少50倍的视网膜感染性。
8.根据权利要求1所述的rAAV病毒体,其中所述基因产物为干扰RNA或适配体。
9.根据权利要求1所述的rAAV病毒体,其中所述基因产物为多肽。
10.根据权利要求7所述的rAAV病毒体,其中所述多肽为神经保护多肽、抗血管生成多肽或增强视网膜细胞功能的多肽。
11.一种药物组合物,包含:
a)权利要求1的重组腺相关病毒病毒体;和
b)药学上可接受的赋形剂。
12.一种将基因产物递送至个体的视网膜细胞的方法,所述方法包括向所述个体施用根据权利要求1所述的重组腺相关病毒(rAAV)病毒体。
13.根据权利要求11所述的方法,其中所述基因产物为多肽。
14.根据权利要求11所述的方法,其中所述基因产物为短干扰RNA或适配体。
15.根据权利要求13所述的方法,其中所述多肽为神经保护因子、抗血管生成多肽、抗凋亡因子或增强视网膜细胞功能的多肽。
16.根据权利要求13所述的方法,其中所述多肽为胶质源性神经营养因子、成纤维细胞生长因子2、神经营养因子、睫状神经营养因子、神经生长因子、脑源性神经营养因子、表皮生长因子、视紫红质、X连锁凋亡抑制蛋白、视网膜劈裂蛋白、RPE65、色素性视网膜炎GTP酶相互作用蛋白-1、外周蛋白、外周蛋白-2、视紫红质或音猬因子。
17.一种治疗视网膜疾病的方法,所述方法包括向有需要的个体施用有效量的根据权利要求1所述的重组腺相关病毒(rAAV)病毒体。
18.根据权利要求17所述的方法,其中所述施用是通过眼内注射。
19.根据权利要求17所述的方法,其中所述施用是通过玻璃体内注射。
20.根据权利要求17所述的方法,其中所述眼病为青光眼、色素性视网膜炎、黄斑变性、视网膜劈裂症、利伯氏先天性黑内障、糖尿病性视网膜病、全色盲或色盲。
21.一种包含编码变异腺相关病毒(AAV)衣壳蛋白的核苷酸序列的分离核酸,其中相对于相应亲本AAV衣壳蛋白,所述变异AAV衣壳蛋白在衣壳蛋白GH环中包含约5个氨基酸至约11个氨基酸的***,并且其中当存在于AAV病毒体中时,所述变异衣壳蛋白提供增强的视网膜细胞AAV病毒体感染性。
22.根据权利要求21所述的分离核酸,其中所述***位点介于AAV2的氨基酸587和588之间,介于AAV1的氨基酸590和591之间,介于AAV5的氨基酸575和576之间,介于AAV6的氨基酸590和591之间,介于AAV7的氨基酸589和590之间,介于AAV8的氨基酸590和591之间,介于AAV9的氨基酸588和589之间,或介于AAV10的氨基酸588和589之间。
23.一种包含根据权利要求21所述的核酸的经分离、遗传修饰的宿主细胞。
24.一种变异腺相关病毒(AAV)衣壳蛋白,其中所述变异AAV衣壳蛋白包含约5个氨基酸至约11个氨基酸的***,其中所述氨基酸***在天然AAV衣壳的GH环中,其中所述***为式I、式IIa、式III或式IV的肽。
25.根据权利要求24所述的变异AAV衣壳蛋白,其中所述***包含选自LGETTRP(SEQIDNO:13)、NETITRP(SEQIDNO:14)、KAGQANN(SEQIDNO:15)、KDPKTTN(SEQIDNO:16)、KDTDTTR(SEQIDNO:57)、RAGGSVG(SEQIDNO:58)、AVDTTKF(SEQIDNO:59)和STGKVPN(SEQIDNO:60)的氨基酸序列。
26.根据权利要求24所述的变异AAV衣壳蛋白,其中所述***位点介于AAV2的氨基酸587和588之间,介于AAV1的氨基酸590和591之间,介于AAV5的氨基酸575和576之间,介于AAV6的氨基酸590和591之间,介于AAV7的氨基酸589和590之间,介于AAV8的氨基酸590和591之间,介于AAV9的氨基酸588和589之间,或介于AAV10的氨基酸588和589之间。
27.一种包含编码根据权利要求24所述的变异AAV衣壳蛋白的核苷酸序列的核酸。
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