CN114008194A - 用于基因疗法的经修饰的腺相关病毒(aav)颗粒 - Google Patents
用于基因疗法的经修饰的腺相关病毒(aav)颗粒 Download PDFInfo
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Abstract
本发明涉及一种用于基因递送和基因疗法的改进的腺相关病毒(AAV)颗粒。提供了包含经修饰的衣壳的腺相关病毒(AAV)颗粒。本发明还涉及通过去除腺相关病毒(AAV)衣壳中的天然结合位点和将配体结合位点引入所述衣壳以提供仅转导特定目标细胞的AAV来产生本发明的改进的AAV颗粒的方法。本发明的另一个方面涉及用于疾病治疗的经修饰的AAV颗粒和用于治疗疾病的方法,其包括向需要的受试者施用经修饰的AAV颗粒。本发明的又一个方面涉及本发明的AAV颗粒,其用于细胞转染,例如作为基础研究的基因递送工具。
Description
本发明涉及用于基因递送和基因疗法的改进的腺相关病毒(AAV)颗粒。提供了包含经修饰的衣壳的腺相关病毒(AAV)颗粒。本发明还涉及通过去除腺相关病毒(AAV)衣壳中天然结合位点和将配体结合位点引入所述衣壳以提供仅转导特定目标细胞的AAVs来产生本发明的改进的AAV颗粒的方法。本发明的另一个方面涉及用于疾病治疗的经修饰的AAV颗粒和用于治疗疾病的方法,其包括向需要的受试者施用经修饰的AAV颗粒。本发明的又一个方面涉及用于细胞转染的本发明的AAV颗粒,例如作为基础研究的基因递送工具。
背景技术
将携带遗传信息的分子引入细胞中是现代医学和基础研究中一个有用的工具。优选的方法包括源自病毒的基因递送载体的使用,所述病毒包括腺病毒、逆转录病毒、痘苗病毒和腺相关病毒。其中,腺相关病毒(AAV)是用于基因治疗方法的优选病毒。野生型AAVs是尺寸相对较小的DNA病毒,其以稳定和位点特异性的方式整合至它们感染的细胞的基因组中。重要的是,迄今为止还未发现与AAV感染相关的人类疾病。因此,腺相关病毒(AAV)因缺乏致病性而成为首选的基因疗法载体。有趣的是,超过100个临床试验正在使用基于AAV的载体,而且重要的是,第一个AAV基因疗法最近获得了FDA的批准,即用于治疗遗传性视网膜疾病的Voretigene neparvovec。
腺相关病毒是包含无包膜二十面体衣壳的细小病毒科依赖病毒属的成员。AAV基因组长度为约4.7千碱基并由线性单链脱氧核糖核酸(ssDNA)组成,其可以是正义或反义的。基因组包含位于DNA链两端的末端反向重复序列(ITRs),和两个开放阅读框(ORFs):rep和cap。rep框由编码AAV生命周期所需的非结构复制(Rep)蛋白的四个重叠基因组成。cap框包含结构VP衣壳蛋白的重叠核苷酸序列:VP1、VP2和VP3,该VP衣壳蛋白一起相互作用以形成二十面体对称的衣壳。末端145nt是自身互补的并组织化,使得可以形成一个用以形成T型发夹的能量稳定的分子内双链体。这些发夹结构充当病毒DNA复制的起点,作为细胞DNA聚合酶复合物的引物。在AAV感染哺乳动物细胞后,rep基因(即Rep78和Rep52)分别从P5启动子和P19启动子表达,并且两种Rep蛋白均在病毒基因组的复制中起作用。rep ORF中的剪接事件导致四种Rep蛋白(即Rep78、Rep68、Rep52和Rep40)的表达。
通常,病毒可以多种方式进入细胞,或者通过在质膜处的直接膜融合/穿入反应,或者通过经历内吞作用,随后在早期核内体中经历类似的细胞膜破裂。病毒内化最常见的形式是通过网格蛋白介导的内吞作用。此过程可以分成几个步骤,其包括:(1)网格蛋白有被小窝的成核;(2)有被小窝中的货物捕获;(3)曲率诱导和膜内陷;和(4)囊泡破裂和脱壳。囊泡将其病毒内容物递送至早期核内体。有趣的是,内吞作用的全过程在体内发生约几秒钟。通过网格蛋白介导的内吞作用内化的病毒的示例是dsDNA病毒,例如腺病毒科、腺病毒2型、腺病毒5型、腺病毒8型、腺病毒37型、犬腺病毒2型(CAV-2)、腺相关病毒2型和腺相关病毒5型(依赖病毒属)。
病毒也可通过穴样内陷内化,穴样内陷是形成50-70nm质膜瓶状内陷的特殊脂质筏。小窝蛋白形成穴样内陷的结构骨架。通过穴样内陷的内化不是组成型过程,而是仅在细胞刺激时发生。与它们的宿主细胞受体结合的内化病毒被递送到早期核内体。穴样内陷代表低容量但高度调节的通路。此外,一些病毒使用几种途径内化,该途径不使用网格蛋白或小窝蛋白壳(coat),并且有时被细菌和病毒劫持以进入宿主细胞。这些途径可以进一步通过它们对各种分子(例如胆固醇、DNM2/发动蛋白-2、或小GTPases或酪氨酸激酶)的依赖性定义。
由于AAV载体稳定且位点特异性的整合到感染细胞的基因组中,并且缺乏致病性,因此AAV载体正被探索作为靶向基因疗法的载体。
Kern等(于:Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2Capsids,Journal of Virology Sep 2003,77(20)11072-11081)公开了腺相关病毒(AAV)2型(AAV-2)的细胞感染是通过与硫酸乙酰肝素蛋白聚糖的结合介导的且可通过肝素竞争。AAV-2衣壳蛋白的突变分析表明一组碱性氨基酸(精氨酸484、487、585和588以及赖氨酸532)有助于肝素和Hela细胞结合。这些氨基酸位于AAV-2衣壳的三倍刺突(threefold spike)区域的三个簇中。R484和R585突变的重组AAV-2在小鼠体内的组织分布表明,相比于野生型重组AAV感染,肝脏感染显著减少,但心脏感染继续。他们认为尽管肝素结合影响AAV-2的感染性,但似乎不是必需的。
US 5,756,283公开了重组AAV载体,其包含在调控序列控制下的选择的转基因。感染的细胞与促进单链重组病毒转化为其双链形式的试剂接触,从而提高重组AAV转导入靶细胞的效率。
EP 1664314 B1描述了基于参与肝素结合的衣壳蛋白结构域和相应氨基酸残基的鉴定,导致肝素结合功能降低或消除的携带衣壳蛋白修饰的重组AAV载体。
WO 2017/143100 A1描述了修饰AAV衣壳多肽的方法,使得相比于非变体亲本衣壳多肽,修饰的AAV衣壳多肽在人类肝脏组织或肝细胞中表现出增强的中和特征、提高的转导和/或向性。
人类基因疗法的安全性和有效性仍然是相当大的争论焦点。目前载体的问题包括某些组织的非预期转导和不利的免疫反应。目前使用AAV颗粒进行基因递送的方法存在问题,其原因在于缺乏有效转导,即通常需要非常高滴度的AAV载体才有效。目前方法的另一个局限性在于,许多AAV载体在转导某些特定的细胞类型时是无效的,并且AAV载体通过不适当的细胞类型的转导可具有脱靶效应。
鉴于上述局限性,本发明的基本任务是提供用于基因递送的改进的腺相关病毒(AAV)颗粒。上述任务可通过设计转导特定目标细胞和/或可以较低滴度递送但仍然有效的腺相关病毒(AAV)衣壳来实现。上述问题可通过修饰病毒衣壳以接受配体连接并将目标配体连接于所述衣壳来解决。任选地,可在修饰病毒以接受配体连接之前去除AAV衣壳中的天然结合位点。
根据本发明的第一个方面,通过提供包含经修饰的衣壳的腺相关病毒(AAV)颗粒解决上述问题,其中所述经修饰的衣壳包含至少一种修饰,其选自所述衣壳中天然结合位点的去除,和配体结合位点向所述衣壳中的引入。
术语“腺相关病毒载体”、“AAV载体”、“腺相关病毒”、“AAV病毒”、“AAV病毒粒子”、“AAV病毒颗粒”和“AAV颗粒”在本文中可互换使用,其是指由至少一种AAV衣壳蛋白和衣壳包裹的重组病毒基因组组成的病毒颗粒。包含重组病毒基因组的AAV颗粒通常被称为“AAV载体颗粒”或“AAV载体”,其中所述重组病毒基因组具有异源多核苷酸和包含AAV末端反向重复序列侧邻的递送至哺乳动物细胞的启动子的转录调控区。
在一个优选的实施方案中,本发明的腺相关病毒(AAV)颗粒选自AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11和AAV12。迄今为止,最常用的基因转移系统是病毒的衍生物,例如腺相关病毒2型(AAV-2)。优选的是AAV-2和/或AAV-9。
目前,已鉴定出超过100种AAV血清型,其衣壳蛋白与可转导不同细胞类型的特定细胞表面受体的结合能力不同。AAV2是克隆至细菌质粒中的第一种血清型,并已被用作鉴定其他血清型的对照。已经全面测试了12种血清型(AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11和AAV12)转导特定细胞类型的能力,并在结合特定细胞表面受体以进行细胞附接的衣壳蛋白基序之间进行了区分。在本发明的背景下,选自AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12的AAV颗粒是优选的。但是,应当理解在本发明的背景下,可以使用任何其他AAV颗粒。
AAV的衣壳由含有独特VP1 N-末端、VP1/VP2共同部分以及VP1、VP2和VP3的共同部分的三个重叠的衣壳蛋白(VP1、VP2、VP3)组成。
本发明的腺相关病毒(AAV)颗粒是进一步优选的,其中所述AAV颗粒的衣壳中的至少一种蛋白被修饰,优选地,其中至少一种蛋白是VP1、VP2和/或VP3。或者,所述衣壳中的蛋白VP1、VP2和/或VP3的两种被修饰,或所述衣壳中的全部三种蛋白VP1、VP2和VP3被修饰。优选地,所述衣壳中至少一种待修饰的蛋白的至少一部分(例如一个氨基酸)被修饰。但是,也可能修饰所述衣壳中蛋白VP1、VP2和VP3的多个部分,例如多个氨基酸,如2、3、4、5、6、7、8、9、10或任何其他数量的部分或氨基酸。优选地,VP1的精氨酸484、487、585和588和赖氨酸532中的至少一个和/或VP2或VP3中类似的精氨酸可通过用不同的氨基酸例如丙氨酸替换来去除。
进一步优选的是,衣壳修饰包含至少一个天然结合位点的去除和至少一个配体结合位点的引入。或者,所述AAV的至少一个天然结合位点可保持不变,即不被去除,但引入至少一个配体结合位点。
如果所述衣壳中的所述天然结合位点存在且未被去除,并通过修饰向所述衣壳中引入至少一个另外的配体结合位点,则本发明的AAV颗粒在所用病毒颗粒的较低滴度下具有较高的感染率。相反,如果在通过修饰向所述衣壳中引入至少一个另外的配体结合位点之前,所述衣壳中的所述天然结合位点被去除,则本发明的AAV颗粒的向性被改变。
优选的是本发明的腺相关病毒(AAV)颗粒,其中所述天然结合位点是能够与硫酸乙酰肝素蛋白聚糖结合的结合位点,并且优选地通过用不同的氨基酸例如丙氨酸替换VP1的精氨酸585或精氨酸588中的至少一个和/或VP2或VP3中类似的精氨酸去除。
此外,优选的是本发明的腺相关病毒(AAV)颗粒,其中所述配体结合位点是能够与配体共价连接的结合位点。进一步优选的是,所述配体结合位点包含苄基鸟嘌呤基团,苄基胞嘧啶基团,氯代烷烃基团,二苯并环辛炔基团,叠氮基团,膦或其组合。或者,可使用任何卤代烷烃基团。此外,优选的是苄基鸟嘌呤或其他基团连接至可用的赖氨酸残基。引入的所述配体结合位点优选连接至所述可用的赖氨酸残基的ε-氨基或伯胺。
进一步优选的是本发明的腺相关病毒(AAV)颗粒,其进一步包含与所述苄基鸟嘌呤基团、所述苄基胞嘧啶基团、所述氯代烷烃基团、所述二苯并环辛炔基团、所述叠氮基团和/或所述膦,特别是HaloTagTM、SNAP-tagTM或CLIP-tagTM,或者膦或叠氮基或二苯并环辛炔基团连接的配体。本发明优选使用能够以高亲和力结合其特异性配体的标签,例如SNAP-tag、CLIP-tag、Halo Tag、Lumio Tag等。
苄基鸟嘌呤、苄基胞嘧啶和氯代烷烃被“自杀”酶(例如SNAP)识别。在本发明的背景下,可进一步使用苄基鸟嘌呤或苄基胞嘧啶衍生物。苄基鸟嘌呤衍生物或苄基胞嘧啶衍生物被理解为意指苄基鸟嘌呤或苄基胞嘧啶基团,其被修饰但仍被自杀酶识别。
标签分子可以是能够与另外的分子特异性结合的任何分子或生物分子。示例可包括SNAP-tag、CLIP-tag、Lumio-Tag或Halo-Tag。例如,亲和标签可以是SNAP-tag,烷基鸟嘌呤-DNA烷基转移酶的突变体。重要的是,SNAP-tag的底物之一是苄基鸟嘌呤。本发明可用的市售产品包括,例如来自Promega的HaloTag、来自Life Technologies的Lumio Tag和来自NEB的SNAP/CLIP Tags。
本领域技术人员熟知将配体连接至衣壳的进一步方法,例如抗-标签抗体、链霉亲和素-生物素或化学交联。在本发明的背景下可以使用任何已知的方法。例如,非天然氨基酸可被掺入衣壳以及配体中。随后,交联剂(例如生物正交交联剂)可被用于将配体共价连接至衣壳。在另一个实施例中,膦或二苯并环辛炔基团可被掺入衣壳中和叠氮基团可被掺入配体中,反之亦然。然后配体可通过Staudinger反应或应变促进的点击反应共价连接至衣壳。
任何种类的配体可连接至所述配体结合位点。优选地,待连接的配体选自蛋白质配体,例如生长因子或细胞因子;毒素亚单位,例如霍乱毒素B亚单位;凝集素,例如同工凝集素B4或麦胚凝集素;粘附因子,例如乳凝集素;抗体,例如抗CD-34抗体;肽,例如δ啡肽阿片受体配体;和基因编辑核酸酶,例如Cas9。
被包装在本发明的腺相关病毒(AAV)颗粒内部的核酸分子可以是任何类型的核酸分子。优选地,所述核酸分子是编码胞内抗体(例如以中和细胞内的某些蛋白质)、编码肽毒素(例如以阻断疼痛途径中的离子通道)的核酸分子、编码光发生致动器(optogeneticactuator)(例如以使用光开启或关闭神经元活性)的核酸分子、编码药物遗传学工具(例如以使用不具有干扰药理作用的化学配体开启或关闭神经元信号传导)的核酸分子、编码用于精确基因编辑的基于CRISPR编辑器的核酸分子、编码CRISPR表观遗传工具以调节基因表达的核酸分子和/或编码自杀基因以诱导细胞死亡的核酸分子。
优选地,当配体是基因编辑核酸酶(例如Cas9)时,本发明的腺相关病毒(AAV)颗粒还携带作为货物的核酸分子,例如gRNA和/或待插入宿主基因组中的特定DNA。
本领域内技术人员知道除Cas9之外的其他基因编辑核酸酶,例如Cpf1、TALEN、ZFN或归巢核酸内切酶。此外,使用DNA向导Argonaute干扰系统(DAIS)进行设计可能是方便的。基本上,在至少一种外源寡核苷酸(DNA向导)的存在下,所述Argonaute(Ago)蛋白可从被引入所述细胞的多核苷酸中异源表达,以提供所述Ago蛋白对预选基因座的切割特异性。WO2013/176915和WO2014/191128中分别描述了TALEN和Cas9系统。锌指核酸酶(ZFNs)最初被记载于Kim,YG;Cha,J.;Chandrasegaran,S.(“Hybrid restriction enzymes:zincfinger fusions to Fok I cleavage domain”(1996).Proc Natl Acad Sci USA 93(3):1156-60)。Cpf1是Zhang等人(Cpfl is a single RNA-guided Endonuclease of a Class2CRIPR-Cas System.(2015).Cell;163:759-771)描述的2类CRISPR Cas系统。Argonaute(AGO)基因家族最初记载于Guo S,Kemphues KJ.(Par-1,a gene required forestablishing polarity in C.elegans embryos,encodes a putative Ser/Thr kinasethat is asymmetrically distributed.(1995).Cell;81(4):611-20)。
本发明的另一个方面涉及根据本发明所述的AAV颗粒,其用于疾病治疗。
根据本发明使用的AAV颗粒可以治疗或预防任何种类的疾病。优选地,根据本发明使用的AAV颗粒所治疗或预防的疾病是可通过基因疗法治疗的疾病,例如癌症、单基因遗传病(例如遗传性视网膜疾病)、遗传性皮肤病(例如Olmsted综合征或家族性原发性局限性皮肤淀粉样变性)、传染病、肾上腺脑白质营养不良、α-1抗胰蛋白酶缺乏症、芳香族L-氨基酸缺乏症、贝敦氏症、贝克型肌营养不良、β地中海贫血、卡纳万病、慢性肉芽肿病、克里格勒-纳贾尔综合征、囊性纤维化、杜兴氏肌营养不良、法布瑞氏症、家族性腺瘤性息肉病、家族性高胆甾醇血、家族性卵磷脂-胆固醇乙酰转移酶缺乏症、范可尼贫血症、半乳糖唾液酸贮积症、高歇氏病、回旋状萎缩(gyrate atrophy)、血友病A和B、Hurler综合征(粘多糖症I型)、Hunter综合征(粘多糖症II型)、亨廷顿舞蹈病、结合性大疱性表皮松解症、婴儿晚发型神经元蜡样脂褐质沉积症、白细胞粘附缺陷病、肢带肌营养不良、脂蛋白脂肪酶缺乏症、异染性脑白质营养不良、Sly综合征(粘多糖症VII型)、Netherton综合征、鸟氨酸氨甲酰基转移酶缺乏症、庞贝病、嘌呤核苷磷酸化酶缺乏症、隐性营养不良性大疱性表皮松解症、sanfilippo A(粘多糖症IIIA型)、sanfilippo B(粘多糖症IIIB型)、镰状细胞病、重度联合免疫缺陷病、脊髓性肌萎缩、戴-萨克斯氏病、维斯科特-奥尔德里奇综合征、冯吉尔克症(糖原贮积病Ia型)、X连锁肌小管性肌病、终末期肾病贫血、心绞痛(稳定型、不稳定型、难治型)、冠状动脉狭窄、严重肢体缺血、心力衰竭、间歇性跛行、心肌缺血、外周血管疾病、肺动脉高血压、静脉溃疡、腺病毒感染、巨细胞病毒感染、Epstein-Barr病毒感染、乙型肝炎感染、丙型肝炎感染、HIV/AIDS、流感、乙型脑炎、疟疾、小儿呼吸系统疾病、呼吸道合胞病毒、破伤风、肺结核、妇科癌症、乳腺癌、卵巢癌、宫颈癌、外阴癌、神经系统癌症、恶性胶质瘤、软脑膜癌病、神经胶质瘤、星形细胞瘤、成神经细胞瘤、视网膜母细胞瘤、胃肠癌、结肠癌、结直肠癌、肝转移、肝炎后肝癌、胰腺癌、胆囊癌、肝细胞癌、泌尿生殖系统癌、前列腺癌、肾癌、膀胱癌、肛门生殖器肿瘤、皮肤癌、黑色素瘤(恶性/转移性)、头颈癌、鼻咽癌、鳞状细胞癌、食道癌、肺癌、腺癌、小细胞/非小细胞、间皮瘤、血液癌、白血病、淋巴瘤、多发性骨髓瘤、肉瘤、生殖细胞癌、李-佛美尼综合征、甲状腺癌、阿尔茨海默病、肌萎缩性脊髓侧索硬化症、腕管综合征、慢性创伤性脑损伤、肘管综合征、糖尿病神经病变、癫痫、巨轴索神经病、婴儿晚发型神经元蜡样脂褐质沉积症、多发性硬化、重症肌无力、疼痛、帕金森病、周围神经病变、脊髓性肌萎缩2型、全色盲、年龄相关性黄斑变性、无脉络膜症(choroideraemia)、糖尿病性黄斑水肿、青光眼、莱伯先天性黑朦症、黄斑毛细血管扩张2型、色素性视网膜炎、浅表性角膜混浊、遗传性视网膜劈裂症、关节炎(类风湿、炎性、退行性)、变性关节病、直肠的严重性炎症性疾病、溃疡性结肠炎、慢性肾病、糖尿病性溃疡/足溃疡、逼尿肌过度活动、勃起功能障碍、骨折、听力损失、遗传性包涵体肌病、移植物抗宿主疾病/移植患者、口腔粘膜炎、腮腺唾液功能减退、系统性硬皮病、I型糖尿病和/或创伤愈合。
本文所用术语“预防和/或抑制”应包括治疗已经存在的疾病。治疗、预防和/或抑制是指包括例如治疗、延缓或减轻疾病进展,减轻疾病或病症的症状或治愈疾病或病症。“有效量”是可缓解所治疗疾病(如上述任何疾病)的症状的本发明的AAV颗粒的量。缓解是指包括例如预防、治疗、减轻疾病或病症的症状或治愈疾病或病症。本发明还包括用于治疗处于疾病发展和/或进展风险中的受试者的方法,其中向患者施用治疗有效量的本发明的AAV颗粒。处于疾病风险中可由例如易患疾病的表型症状引起。如本文所用,当在受试者的上下文中使用术语“预防(prevention)”或“预防性(preventing)”是指停止、阻碍和/或减缓疾病(特别是与疾病相关的症状)的发展或发作。
本发明的又另一个实施方案涉及上述AAV颗粒用于治疗疾病,其中所述AAV以液体、干燥或半固体形式(例如,以片剂、包衣片剂、泡腾片剂、胶囊、粉末、颗粒、糖衣片剂、锭剂、丸剂、安瓿、滴剂、栓剂、乳剂、软膏剂、凝胶、酊剂、糊剂、乳膏、湿敷料、漱口液、植物汁、鼻试剂、吸入混合物、气雾剂、漱口水、口腔喷剂、鼻腔喷剂或室内喷剂的形式)施用于受试者。
本发明的另一个方面涉及产生改进的腺相关病毒(AAV)颗粒的方法,其包括向所述AAV的衣壳中引入至少一种修饰的步骤,优选地,其中所述修饰包括向所述衣壳中引入至少一个配体结合位点,任选地,其中所述衣壳中的天然结合位点被去除,例如预先被去除。
如上所述,如果所述衣壳中的所述天然结合位点存在且未被去除,并通过修饰向所述衣壳中引入至少一个另外的配体结合位点,则本发明的腺相关病毒(AAV)颗粒在所用病毒颗粒的较低滴度下具有较高的感染率。相反,如果在通过修饰向所述衣壳中引入至少一个另外的配体结合位点之前,所述衣壳中的所述天然结合位点被去除,则本发明的腺相关病毒(AAV)颗粒的向性被改变。
通过上述方法产生的腺相关病毒(AAV)颗粒优选地选自AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12及其混合物。
通过上述方法产生的腺相关病毒(AAV)颗粒的任何蛋白可被修饰。优选地,在上述方法中,所述衣壳中蛋白VP1、VP2或VP3中的至少一种被修饰。或者,所述衣壳中蛋白VP1、VP2和/或VP3的两种被修饰,或所述衣壳中的全部三种蛋白VP1、VP2和VP3被修饰。优选地,所述衣壳中至少一种待修饰的蛋白的至少一部分(例如一个氨基酸)被修饰。但是,也可能修饰所述衣壳中蛋白VP1、VP2和VP3的多个部分,例如多个氨基酸,如2、3、4、5、6、7、8、9、10或任何其他数量的部分或氨基酸。优选地,VP1的精氨酸484、487、585和588和赖氨酸532中的至少一个和/或VP2或VP3中类似的精氨酸通过用不同的氨基酸例如丙氨酸替换来去除。
蛋白质(例如VP1、VP2或VP3)可通过使特定氨基酸反应进行化学修饰。这样的修饰的示例是本领域内众所周知的并被总结于例如R.Lundblad,Chemical Reagents forProtein Modification,3rd ed.CRC Press,2005,其通过引用并入本文。氨基酸的化学修饰包括但不限于通过酰化作用、酰胺化、赖氨酸的吡哆醇化、还原烷基化、用2,4,6-三硝基苯磺酸(TNBS)对氨基的三硝基苄化、羧基的酰胺修饰和通过将半胱氨酸过甲酸氧化为磺基丙氨酸的巯基修饰、水银衍生物的形成、混合的二硫化物与其他硫醇化合物的组成物、与马来酰亚胺的反应、碱性pH下使用碘乙酸或碘乙酰胺的羧甲基化和使用氰酸盐的甲氨酰化的修饰,但不限于此。对此,本领域技术人员可参考Current Protocols In ProteinScience,Eds.Coligan(John wiley&Sons NY1995-2000)中第15章以了解涉及蛋白质的化学修饰的更广泛方法。
上述用于产生改进的腺相关病毒(AAV)颗粒的方法是优选的,其中所述衣壳修饰包括天然结合位点的去除和配体结合位点的引入。或者,所述AAV的天然结合位点可以保持不变,即不被去除,但引入至少一个配体结合位点。
在一个优选的实施方案中,通过上述用于产生改进的腺相关病毒(AAV)颗粒的方法去除所述天然结合位点,其中所述结合位点是能够与硫酸乙酰肝素蛋白聚糖结合的天然结合位点,并且优选地通过用不同的氨基酸例如丙氨酸替换VP1的精氨酸585和588中的至少一个和/或VP2或VP3中类似的精氨酸被去除。
更优选的是,所引入的配体结合位点是一个能够与配体共价连接的结合位点,并且优选地选自连接至可用的赖氨酸残基的苄基鸟嘌呤基团,更优选地通过使所述衣壳与苄基鸟嘌呤N-羟基琥珀酰亚胺(BG-NHS),和/或苄基胞嘧啶N-羟基琥珀酰亚胺(BC-NHS)反应。
本发明优选地使用能够以高亲和力与它们的特异性配体结合的标签,例如SNAP-tag、CLIP-tag、Halo-Tag、Lumio-Tag等。上述方法中所引入的标签分子可以是能够与另外的分子特异性结合的任何分子或生物分子。这些示例可包括SNAP-tag、CLIP-tag、Lumio-Tag或Halo-Tag。例如,亲和标签可以是SNAP-tag,烷基鸟嘌呤-DNA烷基转移酶的突变体。重要的是,SNAP-tag的底物之一是苄基鸟嘌呤。本发明可用的市售产品包括,例如来自Promega的HaloTag、来自Life Technologies的Lumio Tag和来自NEB的SNAP/CLIP Tags。所引入的所述配体结合位点优选连接至所述可用赖氨酸残基的ε-氨基或伯胺。
因此,上述用于产生改进的腺相关病毒(AAV)颗粒的方法是进一步优选的,其中所述方法进一步包括将配体连接至所述苄基鸟嘌呤和/或所述苄基胞嘧啶基团(特别是HaloTagTM、SNAP-tagTM或CLIP-tagTM)的步骤。
待连接的所述配体可以是任何种类的配体,但优选地选自蛋白质配体,例如生长因子或细胞因子;毒素亚单位,例如霍乱毒素B亚单位;凝集素,例如同工凝集素B4或麦胚凝集素;粘附因子,例如乳凝集素;抗体,例如抗CD-34抗体;肽,例如δ啡肽阿片受体配体;和基因编辑核酸酶,例如Cas9。
然后,本发明的另一个方面涉及用于治疗可通过基因疗法治疗的疾病的方法,其包括向需要的受试者施用根据本发明所述的AAV颗粒。
在本发明的背景下,某些实施方案中所用的术语“受试者”优选地是指哺乳动物,例如小鼠、大鼠、豚鼠、兔、猫、狗、猴,或优选人类。术语“患者”优选地是指哺乳动物,例如小鼠、大鼠、豚鼠、兔、马、牛、奶牛、猫、狗、猴,或优选人类,例如需要诊断、预后或治疗的人类患者。本发明的受试者可能具有患病的危险,例如细菌感染、病毒感染、真菌感染或寄生虫感染。与本发明背景相关的医学适应症的更详细的描述在本文别处提供。
用本发明的AAV颗粒治疗的细胞和/或受试者优选地来源于哺乳动物,例如来源于人类。然而,根据进入细胞的机制的相似性,本发明也可有利地用于兽医学、细胞培养程序或甚至用于植物细胞疾病。优选地,待治疗的所述细胞是哺乳动物细胞、原核细胞或植物细胞。最优选地,待治疗的所述细胞是人类细胞。
本发明的又另一个实施方案涉及用于治疗疾病的上述方法,其包括向需要的受试者施用根据本发明所述的AAV颗粒,其中所述AAV颗粒以液体、干燥或半固体形式(例如,以片剂、包衣片剂、泡腾片剂、胶囊、粉末、颗粒、糖衣片剂、锭剂、丸剂、安瓿、滴剂、栓剂、乳剂、软膏剂、凝胶、酊剂、糊剂、乳膏、湿敷、漱口液、植物汁、鼻试剂、吸入混合物、气雾剂、漱口水、口腔喷剂、鼻腔喷剂或室内喷剂的形式)施用于受试者。
通过包括向受试者施用AAV颗粒的用于治疗疾病的上述方法治疗的疾病是优选地选自以下的疾病:癌症、单基因遗传病(例如遗传性视网膜疾病)、遗传性皮肤病(例如Olmsted综合症或家族性原发性局限性皮肤淀粉样变性)、传染病、肾上腺脑白质营养不良、α-1抗胰蛋白酶缺乏症、芳香族L-氨基酸缺乏症、贝敦氏症、贝克型肌营养不良、β地中海贫血、卡纳万病、慢性肉芽肿病、克里格勒-纳贾尔综合征、囊性纤维化、杜兴氏肌营养不良,法布瑞氏症、家族性腺瘤性息肉病、家族性高胆固醇血症、家族性卵磷脂-胆固醇乙酰转移酶缺乏症、范可尼贫血、半乳糖唾液酸贮积症、戈谢病、回旋状萎缩、血友病A、血友病B、Hurler综合征(粘多糖症I型)、Hunter综合征(粘多糖症II型)、亨廷顿舞蹈病、结合性大疱性表皮松解、婴儿晚发型神经元蜡样脂褐质沉积症、白细胞粘附缺陷病、肢带肌营养不良、脂蛋白脂肪酶缺乏症、异染性脑白质营养不良、Sly综合征(粘多糖症VII型)、Netherton综合征、鸟氨酸转氨甲酰酶缺乏症、庞贝氏病、嘌呤核苷磷酸化酶缺乏症、隐性营养不良性大疱性表皮松解症、sanfilippoA(粘多糖症IIIA型)、sanfilippoB(粘多糖症IIIB型)、镰状细胞病、重度联合免疫缺陷病、脊髓性肌萎缩、戴-萨克斯氏病、维斯科特-奥尔德里奇综合征、冯吉尔克症(糖原贮积病Ia型)、X连锁肌小管性肌病、终末期肾病贫血、心绞痛(稳定型、不稳定型、难治型)、冠状动脉狭窄、严重肢体缺血、心力衰竭、间歇性跛行、心肌缺血、外周血管疾病、肺动脉高血压、静脉溃疡、腺病毒感染、巨细胞病毒感染、Epstein-Barr病毒感染、乙型肝炎感染、丙型肝炎感染、HIV/AIDS、流感、乙型脑炎、疟疾、儿科呼吸系统疾病、呼吸道合胞病毒、破伤风、肺结核、妇科癌症、乳腺癌、卵巢癌、宫颈癌、外阴癌、神经系统癌症、恶性胶质瘤、软脑膜癌病、神经胶质瘤、星形细胞瘤、成神经细胞瘤、视网膜母细胞瘤、胃肠癌、结肠癌、结直肠癌、肝转移、肝炎后肝癌、胰腺癌、胆囊癌、肝细胞癌、泌尿生殖系统癌、前列腺癌、肾癌、膀胱癌、肛门生殖器肿瘤、皮肤癌、黑色素瘤(恶性/转移性)、头颈癌、鼻咽癌、鳞状细胞癌、食道癌、肺癌、腺癌、小细胞/非小细胞、间皮瘤、血液癌、白血病、淋巴瘤、多发性骨髓瘤、肉瘤、生殖细胞癌、李-佛美尼综合征、甲状腺癌、阿尔茨海默病、肌萎缩性脊髓侧索硬化症、腕管综合征、慢性创伤性脑损伤、肘管综合征、糖尿病性神经病、癫痫、巨轴索神经病、婴儿晚发型神经元蜡样脂褐质沉积症、多发性硬化、重症肌无力、疼痛、帕金森病、周围神经病变、脊髓性肌萎缩2型、全色盲、年龄相关性黄斑变性、无脉络膜症、糖尿病性黄斑水肿、青光眼、莱伯先天性黑朦症、黄斑毛细血管扩张2型、色素性视网膜炎、浅表性角膜混浊、遗传性视网膜劈裂症、关节炎(类风湿、炎性、退行性)、变性关节病、直肠的严重性炎症性疾病、溃疡性结肠炎、慢性肾病、糖尿病性溃疡、足溃疡、逼尿肌过度活动、勃起功能障碍、骨折、听力损失、遗传性包涵体肌病、移植物抗宿主疾病/移植患者、口腔粘膜炎、腮腺唾液功能减退、系统性硬皮病、I型糖尿病以及创伤愈合,或其组合。
进一步优选的是用于治疗疾病的方法,其包括向需要的受试者施用根据本发明所述的AAV颗粒,其中所述AAV颗粒以药物组合物形式(例如与药学上可接受的添加剂、载体、稀释剂、溶剂、过滤剂、润滑剂、赋形剂、粘合剂或稳定剂组合)施用于所述受试者或细胞。优选地,所述组合物以喷雾、包衣、泡沫、洗剂、凝胶、漱口水、口服制剂或注射剂形式施用于所述受试者。所述组合物可以全身、口服或通过任何其他临床/医学上可接受的方法施用于所述受试者。
然后,本发明的另一个方面涉及一种药物组合物,其包含根据本发明所述的AAV颗粒以及至少一种药学上可接受的载体和/或稀释剂,即与药学上可接受的添加剂、载体、稀释剂、溶剂、过滤剂、润滑剂、赋形剂、粘合剂或稳定剂组合。优选地,所述组合物以喷雾、包衣、泡沫、洗剂、凝胶、漱口水、口服制剂或注射剂形式施用于所述受试者。所述组合物可以全身、口服或通过任何其他临床/医学上可接受的方法施用于所述受试者。
然后,本发明的另一个方面涉及一种试剂盒,其包含:
a)根据本发明所公开和/或使用的AAV颗粒,或包含根据本发明所公开的AAV颗粒的药物组合物,
b)向所述目标应用所述AAV颗粒或所述药物组合物的书面说明;和
任选地,保存使用的AAV颗粒或组合物的容器以及书面说明。
本发明的另一个方面涉及上述试剂盒在需要所述治疗的受试者中预防、治疗和/或抑制病毒感染的用途。
本发明的又另一个方面涉及根据本发明所述的AAV颗粒,其用于细胞转染,例如在研究中作为基因递送工具。所述用途也可用于美容目的,并且本发明包括类似于本文公开的医学治疗的用于美容治疗的方法。为此,向受试者或细胞施用根据本发明所述的AAV颗粒还可以化妆品组合物形式实现,例如与化妆品上安全且可接受的添加剂、载体、稀释剂、溶剂、过滤剂、润滑剂、赋形剂、粘合剂或稳定剂组合。优选地,所述组合物以喷雾、包衣、泡沫、洗剂、凝胶、漱口水、口服制剂或注射剂形式施用于所述受试者。所述组合物可以全身、口服或通过任何其他临床/医学上可接受的方法施用于所述受试者。
本领域技术人员知道用于在体外和在体内转移基因的使用源自AAV的载体的方法,例如WO93/09239、US4797368、US 5139941和EP 488 528中描述的那些。
本发明的另一个方面涉及一种试剂盒,其包含:
a)用于细胞转染的AAV颗粒,
b)使用AAV颗粒用于细胞转染的书面说明;和
任选地,保存AAV颗粒的容器以及书面说明。
本发明的每个方面的优选特征加以必要的修改也可用于其他方面的每个方面。本文提及的现有技术文件以法律允许的最大范围并入。尽管已经详细描述了本发明及其优势,但是应当理解,在不偏离所附权利要求限定的本发明的精神和范围的情况下,可以进行各种改变、替换和变更。
本发明将在以下实施例和附图中进一步阐明,这些实施例和附图仅用于说明目的,并不旨在以任何方式限制本发明。对于本发明的目的,本文引用的所有参考文献通过全文引用方式并入本文。
图1显示SNAP标记的配体与具有BG-修饰的病毒的示意图。
图2显示根据本发明所述的△HSPG病毒颗粒不再具有感染活性(深色图);在荧光报告基因小鼠模型中的感觉神经元上测试构建体。小图显示了细胞的相衬显微镜图像。
图3显示当在类似于图2的荧光报告基因小鼠模型中的感觉神经元上测试时,麦胚凝集素(WGA)融合体将病毒转导效率完全恢复至100%(荧光细胞)。
图4显示神经营养因子NGF(A)、NT3(B)和BDNF(C)将病毒递送至不同的神经元群体。小图显示细胞的显微镜图像,在类似于图2的荧光报告基因小鼠模型中的感觉神经元上测试构建体。
图5显示当注射入皮肤时,霍乱毒素B亚单位将病毒逆向转运至神经元细胞体。小图显示细胞的显微镜图像,在类似于图2的荧光报告基因小鼠模型中的感觉神经元上测试构建体。
图6显示注射根据本发明的具有NGF配体IV的病毒三周后(A),用抗TrkA(NGF的受体,B)的抗体染色的三叉神经节中的感觉神经元组织。(C)中可看到至少80%的重叠。
图7显示用针对NF200和IB4的抗体对图6的切片进行染色,其主要标记其他神经元(分别机械性感受器(绿色/灰色)和非肽能伤害性感受器(蓝色/深灰色))。红色(浅灰色)的感染细胞主要不同于绿色和蓝色细胞。
图8显示用配体化病毒进行的基因递送更有效。WGA修饰的构建体导致递送的大幅增多(B)。A)正常AAV9变体PHP.S;B)用WGA修饰的A)的PHP.S变体。
实施例
在本发明的背景下进行的实验的目的是改造腺相关病毒(AAV)衣壳,以使病毒仅转导目标细胞。这可通过去除天然AAV衣壳蛋白中对于细胞的天然结合位点实现。经修饰的病毒随后被适当地(特别是化学地)修饰以接受选择性控制的配体连接。然后这些所需的配体共价连接至病毒,并在细胞上进行体外试验以及在小鼠中进行体内试验。尽管使用AAV2,但这些实施例也可容易地应用于其他AAV衣壳。
1.AAV2中天然结合位点的去除
AAV2通过精氨酸585和588与硫酸乙酰肝素蛋白聚糖结合。这些位点被突变为丙氨酸以产生删除△HSPG。
质粒pTAV2-0包含源自pAV-2的完整AAV-2基因组,其包括两个末端反向重复序列,克隆至pBluescript II的BamHI位点中。产生含有AAV-2的合适片段的亚质粒,并将其用作定点诱变反应的模板。根据制造商的方案,通过使用Stratagene(Amsterdam,TheNetherlands)Quikchange定点诱变试剂盒进行诱变。对于每个突变体,设计两个互补的PCR引物以包含置换的序列,突变的每侧上侧邻15至20个同源碱基对。通过DNA测序鉴定突变质粒。然后,将含有合适突变的片段亚克隆至含有蛋白质其余部分的质粒骨架(例如pTAV2-0)中,并对完整片段测序以检查其他PCR突变。
2.用于接受配体的△HSPG的化学修饰
通常,配体对蛋白质的选择性连接(例如蛋白标记)通过将生物正交基团并入蛋白质中,随后进行化学选择性修饰完成。该方法也被称为“标记-和-修饰(tag-and-modify)”。已经开发了多种生物正交反应,其可被分为:(1)通过羰基的缩合反应,(2)通过叠氮化物的“点击”反应,(3)反电子需求Diels-Alder环加成反应(DAINV)和其他环加成反应,(4)过渡金属催化的偶联和脱笼反应,和(5)半胱氨酸残基处的标记反应。
随后通过病毒与苄基鸟嘌呤NHS酯(SNAP tag底物或BG--NHS)反应将苄基鸟嘌呤(BG)连接至暴露的赖氨酸。为此,在室温下,使用针头将非水性DMSO添加至具有干燥SNAPtag配体BG-NHS的小瓶直至所需的最终浓度(例如20mM)。将待氨基-官能化的蛋白质在溶剂(PBS)中被稀释至所需的最终浓度。混合两种制剂并在室温下孵育180分钟,随后通过使用离心100Kda MWCO过滤单元去除未反应的组分。
3.配体的共价连接
使用该系统有两个步骤:作为融合体的目标蛋白的克隆和表达,以及使用选择的SNAP-tag底物标记融合体。SNAP-tag是基于人类O6-烷基鸟嘌呤-DNA-烷基转移酶(hAGT)的小蛋白,是一种DNA修复蛋白。在这种情况下,SNAP-tag底物是连接至苄基接头的鸟嘌呤离去基团。在标记反应中,底物的取代苄基与SNAP-tag共价连接。
SNAP-tag蛋白标记系统能够将几乎任何分子特异性地共价连接至目标蛋白(对于本发明,参见下文4.)。
在悬浮培养物的大肠杆菌或哺乳动物细胞中产生具有C端SNAP标签的重组配体。对于共价连接,然后通过添加饱和浓度的配体并在室温下孵育过夜,然后SNAP-标记的配体被连接至BG-修饰的病毒(参见图1)。使反应通过离心100Kda MWCO过滤单元来去除过量的未反应配体。
4.体外和体内试验
在本发明的背景下,用多个种类的配体(即蛋白质配体,例如生长因子或细胞因子;毒素亚单位,例如霍乱毒素B亚单位;凝集素,例如同工凝集素B4或麦胚凝集素;粘附因子,例如乳凝集素;抗体,例如抗CD-34抗体(干细胞标志物);和肽,例如δ啡肽阿片受体配体)测试上述策略。
首先显示根据本发明所述的△HSPG病毒颗粒在荧光报告基因小鼠模型中的感觉神经元上测试时不再具有感染活性(图2)。当在相同的荧光报告基因小鼠模型中的感觉神经元上测试时,麦胚凝集素(WGA,凝集素)融合体将病毒转导效率完全恢复至100%(图3)。
然后,测试几种因子,依据荧光报告基因小鼠模型中所用的因子,神经营养因子NGF、NT3和BDNF(蛋白质配体)将病毒递送至不同的特定神经元群体(图4)。霍乱毒素B亚单位(毒素)特异性地将病毒逆向引导至神经元细胞体(即细胞区室/部分特异性的)(图5)。在相似的试验中,乳凝集素(粘附因子)特异性地将病毒引导至暴露磷脂酰丝氨酸的巨噬细胞和神经元,而δ啡肽(肽)特异性地将病毒引导至表达Mu和δ阿片受体的神经元。
在图6所示实验中,将具有NGF配体IV的病毒注射至三叉神经节中,然后在三周后获取感觉神经元组织并分析。用抗TrkA(NGF的受体)的抗体染色切片的组织,发现非常好的重叠。TrkA抗体不是完美的,因此80%重叠是非常有意义的。
在图7所示实验中,来自图6的切片用抗NF200和IB4的抗体染色,所述抗体标记其他神经元(分别机械性感受器和非肽能伤害感受器)。同样,这些标志物并不完美,但可以看出绿色和蓝色细胞不同于红色的感染细胞。
作为阴性对照,将IL31配体病毒引入IL31受体敲除小鼠中不导致感染。
综上所述,当在体外应用于培养的细胞时和当在小鼠中体内注射时,所有配体标记的病毒均成功地且特异性地转导了仅表达相应受体的细胞,即可全身或局部注射且选择性地靶向不同的细胞群。
使用配体化-AAV的其他体内试验
A)靶向TrkA+伤害感受器
在此实施例中,使用配体化AAV靶向周围神经系统中的TrkA+伤害感受器。结合但不激活TrkA的NGFR121W配体缀合于如上所述的具有tdTomato货物的△HSPG-AAV2。该构建体被皮下、神经内、眼眶后或腹膜内注射至小鼠中。三周后,使用TrkA抗体检测并定量荧光。
由此发现,对于眼眶后应用,80%的TrkA+细胞被NGF-AAV感染。83%的NGF-AAV感染细胞是TrkA+。还发现不同的施用途径在其高效结果方面没有显著差异。
B)靶向IL31RA+痒感感受器
在此实施例中,使用配体化AAV靶向IL31RA。结合但不激活IL31RA的IL31K134A配体缀合于如上所述的具有tdTomato货物的△HSPG-AAV2。将构建体注射至野生型和IL31RA敲除小鼠中。三周后,通过使用角蛋白14抗体检测荧光。由此发现靶向的细胞对K14基本上完全阳性。重要的是在IL31RA敲除小鼠中没有检测到荧光。
C)使用具有同工凝集素B4的AAV的靶向
在此实施例中,同工凝集素B4(IB4)缀合于如上所述的具有tdTomato货物的△HSPG-AAV2。IB4可用作脉管系统、非肽能伤害感受器和/或小神经胶质细胞的标志物。该构建体被皮下、神经内或脊柱内注射。三周后,检测荧光。由此发现不论施用途径如何,靶向的细胞基本上是完全阳性的。
D)使用具有麦胚凝集素的AAV的靶向
在此实施例中,麦胚凝集素(WGA)缀合于如上所述的具有tdTomato货物的△HSPG-AAV2。WGA与N-乙酰葡糖胺和大多数神经元的膜结合并被用作(跨突触)示踪物。将构建体注射至小鼠中,P1新生小鼠静脉内注射,或成年小鼠皮质内注射。三周后,检测荧光。
由此发现,使用配体化病毒的基因递送更有效(参见图8)。培养的DRG神经元被AAV9变体PHP.S(1E+9VG)感染,并且发现上述WGA修饰的构建体导致递送的大幅增加(参见图8B)。
Claims (16)
1.腺相关病毒(AAV)颗粒,其包含经修饰的衣壳,其中所述经修饰的衣壳包含选自所述衣壳中天然结合位点的去除和配体结合位点向所述衣壳的引入的至少一种修饰。
2.根据权利要求1所述的AAV颗粒,其中所述AAV选自AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11和AAV12。
3.根据权利要求1或2所述的AAV颗粒,其中所述衣壳中的至少一种蛋白质被修饰,优选地,其中所述至少一种蛋白质是VP1、VP2和/或VP3。
4.根据权利要求1至3中任一项所述的AAV颗粒,其中所述衣壳的所述修饰包括至少一个天然结合位点的去除和至少一个配体结合位点的引入。
5.根据权利要求1至4中任一项所述的AAV颗粒,其中所述天然结合位点是能够与硫酸乙酰肝素蛋白聚糖结合的天然结合位点,并且优选地通过用不同的氨基酸例如丙氨酸替换VP1的精氨酸585和588中的至少一个和/或VP2或VP3中类似的精氨酸而去除。
6.根据权利要求1至5中任一项所述的AAV颗粒,其中所述配体结合位点是适合于配体的共价连接的配体结合位点,并且优选地选自苄基鸟嘌呤基团、苄基胞嘧啶基团、氯代烷烃基团、叠氮基团、二苯并环辛炔基团、膦或其组合。
7.根据权利要求6所述的AAV颗粒,其进一步包含与所述苄基鸟嘌呤基团、所述苄基胞嘧啶基团、所述氯代烷烃基团、所述叠氮基团、所述二苯并环辛炔基团和/或所述膦,特别是HaloTagTM、SNAP-tagTM或CLIP-tagTM、叠氮基、二苯并环辛炔基团或膦连接的配体。
8.根据权利要求6或7所述的AAV颗粒,其中所述配体选自蛋白配体、毒素亚单位、凝集素、粘附因子、抗体、肽和基因编辑核酸酶。
9.一种产生改进的腺相关病毒(AAV)颗粒的方法,其包括以下步骤:向所述AAV的衣壳中引入至少一个修饰,其中所述衣壳中天然结合位点被去除,和/或向所述衣壳中引入至少一个配体结合位点。
10.根据权利要求9所述的方法,其中所述天然结合位点是能够与硫酸乙酰肝素蛋白聚糖结合的天然结合位点,并且优选地通过用不同的氨基酸例如丙氨酸替换VP1的精氨酸585和588中的至少一个和/或VP2或VP3中类似的精氨酸来去除。
11.根据权利要求9或10所述的方法,其中所述配体结合位点是适合于配体共价连接的配体结合位点,并且优选地选自苄基鸟嘌呤基团、苄基胞嘧啶基团、氯代烷烃基团、叠氮基团、二苯并环辛炔基团、膦或其组合。
12.一种药物组合物,其包含根据权利要求1至8中任一项所述的AAV颗粒,以及至少一种药学上可接受的载体和/或稀释剂。
13.根据权利要求1至8中任一项所述的AAV颗粒或根据权利要求12所述的药物组合物,其用于疾病的治疗,其中所述疾病优选是可通过基因疗法治疗的疾病,例如癌症、单基因遗传病、遗传性皮肤病、传染病、I型糖尿病和创伤愈合。
14.一种治疗可通过基因疗法治疗的疾病的方法,其包括向需要的受试者施用根据权利要求1至8中任一项所述的AAV颗粒或根据权利要求12所述的药物组合物,其中所述疾病优选是可通过基因疗法治疗的疾病,如癌症、单基因遗传病、遗传性皮肤病、传染病、I型糖尿病和创伤愈合。
15.根据权利要求1至8中任一项所述的AAV颗粒用于细胞转染的用途,例如作为基因递送工具。
16.根据权利要求15所述的AAV颗粒的用途,其中所述用途是用于美容目的。
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