CN114591921B - 腺相关病毒的纯化方法和洗脱液 - Google Patents
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Abstract
本发明涉及腺相关病毒(AAV)的纯化方法和亲和柱洗脱液、由该纯化方法获得的AAV及其应用。本发明的亲和柱洗脱液包含精氨酸和镁离子。本发明的纯化方法不仅提高了AAV的产量,还改变了AAV的靶器官递送能力,提高了AAV的肝脏转导特异性。
Description
技术领域
本公开涉及腺相关病毒(AAV)的纯化方法和亲和柱洗脱液、由该纯化方法获得的AAV及其应用。
背景技术
近年来,腺相关病毒(AAV)载体在基因治疗中的应用引起了极大的关注。然而,野生型AAV血清型(例如AAV2、AAV5、AAV8和AAV9)由于组织特异性不佳,会感染哺乳动物的多个组织/器官。为了改善AAV的器官和组织靶向性,我们已构建了一种具有视网膜和肌肉高亲和性的血清型突变体AAVz2(图1A和图1B,SEQ ID NO:1,描述于CN 113121652 A)。此外,利用AAV载体进行的肝脏靶向基因治疗,已在A型血友病和B型血友病的多项临床试验中取得了令人瞩目的成功(参考文献1)。在这些情况下,中等剂量的AAV给药足以安全地恢复血液凝固。然而,将AAV更广泛地应用于其他疾病可能需要更高效的肝细胞基因递送能力和更少的组织/器官脱靶转导。因此,开发具有优异肝细胞靶向性的AAV血清型是一个有前景的方向。
此外,针对AAV病毒的预先存在的中和抗体(Nab)也是其应用在更广泛的人群中的最大障碍之一。循环中的Nab能够屏蔽暴露在衣壳蛋白表面的受体结合位点并阻断AAV进入靶细胞或通过免疫系统加速AAV的耗竭,使AAV载体的治疗效果无效。尽管已有报道在原代人类肝细胞和人类肝细胞的异种移植小鼠模型中,工程化AAV病毒已经改善了肝脏转导,但预先存在的Nab显著降低了这些AAV血清型的活性(参考文献2)。
目前已开发了许多AAV的纯化方法,其中亲和层析、离子交换层析和分子排阻层析等层析技术已广泛应用于各种血清型AAV载体的分离纯化。在亲和层析中,洗脱条件如洗脱液的选择对于AAV的纯化至关重要。
参考文献:
1.Leebeek FWG,Miesbach W.Gene therapy for hemophilia:A review onclinical benefit,limitations,and remaining issues.Blood.2021;138:923-931
2.Perocheau DP,Cunningham S,Lee J,Antinao Diaz J,Waddington SN,Gilmour K,Eaglestone S,Lisowski L,Thrasher AJ,Alexander IE,Gissen P,BaruteauJ.Age-related seroprevalence of antibodies against aav-lk03 in a ukpopulation cohort.Human gene therapy.2019;30:79-87
发明内容
发明人出乎意料地发现,在AAV的纯化中,通过在亲和柱洗脱液中添加一定浓度的精氨酸和镁离子,不仅可以提高AAV的产量,还可以改变AAV的靶器官递送能力,提高AAV的肝脏靶向性。
因此,在第一方面,本公开提供一种纯化腺相关病毒(AAV)的方法,其包括:(a)将含有AAV的溶液加样到亲和层析柱上;(b)用包含精氨酸和镁离子的第一洗脱液从亲和层析柱上洗脱AAV,收集含AAV的洗脱液;以及(c)对步骤(b)获得的含AAV的洗脱液进行超速离心。
在一个实施方式中,与用不含精氨酸和镁离子的洗脱液进行洗脱步骤的方法相比,本公开的方法使AAV的产量提高约10%、约20%、约30%、约40%,甚至约50%或更高。
在一个实施方式中,在第一洗脱液中,精氨酸的浓度为200mM至2000mM,优选400mM至1000mM,更优选500mM至800mM;和/或镁离子的浓度为0.5mM至3mM,优选1mM至3mM,更优选1.5mM至2.5mM。
在一个实施方式中,镁离子由MgCl2或MgSO4提供。
在一个实施方式中,第一洗脱液包含:0.1-2M AcOH、200-2000mM精氨酸、137-600mM NaCl或Na2SO4、0.5-3mM MgCl2或MgSO4,以及0.05%泊洛沙姆188。
在一个实施方式中,第一洗脱液的pH为2.5~3.2。当第一洗脱液的pH在该范围内时,可以更好地实现纯化效果。
在一个实施方式中,步骤(b)中还使用包含尿素和镁离子的第二洗脱液进行AAV的洗脱。
在一个实施方式中,镁离子由MgCl2或MgSO4提供。
在一个实施方式中,第二洗脱液包含2-6M尿素和0.5-3mM MgCl2。
在一个实施方式中,超速离心为碘克沙醇密度梯度超速离心或氯化铯密度梯度超速离心。
在一个实施方式中,AAV是真核细胞或原核细胞包装生产的AAV。
在一个实施方式中,AAV是三质粒转染法或昆虫杆状病毒法包装生产的AAV。
在一个实施方式中,AAV是野生型AAV。
在一个实施方式中,AAV是AAVz2。
本公开的纯化方法不仅提高了AAV的产量,还提高了AAV在体内和体外对于肝脏细胞的转导效率,但对于其他组织器官的转导效率并无明显提高,使得肝脏转导特异性显著改善。
在第二方面,本公开提供由根据第一方面所述的方法获得的AAV。
在一个实施方式中,AAV是野生型AAV。
在一个实施方式中,AAV是AAVz2。
在一个实施方式中,与用不含精氨酸和镁离子的洗脱液进行洗脱步骤获得的AAV相比,通过本公开的方法纯化的AAV具有改善的肝脏靶向性。
本公开的AAV具有优异的肝脏特异靶向性,可应用于肝脏相关疾病或需要递送药物至肝脏的其他疾病的预防和治疗,且低脱靶(非靶器官)效应和低免疫原性增强了其安全性潜力。
在第三方面,本公开提供根据第二方面所述的AAV在制备用于治疗疾病的药物中的应用,其中,所述疾病为肝脏疾病或需要递送基因至肝脏中表达的其他疾病。
在第四方面,本公开提供一种药物,其包含:根据第三方面所述的AAV和赋形剂。
在一个实施方式中,药物用于肝脏疾病或需要递送基因至肝脏中表达的其他疾病的预防、诊断和治疗。
在一个实施方式中,药物通过全身途径或局部途径施用,优选静脉内施用、口服施用、鼻腔内施用、翘内施用、肌肉内施用、皮下施用、腹膜内施用或病灶内施用;优选通过全身途径或局部途径施用于肝脏。
在一个实施方式中,肝脏疾病包括:原发或继发性肝癌、肝硬化、肝脓肿、脂肪肝、酒精性肝病、肝移植、甲型肝炎、乙型肝炎、丙型肝炎、丁型肝炎、戊型肝炎、自身免疫性肝炎、药物毒性肝炎以及其他肝炎。
在一个实施方式中,需要递送基因至肝脏中表达的其他疾病包括:A型和B型血友病、溶酶体贮积症(例如MPS II和III)、法布里综合征(Fabry’s disease)、糖原贮积症、贝墩氏症(Batten disease)、高歇氏病(Gaucher’s disease)、沃尔曼氏症(Wolman’sdisease)、威尔逊氏症(Wilson’s disease)、自身免疫性疾病(例如多发性硬化症、重症肌无力、风湿或类风湿性关节炎、红斑狼疮、自身免疫性心脏病)、心血管疾病或肺部疾病(例如高血压、动脉粥样硬化、高胆固醇血症、慢性阻塞性肺疾病)、高血氨症、糖尿病、沙费利波综合征、综合性创伤、肾衰竭、贫血。
在第五方面,本公开提供一种亲和柱洗脱液,其包含:精氨酸和镁离子。
在一个实施方式中,亲和柱洗脱液包含200mM至2000mM、优选400mM至1000mM、更优选500mM至800mM的精氨酸和0.5mM至3mM、优选1mM至3mM、更优选1.5mM至2.5mM的镁离子。
在一个实施方式中,亲和柱洗脱液包含:0.1-2M AcOH、200-2000mM精氨酸、137-600mM NaCl或Na2SO4、0.5-3mM MgCl2或MgSO4,以及0.05%泊洛沙姆188。
在一个实施方式中,亲和柱洗脱液的pH为2.5~3.2。当本公开的亲和柱洗脱液的pH在该范围内时,可以更好地实现纯化效果。
本公开的亲和柱洗脱液通过包含精氨酸和镁离子显著提高了AAV的洗脱产量,并且改善了AAV在体内和体外对于肝脏细胞的特异性转导效率。
在第六方面,本公开提供一种纯化腺相关病毒(AAV)的方法,其包括:通过两步的碘克沙醇密度梯度超速离心纯化AAV。
在一个实施方式中,本公开的方法包括:(i)将含有AAV的溶液装载至碘克沙醇密度梯度上方,进行超速离心,得到一次纯化液;以及(ii)将步骤(i)的一次纯化液装载至碘克沙醇密度梯度上方,进行超速离心,得到纯化的AAV。
在一个实施方式中,本公开的方法在步骤(i)之前还包括步骤:使用不同浓度的碘克沙醇,分别形成步骤(i)和步骤(ii)中的碘克沙醇密度梯度。
在一个实施方式中,不同浓度的碘克沙醇包括:15w/v%、25w/v%、40w/v%、60w/v%的碘克沙醇。
在一个实施方式中,本公开的方法不包括亲和柱层析步骤。
在一个实施方式中,AAV是真核细胞或原核细胞包装生产的AAV。
在一个实施方式中,AAV是三质粒转染法或昆虫杆状病毒法包装生产的AAV。
在一个优选实施方式中,AAV为AAVz2。
本公开的纯化方法提高了AAV在体内和体外对于肝脏细胞的转导效率,但对于其他组织器官的转导效率并无明显提高,使得肝脏转导特异性显著改善。
因此,在第七方面,本公开提供由根据第六方面所述的方法获得的AAV。
在一个优选实施方式中,AAV为AAVz2。
与用亲和柱层析加一步的碘克沙醇超速离心的纯化方法获得的AAV相比,通过本公开的方法纯化的AAV具有改善的肝脏靶向性。
通过本公开的上述纯化方法纯化得到的AAV载体具有优异的肝脏特异靶向性,以及更低的中和抗体水平,体现了更低的免疫原性和更高的安全性。具有优异的肝脏特异靶向性的本公开的AAV载体可应用于肝脏相关疾病或需要递送药物至肝脏的其他疾病的预防和治疗,且低脱靶(非靶器官)效应和低免疫原性增强了其安全性潜力。
在第八方面,本公开提供根据第七方面所述的AAV在制备用于治疗疾病的药物中的应用,其中,所述疾病为肝脏疾病或需要递送基因至肝脏中表达的其他疾病。
在第九方面,本公开提供一种药物,其包含:根据第七方面所述的AAV和赋形剂。
在一个实施方式中,药物用于肝脏疾病或需要递送基因至肝脏中表达的其他疾病的预防、诊断和治疗。
在一个实施方式中,药物通过全身途径或局部途径施用,优选静脉内施用、口服施用、鼻腔内施用、翘内施用、肌肉内施用、皮下施用、腹膜内施用或病灶内施用;优选通过全身途径或局部途径施用于肝脏。
在一个实施方式中,肝脏疾病包括:原发或继发性肝癌、肝硬化、肝脓肿、脂肪肝、酒精性肝病、肝移植、甲型肝炎、乙型肝炎、丙型肝炎、丁型肝炎、戊型肝炎、自身免疫性肝炎、药物毒性肝炎以及其他肝炎。
在一个实施方式中,需要递送基因至肝脏中表达的其他疾病包括:A型和B型血友病、溶酶体贮积症(例如MPS II和III)、法布里综合征(Fabry’s disease)、糖原贮积症、贝墩氏症(Batten disease)、高歇氏病(Gaucher’s disease)、沃尔曼氏症(Wolman’sdisease)、威尔逊氏症(Wilson’s disease)、自身免疫性疾病(例如多发性硬化症、重症肌无力、风湿或类风湿性关节炎、红斑狼疮、自身免疫性心脏病)、心血管疾病或肺部疾病(例如高血压、动脉粥样硬化、高胆固醇血症、慢性阻塞性肺疾病)、高血氨症、糖尿病、沙费利波综合征、综合性创伤、肾衰竭、贫血。
附图说明
图1A显示了AAVz2衣壳蛋白是通过在AAV5衣壳蛋白可变区VIII上的Q574之后插入寡肽“FAPTPGP”而构建的。
图1B显示了通过Chimera 1.15(UCSF)构建AAVz2的VP1蛋白的3D结构的PDB图。插入的寡肽在矩形框中放大显示。
图1C显示了生产纯化后的包装GFP基因的各AAV颗粒的银染显色,每个AAV血清型有3个平行组泳道。
图1D显示了从2×108个细胞(培养基+裂解物)中生产并用相应纯化方法得到的各AAV产量。AAVz2-0和野生型AAV:亲和柱层析(洗脱液不含精氨酸和镁离子)加一步碘克沙醇超速离心纯化得到;AAVz2-1:两步碘克沙醇超速离心纯化得到;AAVz2-2:亲和柱层析(洗脱液包含精氨酸和镁离子)加一步碘克沙醇超速离心纯化得到。n=3。vg:载体基因组;vp:病毒颗粒数。
图2显示了使用不同洗脱液的亲和柱层析纯化的野生型AAV5产量。洗脱峰A和C1:亲和柱层析(洗脱液A+洗脱液C)加一步碘克沙醇超速离心纯化AAV5,以260和280nm紫外吸收峰为代表。洗脱峰B和C2:亲和柱层析(洗脱液B+洗脱液C)加一步碘克沙醇超速离心纯化AAV5。qPCR定量病毒基因组(vg),银染定量病毒颗粒数(vp)。洗脱液A:1M AcOH、500mMNaCl、0.05%泊洛沙姆188,pH=2.5。洗脱液B:1M AcOH,500mM精氨酸,2mM MgCl2、500mMNaCl、0.05%泊洛沙姆188,pH=2.5;洗脱液C(C1或C2):6M尿素,2mM MgCl2。
图3A显示了不同纯化方法的AAV对小鼠肝脏的感染能力。比例尺=200微米。
图3B显示了基于DAPI斑点数统计的GFP阳性肝细胞数与GFP阳性心脏、肺、脾和肾细胞数的比值,AAV的肝脏感染能力相比于心脏、肺、脾和肾脏感染能力的相对定量。n=5只小鼠/组(3雄2雌)。***p<0.001,单向方差分析。
图4显示了尾静脉注射各AAV的小鼠在肝脏、心脏、肺、脾、肾脏和大脑中的GFP基因的mRNA表达。n=4只小鼠/组。*p<0.05,**p<0.01,***p<0.001,单向方差分析。
图5A显示了不同纯化方法获得的AAV对各种骨骼肌的感染能力。GA:腓肠肌;LO:胸最长肌;QU:股四头肌;TR:肱三头肌;ST:胸锁乳突肌;SO:比目鱼肌。比例尺=200微米。
图5B显示了基于GFP阳性肌肉细胞面积与总肌肉横截面积的百分比,对AAV的肌肉转导效率进行定量的结果。n=5只小鼠/组(3雄2雌),*p<0.05,**p<0.01,***p<0.001。单向方差分析。
图6A显示了人肝癌细胞Huh7和正常肝细胞L02被相应的包装GFP基因的AAV载体感染(MOI=1×105vg/细胞)后48小时的拍摄图像。上图:GFP信号;下图:明场。比例尺:100微米。
图6B显示了由各AAV递送的GFP蛋白的表达水平。
图6C显示了图6A中GFP阳性Huh7和L02细胞百分比的统计结果。以Huh7细胞和L02细胞的GFP阳性信号面积与整个明场面积的比值进行计算。*p<0.05,**p<0.01,***p<0.001,n=6孔细胞,单向方差分析。
图6D显示了图6B中各AAV处理的Huh7和L02细胞中GFP蛋白表达量的统计结果。*p<0.05,***p<0.001,n=4孔细胞,单向方差分析。
具体实施方式
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公开所属领域的普通技术人员的通常理解相同的含义。
在本文中,术语“包含”、“具有”、“包括”和“含有”应被解释为开放式术语(即意味着“包括但不限于”)。
在本文中,术语“治疗”包括:(1)抑制病状、疾病或者病症,即,阻止、减少或者延迟疾病的发展或其复发或者其至少一种临床或者亚临床症状的发展;或者(2)缓解疾病,即,引起病状、疾病或者病症或者其临床或者亚临床症状中的至少一种消退。
在本文中,术语“预防”病状、疾病或者病症包括:预防、延迟或者减少受试者中发展的病状、疾病或者病症的至少一种临床或者亚临床症状出现的发生率和/或可能性,该受试者可能患有或易患该病状、疾病或者病症但尚未经历或者表现出该病状、疾病或者病症的临床或亚临床症状。
在本文中,术语“局部施用”或“局部途径”是指具有局部作用的给药。
在本文中,术语“转导”、“转染”和“转化”是指将外源核酸递送到宿主细胞中,然后多核苷酸产物的转录和翻译的过程,该过程包括使用重组病毒将外源多核苷酸引入宿主细胞。
在本文中,术语“基因送递”指的是将外源多核苷酸引入细胞来进行基因传递,包括靶向、结合、摄取、转运、复制子整合和表达。
在本文中,术语“基因表达”或“表达”是指基因转录、翻译和翻译后修饰产生基因的RNA或蛋白产物的过程。
在本文中,术语“感染”是指包含多核苷酸组分的病毒或病毒颗粒将多核苷酸递送至细胞中并产生其RNA和蛋白质产物的过程,也可指病毒在宿主细胞中的复制过程。
在本文中,术语“靶向”或“特异性”是指病毒优先进入一些细胞或组织,然后进一步在细胞中表达病毒基因组或重组转基因携带的序列。
在本文中,与多核苷酸相关的“重组”意味着该多核苷酸是一个通过多步克隆步骤构建的不同于天然多核苷酸的合成产物。重组病毒是包含重组多核苷酸的病毒颗粒。
在本文中,术语“载体”指封装多核苷酸的一个或一系列大分子,其促进多核苷酸在体外或体内递送至靶细胞。载体的种类包括但不限于质粒、病毒载体、脂质体和其他基因递送载体。待递送的多核苷酸有时被称为“转基因”,包括但不限于某些蛋白质或合成多肽(其可以增强、抑制、削弱、保护、触发或预防某些生物学和生理功能)的编码序列、疫苗开发中感兴趣的编码序列(例如表达适于在哺乳动物中引发免疫应答的蛋白质、多肽或肽的多核苷酸)、RNAi组分(例如,shRNA、siRNA、snRNA、microRNA、核酶、反义寡核苷酸和反义多核苷酸,它们可以敲低以异常方式激活的任何内源基因或侵入宿主细胞的外源基因)或可选的生物标记。病毒或细菌的多核苷酸是本领域已知的。RNAi组分通常与其靶基因在序列上具有60-100%的同一性,并导致相应的蛋白质产物减少至少30%(如30%、40%、50%、60%、70%、80%、90%或100%)。
在本文中,术语“多核苷酸”指的是任意长度的核苷酸的聚合形式,包括脱氧核苷酸,核糖核苷酸,其杂合序列和类似物。多核苷酸可包括修饰的核苷酸,比如甲基化或加帽的核苷酸或核苷酸类似物。本文使用的术语多核苷酸可互换地指单链或双链分子。除非另有说明,本文描述的多核苷酸包括双链的形式和已知的或可预测的构成双链形式的两条互补的单链。
在本文中,术语“多肽”和“蛋白质”在本文中同义地是指由20个以上的氨基酸组成的聚合物。这些术语还涵盖合成或人工氨基酸聚合物。
在本文中,“病毒颗粒”是指病毒的衣壳蛋白包装天然的或人工合成的病毒基因组形成的功能性病毒单位,其功能包括感染或转导组织器官及细胞、将病毒基因组递送至组织器官及细胞内并表达出相应的核酸及蛋白产物。
在本文中,术语“反向末端重复(ITR)”包括形成发夹结构并用作顺式元件以介导病毒复制、包装和整合的任何AAV病毒末端重复或合成序列。本文的ITR包括但不限于来自1-11型AAV(禽类AAV、牛AAV、犬AAV、马AAV和绵羊AAV的末端重复序列)。此外,AAV末端重复序列不必具有天然末端重复序列,只要该末端重复序列可用于病毒复制、包装和整合即可。
在本文中,术语“腺相关病毒(AAV)”或“腺相关病毒(AAV)血清型”包括天然的AAV(例如,天然的1-11型AAV、禽AAV、牛AAV、犬AAV、马AAV和绵羊AAV的衣壳蛋白)和其他人工改造的AAV。不同AAV血清型的基因组序列、ITR序列、Rep和Cap蛋白在本领域内是已知的。这些序列可以在文献或在公共数据库查找,例如GenBank数据库,如GenBank(R)登录号NC002077、NC 001401、NC 001729、NC 001863、NC 001829、NC 001862、NC 000883、NC 001701、NC 001510、AF063497、U89790、AF043303、AF028705、AF028704、J02275、JO1901、J02275、XO1457、AF288061、AHO09962、AY028226、AY028223、NC 001358、NC 001540、AF513851、AF513852、AY530579、AY631965、AY631966;其内容通过引用整体并入本文;以及例如Srivistava等,J.Virol(1983)45:555;Chiorini等,J.Virol(1998)71:6823;Chiorini等,J.Virol(1999)73:1309;Bantel-Schaal等,J.Virol(1999)73:939;Xiao等,J.Virol(1999)73:3994;Muramatsu等,Virology(1996)221:208;WO 00/28061;WO 99/61601;WO 98/11244;US 6156303。
本发明人出人意料地发现,通过使用特定的纯化方法(两步的碘克沙醇或氯化铯密度梯度离心)或特定的化学试剂(含精氨酸和镁离子的亲和柱洗脱液),可以提高现有的AAV(如AAVz2,描述于CN 113121652 A)的产量和/或肝脏细胞靶向性,改变AAV在全身组织的分布。
在一个实施方式中,本公开的AAV载体可以装载外源多核苷酸用于将基因递送到靶细胞中。本公开的AAV载体可用于在体外或体内将核酸递送至组织器官及细胞中。本公开的AAV载体优先进行肝脏特异性的基因递送而其他器官(如心脏、肺、脾脏、肾脏和脑)的脱靶传递相对较少,意味着对脱靶组织的感染和潜在不良反应的发生率更低。
在一个实施方式中,由AAV载体递送的外源多核苷酸编码充当报告子的多肽(即报告蛋白)。报告蛋白用于指示被AAV成功感染的细胞。这些报告蛋白包括但不限于绿色荧光蛋白(GFP)、β-半乳糖苷酶、碱性磷酸酶、荧光素酶和氯霉素乙酰转移酶。
在一些实施方式中,递送的核酸包括用于治疗性(例如,医学或兽医学)用途的编码天然蛋白质的核酸,所述天然蛋白质经密码子优化或未经密码子优化。这些蛋白由AAV载体递送至肝脏中表达,可分泌至血液循环中并作用于全身其他组织器官,用以防治相关疾病。此类蛋白包括但不限于:囊性纤维化跨膜调节蛋白(CFTR)、肌营养不良蛋白(包括截短的形式,称为微型肌营养不良蛋白或微肌营养不良蛋白,参见例如Vincent等,Nat Genet(1993)5:130;US 2003017131;Wang等,Proc Natl Acad Sci USA(2000)97:13714-9;Harper等,Nat Med(2002)8:253-61);微型凝集素、整联蛋白β1、层粘连蛋白β2、肌聚糖α和β、肌节蛋白、突触核蛋白、促性腺激素、微型促卵磷脂、Lamin A/C、四个半LIM结构域蛋白1(FHL1)、卵泡抑素、SOD1或SOD2、全长或显性负性肌生长抑制素;血管生成因子(例如VEGF,血管生成素1或2)和血管生成抑制剂(例如内皮抑素和血管抑制素);抗炎多肽(例如,抗炎白细胞介素4、10、11和13,全长和显性突变体Ikappa B、Pinch和ILK基因);凝血因子(例如凝血因子VIII,凝血因子IX,凝血因子X);血影蛋白、酪氨酸羟化酶、芳香族L-氨基酸脱羧酶、瘦素和瘦素受体;神经营养因子(例如BDNF、GDNF、NGF、信号量、Slit1、2和3、FGF7、10和22)以及相应的神经营养蛋白受体;LDL受体、脂蛋白脂肪酶、肾上腺素能受体α和β、B球蛋白、C球蛋白;胰岛素样生长因子,例如IGF-1和IGF-2;腺苷脱氨酶和骨形态发生蛋白超家族(例如BMP1、2、4、6、7和TGF-β,RANKL);BDNF、GDNF、NTF、APP、GRIA1和2、atrophin 1、SLC1A1;SMN1、UBE1、DYNC1H1;RPE65、RPGR、Bestrophin-1、CNGA3、CNGB3、视黄质壳素、视网膜特异性磷脂转运ATPase ABCA4和视网膜特异性ABC转运蛋白。
在一些实施方式中,包装在AAV病毒颗粒中的多核苷酸可以编码人工合成的多肽或蛋白质并将其递送于肝脏细胞中表达、分泌并随血液循环作用于全身。这类多肽或蛋白包括但不限于:Aflibercept(由Rengeron Pharmaceuticals生产的具有抗血管生成作用的重组VEGF可溶性受体);重组白介素1、18和TNF-α拮抗可溶性受体;激活素II型可溶性受体;抗体或单链抗体,包括但不限于抗VEGF抗体(例如贝伐单抗,雷珠单抗和Brolucizumab)、抗硬化蛋白抗体(例如Romosozumab和Blosozumab)、抗RANKL抗体(例如Denosumab)、抗补体成分C5抗体(例如Ravulizumab和Eculizumab)、抗PD-1抗体(例如Nivolumab、Pembrolizumab和Cemiplimab)、PD-L1抗体(例如Avelumab和Atezolizumab)、抗CTLA-4抗体(例如Ipilimumab)、抗CGRP抗体(例如Fremanezumab、Galcanezumab和Erenumab)、抗HER2抗体(例如Trastuzumab和Pertuzumab)、抗EGFR抗体(例如Cetuximab、Panitumumab和Necitumumab)、针对促炎细胞因子的抗体及其受体(例如Sarilumab、Siltuximab、Tocilizumab、Canakinumab、Golimumab、Certolizumab、Adalimumab、Infliximab、Daclizumab和Basiliximab);修饰的酶,例如Cethrin(可从BioAxone BioSciences Inc.获得的神经保护药物用于治疗脊髓损伤);可产生疫苗的抗原或抗原片段(例如冠状病毒病2019(COVID 2019)或严重急性呼吸系统综合症(SARS)冠状病毒的刺突蛋白,甲、乙、丙型肝炎和人免疫缺陷病毒(HIV)的包膜蛋白,多种肿瘤细胞免疫原,例如MAGE抗原,HER2,ErbB2,粘蛋白抗原和雌激素受体)。疫苗可以引发保护性免疫反应预防某些疾病的发作。以肌内注射为代表的将抗体或疫苗递送到受试者体内的方法是本领域技术人员已知的。
在一些实施方式中,由AAV载体递送的核酸可编码基因编辑(例如ZFN、TALEN和CRISPR)的相关原件,包括核酸内切酶或重组酶例如Cas9和Cas13,以及对应的sgRNA。
在一些实施方式中,可递送的异源多核苷酸包含调控元件,如转录/翻译控制信号、复制起点、聚腺苷酸化信号、内部核糖体进入位点(IRES)或2A信号(例如P2A、T2A、F2A)、启动子和增强子(例如,例如CMV启动子或具有脊椎动物β-肌动蛋白、β-球蛋白或β-球蛋白调节元件的其他杂CMV启动子、EF1启动子、泛素启动子、T7启动子、SV40启动子、VP16或VP64启动子)。启动子和增强子的使用取决于它们的组织特异性表达谱。启动子和增强子可以被化学药品或激素(例如强力霉素或他莫昔芬)诱导,以确保在特定时间点的进行基因表达。此外,启动子和增强子可以是天然或人工或嵌合序列,即原核或真核序列。
在一些实施例中,用于基因表达的诱导型调控元件可以是组织特异性或组织嗜性启动子/增强子元件。
在一些实施方式中,本公开的AAV载体应用于需要标记特定细胞(如肝脏细胞)的情况,例如研究实验。
在一个实施方式中,本公开的AAV载体包含报告蛋白,其用于指示或标记被病毒成功感染的细胞。
在一些实施方式中,将本公开的AAV病毒颗粒在体外施用给宿主细胞,然后将细胞植入受试者中。因此,包装在AAV中的异源核酸通过细胞引入受试者体内进行转录和/或翻译,产生从细胞分泌到受试者体内或调节宿主生物活性的蛋白质或RNA产物。
本领域技术人员可以使用已知的标准方法来生产重组和合成的多肽或其蛋白质、生产抗体或抗原结合片段、改变核酸序列、生产转化细胞、构建重组AAV突变体、改造衣壳蛋白、包装表达AAV Rep和/或Cap序列的载体,以及瞬时或稳定转染包装细胞。这些技术是本领域技术人员已知的。参见例如,分子克隆(MOLECULAR CLONING):实验室手册(ALABORATORY MANUAL),第二版,(冷泉港,纽约州,1989年);F.M.AUSUBEL等,CURRENTPROTOCOLS IN MOLECULAR BIOLOGY(Green Publishing Associates,Inc.and JohnWiley&Sons,Inc.,纽约)。
在一个实施方式中,本公开的AAV病毒颗粒被制成药物(例如,注射剂、片剂、胶囊剂、散剂)施用于人或其他哺乳动物。该药物还包含其他成分,例如药物辅料、水溶性或有机溶剂(例如水、甘油、乙醇、甲醇、异丙醇、氯仿、苯酚或聚乙二醇)、盐(例如氯化钠、氯化钾、磷酸盐、乙酸盐、碳酸氢盐、Tris-HCl和Tris-乙酸盐)、延缓溶解试剂(例如石蜡)、表面活性剂、抗微生物剂、脂质体、脂质复合物、免疫抑制剂(例如可的松、泼尼松、环孢霉素)、非甾体抗炎药(NSAID,例如阿司匹林、布洛芬、对乙酰氨基酚)微球、硬质基质、半固体载体、纳米球或纳米颗粒。此外,药物可以通过吸入、全身或局部(例如,静脉内、肌肉内、皮下、经口、腹膜内和病灶内)的给药方式以单剂量或多剂量递送。
在一个实施方式中,本公开的药物以105-1014vg/mL的滴度包含本公开的AAV病毒颗粒。
在一个实施方式中,本公开的药物用于预防和/或治疗疾病,如肝脏疾病,包括但不限于原发或继发性肝癌、肝硬化、肝脓肿、脂肪肝、酒精性肝病、肝移植、甲型肝炎、乙型肝炎、丙型肝炎、丁型肝炎、戊型肝炎、自身免疫性肝炎、药物毒性肝炎以及其他肝炎;以及其他与肝脏间接相关或需要递送药物至肝脏的疾病,例如A型和B型血友病、溶酶体贮积症(例如MPS II和III)、法布里综合征(Fabry’s disease)、糖原贮积症、贝墩氏症(Battendisease)、高歇氏病(Gaucher’s disease)、沃尔曼氏症(Wolman’s disease)、威尔逊氏症(Wilson’s disease)、自身免疫性疾病(例如多发性硬化症、重症肌无力、风湿或类风湿性关节炎、红斑狼疮、自身免疫性心脏病)、心血管疾病或肺部疾病(例如高血压、动脉粥样硬化、高胆固醇血症、慢性阻塞性肺疾病)、高血氨症、糖尿病、沙费利波综合征、综合性创伤、肾衰竭、贫血。
在一些实施方式中,AAV的生产采用本领域人员所熟知的三质粒转染法(质粒1:顺式元件质粒;质粒2:AAV Rep/Cap质粒;质粒3:辅助质粒)。
在一些实施方式中,本公开还提供从细胞生产AAV的方法。这些细胞支持高效转染编码AAV Rep/Cap蛋白、辅助基因和编码天然的病毒基因组或异源蛋白的病毒载体的质粒。
下面结合附图和实施例对本公开作进一步详细的说明。以下实施例仅用于说明本公开而不用于限制本公开的范围。实施例中未注明具体条件的实验方法,系按照本领域已知的常规条件,或按照制造厂商所建议的条件进行操作。
实施例
实施例1.AAV的纯化
通过如CN 113121652 A中所述的亲和柱层析加一步碘克沙醇超速离心的方法,对野生型AAV(例如AAV2、AAV5、AAV8和AAV9)进行纯化(野生型AAV如无特殊说明均采用此纯化方法),具体如下:
1)预处理步骤:在三质粒转染48小时后,收取细胞和上清液,加入0.1%的TritonX-100后,静置30分钟裂解细胞。然后加入5mM杜灭芬(domiphen bromide),静置30分钟沉淀核酸。然后,加入300mM NaCl调节电导率。在9000rpm离心30分钟。取离心后的上清液,浓缩至500mL。
2)亲和柱层析步骤:将浓缩的上清液装载至亲和层析柱上(AAVX树脂,ThermoScientific),用平衡液(20mM Tris-HCl、500mM NaCl、0.05%泊洛沙姆188,pH=7.2)润洗。平衡亲和层析10个柱体积后,进行病毒样品的上样。病毒样品全部上样完成后,用平衡液进行后平衡。平衡10个柱体积后,用洗脱液A(1M AcOH、500mM NaCl、0.05%泊洛沙姆188,pH=2.5)洗脱病毒,收集洗脱液并调节pH至约7。
3)碘克沙醇超速离心步骤:将前一步骤所得溶液装载至15%、25%、40%、60%碘克沙醇密度梯度上方并在65000rpm离心2.5h,收集6mL病毒溶液。
4)浓缩步骤:将前一步骤所得溶液用病毒稀释液(PBS+300mM NaCl+0.05%泊洛沙姆188)浓缩至3mL。
通过与上述野生型AAV相同的纯化步骤,对AAVz2进行纯化,得到AAVz2-0。
通过与上述野生型AAV相同的纯化步骤,对AAVz2进行纯化,得到AAVz2-1,不同之处在于将2)亲和柱层析步骤替换为与3)碘克沙醇超速离心步骤相同的步骤。
通过与上述野生型AAV相同的纯化步骤,对AAVz2进行纯化,得到AAVz2-2,不同之处在于将2)亲和柱层析步骤中使用的洗脱液A替换为洗脱液B(1M AcOH、500mM精氨酸、2mMMgCl2、500mM NaCl、0.05%泊洛沙姆188,pH=2.5)+洗脱液C(6M尿素,2mM MgCl2)。
接着,通过qPCR和银染,对采用不同纯化方法获得的AAV的病毒产量进行定量。
结果显示,用非亲和层析的两步碘克沙醇超速离心方法得到的AAVz2-1的病毒基因组数目和病毒颗粒数与野生型AAV5、8、9以及AAVz2-0相当,且远高于野生型AAV2。出人意料的是,采用了特定洗脱液的AAVz2-2的产量与其他AAV相比显著增加(提高了约50%)(图1C和图1D)。
为了验证洗脱液中精氨酸和镁离子的添加对病毒纯化的影响,分别使用洗脱液A+洗脱液C以及洗脱液B+洗脱液C,采用上述相同步骤对野生型AAV5进行纯化,获得AAV5-2。结果显示,洗脱液中精氨酸和镁离子的添加提高了病毒的产量(图2)。
此外,发明人发现,在200-2000mM的范围内调整洗脱液B中的精氨酸浓度或在0.5-3mM的范围内调整洗脱液B中的MgCl2浓度时,所获得的AAVz2均具有与AAVz2-2基本相同的特性。
实施例2.纯化的AAV对小鼠肝脏的转导效率
为了研究实施例1中不同纯化方法获得的AAV的体内转导趋向,C57BL/6小鼠通过尾静脉注射1×1013vg/kg剂量的包装有GFP编码基因的各种AAV血清型载体,在病毒注射后3周后对肝脏、心脏、肺、脾和肾脏进行GFP和DAPI免疫荧光染色。
结果显示,AAVz2-1和AAVz2-2注射组中分别有高达92.25%和91.8%的肝细胞呈GFP阳性,显著优于AAV5注射组(仅22.64%),也优于AAV8(85.37%)和AAV9(88.11%)注射组(图3A)。并且,与AAV8和AAV9在心脏中也表现出较高转导效率以及对肺、脾和肾的轻度至中度感染性不同,AAVz2-1和AAVz2-2几乎不感染这些组织器官。上述结果表明,AAVz2-1和AAVz2-2具有特异性转导肝脏的能力。
对GFP信号进行定量,通过肝脏中GFP阳性细胞与其他组织/器官(心脏、肺、脾和肾脏)的比值来量化AAV的肝脏转导特异性。如图3B所示,AAVz2-1和AAVz2-2的肝脏相比于心脏、肺、脾和肾脏的相对特异性比AAV5、AAV8、AAV9和AAVz2-0高约4-11倍。这些结果再次证明了AAVz2-1和AAVz2-2的高肝脏靶向能力。
接着,测试了尾静脉注射各AAV的小鼠组织和器官中的GFP基因表达。C57BL/6小鼠通过尾静脉注射包装了GFP转基因的相应AAV血清型,剂量为1×1013vg/kg。对肝脏、心脏、肺、脾、肾脏和大脑组织匀浆进行RT-PCR以检测AAV5、AAV8、AAV9和各种方法纯化的AAVz2所递送的GFP基因mRNA的表达,结果呈现为各组GFP的mRNA相比于AAV5组的相对值。
结果显示,AAVz2-1和AAVz2-2在肝脏中递送GFP的mRNA水平是AAV5的14.7±5.7倍,与AAV8和AAV9相当(图4)。并且,AAVz2-1和AAVz2-2在小鼠的心脏、肺、脾、肾脏和脑中的GFP表达水平与AAV5相似或低于AAV5,且显著低于AAV8和AAV9。此外,AAVz2-0在小鼠的肝脏、心脏、肺、脾、肾和脑中递送GFP的mRNA水平和AAV5接近。
上述结果表明,本公开的纯化方法可提高AAV对肝脏的转导特异性。
实施例3.纯化的AAV对小鼠骨骼肌的转导效率
为了研究实施例1中不同纯化方法获得的AAV对骨骼肌的感染能力,用相对高剂量(5×1013vg/kg)的各AAV(包装GFP基因)尾静脉注射两种性别的C57BL/6小鼠。病毒注射后21天,用GFP对身体多个区域的肌肉,包括后肢(腓肠肌、股四头肌、比目鱼肌)、背部(胸最长肌)、前肢(肱三头肌)和颈部(胸锁乳突肌)进行免疫荧光染色。
如图5A和图5B所示,AAV9显示出对骨骼肌的最高转导效率:约60%的腓肠肌、股四头肌和三头肌细胞,30-40%的比目鱼肌和胸最长肌细胞成功表达GFP;AAV8排在第二位,约15-40%的腓肠肌、股四头肌和最长肌细胞被有效转导;在AAV5注射组中,GFP阳性的股四头肌细胞也达到了19.19%。相比之下,AAVz2-1和AAVz2-2所感染的肌纤维数量可忽略不计,表明它们在体内几乎不转导骨骼肌细胞。这些结果进一步确认了AAVz2-1和AAVz2-2在静脉注射全身给药条件下的肝脏转导特异性。
实施例4.纯化的AAV对人源肝脏细胞系的转导效率
为了进一步研究实施例1中不同纯化方法获得的AAV在人源肝细胞中的转导效率,发明人选择了人类肝细胞癌细胞系Huh7和正常肝细胞系L02,这些细胞系经常应用于肝脏药物递送的研究。
结果显示,在感染复数(MOI)为1×105vg/细胞的情况下,AAVz2-1和AAVz2-2的GFP阳性细胞比例(即GFP信号面积与总细胞面积之比)显著高于AAVz2-0和AAV5(Huh7细胞为21.51倍,L02细胞为20.83倍),且略高于AAV8和AAV9(图6A和图6C)。
用Western blot检测Huh7和L02细胞裂解液中由AAV递送的GFP蛋白的表达,以微管蛋白作为内参。Western blot实验显示,AAVz2-1和AAVz2-2的GFP蛋白表达量显著高于AAVz2-0和AAV5(图6B和图6D)。
上述结果表明,本公开的纯化方法可有效改善AAV对人源肝脏细胞系的感染效率。
实施例5.AAV的中和抗体水平
血清中预先存在的中和抗体(Nab)可能导致用于基因治疗的AAV载体失活。在野生型AAV中,AAV5由于体循环Nab水平最低而具有显著优势,其在某些人群中的Nab阳性率仅为3%。
血友病是一种需要通过AAV载体将凝血因子IX基因递送至肝脏进行治疗的代表性疾病。发明人收集了10名B型血友病患者的血清样本,以1:2的比例梯度稀释,并与指定的编码荧光素酶的AAV载体混合,然后用病毒-血清混合物处理Huh7细胞。将荧光素酶活性抑制至少50%的最高血清稀释比定义为Nab滴度。
如表1所示,4/10的患者针对AAV8和AAV9的Nab滴度高于1:4,这被定义为Nab阳性。然而,没有一个患者对AAV5和AAVz2-1的Nab呈阳性。
表1.血友病B病人血清中各种AAV血清型的中和抗体水平
此外,还检查了21只健康恒河猴的Nab阳性率。结果显示,3/21只猴子对AAV5的Nab呈阳性,4/21只猴子对AAV8的Nab呈阳性,9/21只猴子对AAV9的Nab呈阳性,1/21只猴子对AAVz2-1的Nab呈阳性(表2)。
表2.恒河猴血清中各种AAV血清型的中和抗体水平
上述结果表明AAVz2-1具有低的Nab阳性率。
本公开中提及的所有出版物、专利申请、专利、核酸和氨基酸序列以及其他参考文献均通过引用全文的方式并入本文。
虽然通过参照本公开的某些优选实施方式,已经对本公开进行了图示和描述,但本领域的普通技术人员应该明白,以上内容是结合具体的实施方式对本公开所作的进一步详细说明,不能认定本公开的具体实施只局限于这些说明。本领域技术人员可以在形式上和细节上对其作各种改变,包括做出若干简单推演或替换,而不偏离本公开的精神和范围。
序列表
<110> 上海勉亦生物科技有限公司
<120> 腺相关病毒的纯化方法和洗脱液
<130> PCNCNN223158G
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 731
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> AAVz2衣壳蛋白的氨基酸序列
<400> 1
Met Ser Phe Val Asp His Pro Pro Asp Trp Leu Glu Glu Val Gly Glu
1 5 10 15
Gly Leu Arg Glu Phe Leu Gly Leu Glu Ala Gly Pro Pro Lys Pro Lys
20 25 30
Pro Asn Gln Gln His Gln Asp Gln Ala Arg Gly Leu Val Leu Pro Gly
35 40 45
Tyr Asn Tyr Leu Gly Pro Gly Asn Gly Leu Asp Arg Gly Glu Pro Val
50 55 60
Asn Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn Glu
65 70 75 80
Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp
85 90 95
Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly Asn
100 105 110
Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Phe
115 120 125
Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg Ile
130 135 140
Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp Ser
145 150 155 160
Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser Gln
165 170 175
Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp Thr
180 185 190
Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala
195 200 205
Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp
210 215 220
Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu Pro
225 230 235 240
Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val Asp
245 250 255
Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr
260 265 270
Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp Gln
275 280 285
Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg Val
290 295 300
Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr
305 310 315 320
Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp
325 330 335
Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys
340 345 350
Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly Tyr
355 360 365
Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser Ser
370 375 380
Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly Asn
385 390 395 400
Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser
405 410 415
Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp
420 425 430
Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln
435 440 445
Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp
450 455 460
Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly
465 470 475 480
Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu
485 490 495
Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met Thr
500 505 510
Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile
515 520 525
Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu
530 535 540
Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn Arg
545 550 555 560
Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Phe Ala
565 570 575
Pro Thr Pro Gly Pro Ser Ser Thr Thr Ala Pro Ala Thr Gly Thr Tyr
580 585 590
Asn Leu Gln Glu Ile Val Pro Gly Ser Val Trp Met Glu Arg Asp Val
595 600 605
Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro Glu Thr Gly Ala His
610 615 620
Phe His Pro Ser Pro Ala Met Gly Gly Phe Gly Leu Lys His Pro Pro
625 630 635 640
Pro Met Met Leu Ile Lys Asn Thr Pro Val Pro Gly Asn Ile Thr Ser
645 650 655
Phe Ser Asp Val Pro Val Ser Ser Phe Ile Thr Gln Tyr Ser Thr Gly
660 665 670
Gln Val Thr Val Glu Met Glu Trp Glu Leu Lys Lys Glu Asn Ser Lys
675 680 685
Arg Trp Asn Pro Glu Ile Gln Tyr Thr Asn Asn Tyr Asn Asp Pro Gln
690 695 700
Phe Val Asp Phe Ala Pro Asp Ser Thr Gly Glu Tyr Arg Thr Thr Arg
705 710 715 720
Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu
725 730
<210> 2
<211> 2196
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 编码AAVz2衣壳蛋白的核酸序列
<400> 2
atgtcttttg ttgatcaccc tccagattgg ttggaagaag ttggtgaagg tcttcgcgag 60
tttttgggcc ttgaagcggg cccaccgaaa ccaaaaccca atcagcagca tcaagatcaa 120
gcccgtggtc ttgtgctgcc tggttataac tatctcggac ccggaaacgg tctcgatcga 180
ggagagcctg tcaacagggc agacgaggtc gcgcgagagc acgacatctc gtacaacgag 240
cagcttgagg cgggagacaa cccctacctc aagtacaacc acgcggacgc cgagtttcag 300
gagaagctcg ccgacgacac atccttcggg ggaaacctcg gaaaggcagt ctttcaggcc 360
aagaaaaggg ttctcgaacc ttttggcctg gttgaagagg gtgctaagac ggcccctacc 420
ggaaagcgga tagacgacca ctttccaaaa agaaagaagg ctcggaccga agaggactcc 480
aagccttcca cctcgtcaga cgccgaagct ggacccagcg gatcccagca gctgcaaatc 540
ccagcccaac cagcctcaag tttgggagct gatacaatgt ctgcgggagg tggcggccca 600
ttgggcgaca ataaccaagg tgccgatgga gtgggcaatg cctcgggaga ttggcattgc 660
gattccacgt ggatggggga cagagtcgtc accaagtcca cccgaacctg ggtgctgccc 720
agctacaaca accaccagta ccgagagatc aaaagcggct ccgtcgacgg aagcaacgcc 780
aacgcctact ttggatacag caccccctgg gggtactttg actttaaccg cttccacagc 840
cactggagcc cccgagactg gcaaagactc atcaacaact actggggctt cagaccccgg 900
tccctcagag tcaaaatctt caacattcaa gtcaaagagg tcacggtgca ggactccacc 960
accaccatcg ccaacaacct cacctccacc gtccaagtgt ttacggacga cgactaccag 1020
ctgccctacg tcgtcggcaa cgggaccgag ggatgcctgc cggccttccc tccgcaggtc 1080
tttacgctgc cgcagtacgg ttacgcgacg ctgaaccgcg acaacacaga aaatcccacc 1140
gagaggagca gcttcttctg cctagagtac tttcccagca agatgctgag aacgggcaac 1200
aactttgagt ttacctacaa ctttgaggag gtgcccttcc actccagctt cgctcccagt 1260
cagaacctct tcaagctggc caacccgctg gtggaccagt acttgtaccg cttcgtgagc 1320
acaaataaca ctggcggagt ccagttcaac aagaacctgg ccgggagata cgccaacacc 1380
tacaaaaact ggttcccggg gcccatgggc cgaacccagg gctggaacct gggctccggg 1440
gtcaaccgcg ccagtgtcag cgccttcgcc acgaccaata ggatggagct cgagggcgcg 1500
agttaccagg tgcccccgca gccgaacggc atgaccaaca acctccaggg cagcaacacc 1560
tatgccctgg agaacactat gatcttcaac agccagccgg cgaacccggg caccaccgcc 1620
acgtacctcg agggcaacat gctcatcacc agcgagagcg agacgcagcc ggtgaaccgc 1680
gtggcgtaca acgtcggcgg gcagatggcc accaacaacc agttcgcacc aacaccggga 1740
ccaagctcca ccactgcccc cgcgaccggc acgtacaacc tccaggaaat cgtgcccggc 1800
agcgtgtgga tggagaggga cgtgtacctc caaggaccca tctgggccaa gatcccagag 1860
acgggggcgc actttcaccc ctctccggcc atgggcggat tcggactcaa acacccaccg 1920
cccatgatgc tcatcaagaa cacgcctgtg cccggaaata tcaccagctt ctcggacgtg 1980
cccgtcagca gcttcatcac ccagtacagc accgggcagg tcaccgtgga gatggagtgg 2040
gagctcaaga aggaaaactc caagaggtgg aacccagaga tccagtacac aaacaactac 2100
aacgaccccc agtttgtgga ctttgccccg gacagcaccg gggaatacag aaccaccaga 2160
cctatcggaa cccgatacct tacccgaccc ctttaa 2196
Claims (6)
1.一种纯化腺相关病毒(AAV)的方法,其中,所述方法包括:
(a)将含有AAV的溶液加样到亲和层析柱上,所述亲和层析柱为AAVX树脂;
(b)用第一洗脱液和第二洗脱液从亲和层析柱上洗脱AAV,收集含AAV的洗脱液;
(c)对步骤(b)获得的含AAV的洗脱液进行超速离心;
所述第一洗脱液包含1 M AcOH、400-2000 mM精氨酸、500 mM NaCl、0.5-3 mM MgCl2,以及0.05%泊洛沙姆188,所述第一洗脱液的pH=2.5;
所述第二洗脱液包含6 M尿素和2 mM MgCl2;
所述超速离心为碘克沙醇密度梯度超速离心;
所述AAV为AAVz2,所述AAVz2的氨基酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的方法,其中,所述精氨酸的浓度为400 mM至1000 mM;和/或所述MgCl2的浓度为1 mM至3 mM。
3.根据权利要求1所述的方法,其中,所述精氨酸的浓度为400 mM至1000 mM;和/或所述MgCl2的浓度为1.5 mM至2.5 mM。
4.根据权利要求1所述的方法,其中,所述精氨酸的浓度为500 mM至800 mM;和/或所述MgCl2的浓度为1 mM至3 mM。
5.根据权利要求1所述的方法,其中,所述精氨酸的浓度为500 mM至800 mM;和/或所述MgCl2的浓度为1.5 mM至2.5 mM。
6.由权利要求1至5中任一项所述的方法获得的AAV,其中,所述AAV是AAVz2,所述AAVz2具有肝脏靶向性。
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