CN115160410A - 新型肌肉高效亲和腺相关病毒血清型及相关应用 - Google Patents
新型肌肉高效亲和腺相关病毒血清型及相关应用 Download PDFInfo
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Abstract
本发明提供了一种改造型腺相关病毒(AAV)衣壳蛋白。由这些衣壳蛋白包装的腺相关病毒具有较高的骨骼肌亲和性及基因递送效率、更低的毒副作用和更好的安全性潜力,有望被应用于包括骨骼肌相关疾病的预防、诊断和治疗。
Description
技术领域
本发明属于生物医学技术领域,具体涉及一种新型肌肉高效亲和腺相关病毒血清型及相关应用。
背景技术
肌肉疾病可分为非遗传性肌肉疾病和遗传性肌肉疾病。遗传性肌肉疾病包括Duchenne肌营养不良(DMD)、Emery-Dreifuss肌营养不良(EDMD)、肢带肌营养不良(LGMD)、脊髓性肌萎缩症(SMA)等,目前遗传性肌肉疾病没有特效的治疗方法。
近年来基因治疗的飞速发展,给遗传性肌肉疾病的治疗带来了革命性的变革。腺相关病毒作为基因治疗最有希望的载体之一,具有治疗遗传性肌肉疾病的巨大潜力。在2019年,FDA批准了一款以AAV9为载体的药物Zolgensma用以治疗脊髓性肌萎缩(SMA),这是一种由SMN基因突变造成运动神经元发育异常不能行使功能进而引发的肌无力疾病。该款药物用AAV9递送正常的SMN基因进入病人体内表达出正常功能的SMN蛋白确保运动神经系统正常工作以保障正常的呼吸,进食等运动功能,取得不错的治疗效果。
然而,对于其他许多肌肉疾病例如Duchenne肌营养不良(DMD)、Emery-Dreifuss肌营养不良(EDMD)等,仍然缺乏高效感染肌肉的AAV载体;并且野生型AAV在灵长类动物中都有中和抗体,会限制AAV载体的适用范围。
换言之,AAV在基因治疗中显示出巨大的潜力,但依旧面临诸多的挑战,如AAV衣壳的免疫反应、无效的病毒生产和纯化方法、缺少组织器官特异性以及缺乏评估人类临床试验中AAV转导效率的可靠系统。近年来,人们发现AAV载体的遗传修饰可以克服其在临床应用的一些障碍,对AAV衣壳进行修饰能够增强转基因表达并且阻止对AAV衣壳和转基因的免疫反应。在本领域中,已经有AAV9与结合肽结合的新型腺相关病毒衣壳多肽用于治疗与肌肉相关的疾病的报导。然而,肌肉细胞对野生型AAV5的转导敏感性很低,并且本领域尚未有改造AAV5衣壳提高肌肉靶向性的相关报导。
因此,本领域迫切需要开发出一种可用于治疗与肌肉相关的疾病的针对肌肉细胞具有高转染性和低中和抗体反应性的AAV5突变体(即AAV5突变型血清型)。
发明内容
本发明的目的就是提供一种可用于治疗与肌肉相关的疾病的针对肌肉细胞具有高转染性和低中和抗体反应性的AAV5突变体(即AAV5突变型血清型)。
在本发明的第一方面,提供了一种腺相关病毒5(AAV5)衣壳突变体,所述的AAV5衣壳突变体具有选自下组特性:
(a)特异性靶向肌肉细胞;
(b)肌肉细胞高转导率,所述的肌肉细胞高转导率是指:所述的AAV5衣壳突变体在肌肉细胞中的转导率是其他AAV衣壳的2-3倍(较佳地可达到3倍);和
(c)低免疫原性,所述的低免疫原性是指:所述AAV5衣壳突变体在免疫应答中所产生的中和抗体滴度值≤1:4(较佳地≤1:2,更佳地≤1:1)。
在另一优选例中,所述的肌肉细胞包括:具有慢肌纤维(I型)和/或快肌纤维(II型)的肌肉细胞。
在另一优选例中,所述的肌肉细胞包括:小鼠胫前肌(Tibial anterior,TA)、比目鱼肌(Soleus,SO)、腓肠肌(Gastrocnemius,GA)、膈肌或肱二头肌等I型或II型肌肉。
在另一优选例中,所述的AAV5衣壳突变体中具有寡肽序列PGPSPAD(SEQ ID NO:3)。
在另一优选例中,所述AAV5衣壳突变体具有选自下组的氨基酸序列:
(i)在对应于野生型AAV5衣壳蛋白的氨基酸序列的第560至585位(较佳地为第Q574位)后插入一段PGPSPAD(SEQ ID NO:3),从而获得的氨基酸序列;
(ii)在(i)的氨基酸序列的基础上,进行一个或多个氨基酸残基的替换、缺失、改变或插入,或在其N端或C端添加1至10个氨基酸残基(较佳地1至5个氨基酸残基,更佳地1至3个氨基酸残基),并且保留与(i)具有≥85%(优选地≥90%,更优选地≥95%,例如≥96%、≥97%、≥98%或≥99%)的序列同一性的氨基酸序列。
在另一优选例中,所述的野生型AAV5衣壳蛋白的氨基酸序列如SEQ ID NO:4所示。
在另一优选例中,所述AAV5衣壳突变体具有选自下组的氨基酸序列:
(i)如SEQ ID NO:2所示的氨基酸序列;和
(ii)在如SEQ ID NO:2所示序列的基础上,进行一个或多个氨基酸残基的替换、缺失、改变或插入,或在其N端或C端添加1至10个氨基酸残基(较佳地1至5个氨基酸残基,更佳地1至3个氨基酸残基),从而获得的氨基酸序列;并且所述获得的氨基酸序列与如SEQ IDNO:2所示序列具有≥85%(优选地≥90%,更优选地≥95%,例如≥96%、≥97%、≥98%或≥99%)的序列同一性。
在本发明的第二方面,提供了一种AAV病毒颗粒,所述AAV病毒颗粒包括:
(i)如本发明第一方面所述的AAV5衣壳突变体;和
(ii)包装于所述AAV5衣壳突变体中的核酸,所述核酸包括重组病毒基因核酸序列。
在另一优选例中,所述的核酸中包括一外源核苷酸构建物,
在另一优选例中,所述的外源核苷酸构建物从5’端到3’端依次具有以下元件:5’ITR、启动子、目的基因序列、poly(A)序列、3’ITR。
在另一优选例中,所述启动子是CMV启动子、肌生成素(myogenin)启动子或α骨架肌动蛋白(alpha-skeletal actin)启动子。
在另一优选例中,所述目的基因为外源报告基因,包括但不限于:荧光蛋白家族、β-半乳糖苷酶、碱性磷酸酶、荧光素酶和氯霉素乙酰转移酶、β-葡萄糖醛酸、分泌型碱性磷酸酶,或其组合。
在另一优选例中,所述荧光蛋白家族包括绿色荧光蛋白。
在另一优选例中,所述目的基因为治疗性(例如,医学或兽医学)用途的编码天然蛋白质的核酸,所述的治疗包括治疗骨骼肌相关疾病。
在另一优选例中,所述的天然蛋白质是经密码子优化或未经密码子优化的。
在另一优选例中,所述的天然蛋白质选自下组:囊性纤维化跨膜调节蛋白(CFTR)、全长或截短形式的肌营养不良蛋白、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。
在另一优选例中,截短形式的肌营养不良蛋白包括微型肌营养不良蛋白或微肌营养不良蛋白。
在另一优选例中,所述的目的基因为编码抗体或抗原的基因。
在另一优选例中,所述的抗体或抗原选自下组:VEGF抗体、抗硬化蛋白抗体和抗RANKL抗体、抗补体成分C5抗体、抗PD-1抗体和PD-L1抗体、抗CTLA-4抗体、抗CGRP抗体、抗HER2抗体和抗EGFR抗体、针对促炎细胞因子的抗体及其受体、冠状病毒病2019(COVID2019)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、多种肿瘤细胞免疫抗原,或其组合。
在另一优选例中,所述的VEGF抗体包括贝伐单抗、雷珠单抗或溴珠单抗(Brolucizumab)。
在另一优选例中,所述的抗硬化蛋白抗体包括罗莫珠单抗(Romosozumab)、布洛珠单抗(Blosozumab)。
在另一优选例中,所述的抗RANKL抗体包括地诺单抗(Denosumab)。
在另一优选例中,所述的抗补体成分C5抗体包括拉夫珠单抗(Ravulizumab)、依库丽单抗(Eculizumab)。
在另一优选例中,所述的抗PD-1抗体包括纳武单抗(Nivolumab)、派姆单抗(Pembrolizumab)、西米普利单抗(Cemiplimab)。
在另一优选例中,所述的PD-L1抗体包括阿维单抗(Avelumab)、阿替利珠单抗(Atezolizumab)。
在另一优选例中,所述的抗CTLA-4抗体包括伊匹单抗(Ipilimumab)。
在另一优选例中,所述的抗CGRP抗体包括夫瑞奈组单抗(Fremanezumab)、伽奈珠单抗(Galcanezumab)、埃仑单抗(Erenumab)。
在另一优选例中,所述的抗HER2抗体包括曲妥珠单抗(Trastuzumab)、帕妥珠单抗(Pertuzumab)。
在另一优选例中,所述的抗EGFR抗体包括西妥昔单抗(Cetuximab)、帕尼单抗(Panitumumab)、耐昔妥珠单抗(Necitumumab)。
在另一优选例中,所述的针对促炎细胞因子的抗体及其受体包括沙立芦人单抗(Sarilumab)、司妥昔单抗(Siltuximab)、托珠单抗(Tocilizumab)、卡那单抗(Canakinumab)、戈利木单抗(Golimumab)、塞妥珠单抗(Certolizumab)、阿达木单抗(Adalimumab)、英夫利昔单抗(Infliximab)、达利珠单抗(Daclizumab)、巴利昔单抗(Basiliximab)。
在另一优选例中,所述的多种肿瘤细胞免疫抗原包括MAGE抗原、HER2、ErbB2、粘蛋白抗原和雌激素受体。
在另一优选例中,所述的目的基因包括RNAi组分。
在另一优选例中,所述的RNAi组分包括siRNA、shRNA、snRNA、microRNA、核酶、反义寡核苷酸和反义多核苷酸,或其组合。
在本发明的第三方面,提供了一种用于生产如本发明第二方面所述的AAV病毒颗粒的基因工程细胞,所述基因工程细胞中含有:
(i)第一核酸构建物,所述的第一核酸构建物中含有重组病毒基因核酸序列;和
(ii)第二核酸构建物,所述的第二核酸构建物中含有AAV包装所需的rep和cap基因,其中cap基因编码如本发明第一方面所述的AAV5衣壳突变体。
在另一优选例中,所述基因工程细胞是真核细胞。
在另一优选例中,所述基因工程细胞选自下组:293T细胞、HEK293细胞、Sf9细胞,或BHK细胞。
在另一优选例中,所述基因工程细胞中还含有:
(iii)第三核酸构建物,所述的第三核酸构建物是辅助质粒,其具有辅助病毒包装,以形成病毒颗粒的功能。
在另一优选例中,所述辅助质粒来源于腺病毒(Ad)、单纯疱疹病毒(HSV),或者是其他具有辅助功能的辅助质粒。
在另一优选例中,所述的第一核酸构建物中还可含有外源报告基因。
在另一优选例中,所述的外源报告基因选自下组:绿色荧光蛋白、β-半乳糖苷酶、碱性磷酸酶、荧光素酶和氯霉素乙酰转移酶,或其组合。
在另一优选例中,所述的第一核酸构建物、第二核酸构建物和/或第三核酸构建物可暂时存在于所述基因工程细胞中,也可稳定整合进所述基因工程细胞的基因组中。
在另一优选例中,如本发明第二方面所述的AAV病毒颗粒可在所述基因工程细胞内部包装形成。
在另一优选例中,所述的基因工程细胞中包括:
(i)所述AAV5衣壳突变体的核苷酸编码序列,如SEQ ID NO:1或其简并序列所示;
(ii)任选的AAV Rep蛋白的核苷酸编码序列;
(iii)重组病毒基因核酸序列;
(iv)具有支持AAV复制的辅助功能的核苷酸序列。
在本发明的第四方面,提供了如本发明第一方面所述的AAV5衣壳突变体或如本发明第二方面所述的AAV病毒颗粒在制备药物中的用途,所述的药物选自下组:
(i)用于治疗骨骼肌相关疾病的药物组合物;
(ii)其他用于全身性治疗的药物组合物;和/或
(iii)用于免疫防疫的疫苗组合物。
在另一优选例中,所述的骨骼肌相关疾病包括:Duchenne肌营养不良(DMD)、Emery-Dreifuss肌营养不良(EDMD)、肢带肌营养不良(LGMD)、重症肌无力、先天性肌无力综合症、肌萎缩性侧索硬化症(ALS)、I型和II型肌强直性营养不良等。
在另一优选例中,当所述药物是用于治疗骨骼肌相关疾病的药物组合物时,所述AAV5衣壳突变体中包裹有,或所述AAV病毒颗粒中含有治疗性(例如,医学或兽医学)用途的编码天然蛋白质的核酸,所述的天然蛋白质选自下组:囊性纤维化跨膜调节蛋白(CFTR)、全长或截短形式的肌营养不良蛋白、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。
在另一优选例中,所述用于全身性治疗的药物组合物是指用于肿瘤靶向治疗的药物。
在另一优选例中,当所述药物是用于肿瘤靶向治疗的药物时,所述AAV5衣壳突变体中包裹有,或所述AAV病毒颗粒中含有编码靶向的抗体或抗原的基因序列。
在本发明的第五方面,提供了一种药物组合物,所述药物组合物包括以下组分:
(i)如本发明第二方面所述的AAV病毒颗粒;和
(ii)药学上可接受的载体。
在另一优选例中,所述组分(i)占所述药物组合物总重量的0.1-99.9wt%,较佳地10-80wt%,更佳地30-60wt%。
在另一优选例中,所述药物组合物是液态剂型的。
在另一优选例中,所述药物组合物是注射剂。
在另一优选例中,所述药物组合物的剂型包括但不限于:肌肉注射剂、腹腔注射剂、静脉注射剂,或其组合。
在另一优选例中,所述药学上可接受的载体包括但不限于:溶剂、分散介质、包衣、抗细菌或抗真菌剂、等渗剂和吸收延迟剂等。
在另一优选例中,所述药学上可接受的载体包括盐水,所述盐水包括但不限于:缓冲盐水、生理盐水、磷酸盐缓冲液、柠檬酸盐缓冲液、乙酸盐缓冲液、碳酸氢盐缓冲液、蔗糖溶液、盐溶液、聚山梨醇酯溶液,或其组合。
在另一优选例中,所述药学上可接受的载体可进一步包含添加剂,所述添加剂包括但不限于:稳定剂、防腐剂、有助于细胞摄取的转染促进剂,或其组合。
在另一优选例中,所述稳定剂包括但不限于:谷氨酸钠、甘氨酸、EDTA、白蛋白(例如人血清白蛋白),或其组合。
在另一优选例中,所述防腐剂包括但不限于:2-苯氧基乙醇、苯甲酸钠、山梨酸钾、羟基苯甲酸甲酯、苯酚、硫柳汞、抗生素,或其组合。
在另一优选例中,所述转染促进剂包括钙离子。
在另一优选例中,所述药物组合物在治疗骨骼肌相关疾病的应用中,可单独施用,或与其他用于治疗所述骨骼肌相关疾病的药物联合施用。
在本发明的第六方面,提供了一种预防和/或治疗骨骼肌相关疾病的方法,包括步骤:向有所需要的对象施用有效量的如本发明第二方面所述的AAV病毒颗粒,或如本发明第五方面所述的药物组合物。
在另一优选例中,所述施用包括在体注射。
在另一优选例中,所述在体注射的方式包括但不限于:肌肉注射、腹腔注射、静脉注射,或其组合。
在另一优选例中,所述的肌肉注射包括向I、II型骨骼肌肉的注射。
在另一优选例中,所述对象包括人或非人哺乳动物。
在另一优选例中,所述非人哺乳动物包括但不限于:非人灵长类动物、绵羊、狗、猫、马、牛、鸡、大鼠、小鼠等。
在本发明的第七方面,提供了一种免疫接种方法,包括步骤:向有所需要的对象施用有效量的如本发明第二方面所述的AAV病毒颗粒,或如本发明第五方面所述的药物组合物。
在本发明的第八方面,提供了一种靶向治疗肿瘤的方法,包括步骤:向有所需要的对象施用有效量的如本发明第二方面所述的AAV病毒颗粒,或如本发明第五方面所述的药物组合物。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
附图说明
图1为本发明实施例中AAV5突变体病毒文库的构建流程。
图2为本发明实施例中肌肉靶向型AAV5突变体AAVc1的筛选流程。
图3为本发明实施例中四种病毒AAV5、AAV8、AAV9、AAVc1用实时荧光定量PCR检测的滴度值。
图4为本发明实施例中四种血清型AAV5、AAV8、AAV9、AAVc1用银染检测的总蛋白量。
图5为本发明实施例中四种血清型AAV5、AAV8、AAV9、AAVc1的病毒滴度与AAV总蛋白量的比值,即包装病毒的实心率(实心与空壳的比例)。
图6为本发明实施例中四种血清型AAV5、AAV8、AAV9、AAVc1携带GFP报告基因感染C2C12细胞荧光图;比例尺100μm。
图7为本发明实施例中四种血清型AAV5、AAV8、AAV9、AAVc1携带GFP阳性细胞细胞密度定量(n=5;***p<0.001;**p<0.01;*p<0.05)。
图8为本发明实施例4中两种血清型AAV9、AAVc1携带GFP报告基因肌肉注射小鼠不同类型肌肉胫前肌(TA),比目鱼肌(SO)和腓肠肌(GA)的GFP免疫荧光图。
图9为本发明实施例中两种血清型AAV9、AAVc1在小鼠不同类型肌肉(TA、SO、GA)中GFP免疫荧光阳性肌肉细胞比例(n=5;***p<0.001;**p<0.01;*p<0.05)。
图10为本发明实施例中两种血清型AAV9、AAVc1在小鼠不同类型肌肉(TA、SO、GA)中GFP免疫荧光强度(n=5;***p<0.001;**p<0.01;*p<0.05)。
具体实施方式
本发明人经过广泛而深入的研究,经过大量的筛选,首次开发了一种具有高效肌肉感染能力的AAV血清型。具体地,本发明通过肽展示的方法改造获得一种AAV5突变体AAVc1,其在对应于AAV5的cap序列的氨基酸第Q574位后插入一段含有7个氨基酸片段的寡肽Pro-Gly-Pro-Ser-Pro-Ala-Asp(PGPSPAD)。实验结果表明,该AAV5突变体AAVc1具有较强的肌肉感染效率和较低的中和抗体反应性,在基因治疗领域具有巨大的应用潜能。在此基础上完成了本发明。
术语
为了更容易理解本发明,以下具体定义了某些技术和科学术语。除非在本文中另有明确定义,本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。在描述本发明之前,应当理解本发明不限于所述的具体方法和实验条件,因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案,并且不意图是限制性的,本发明的范围将仅由所附的权利要求书限制。
除非另外定义,否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用,在提到具体列举的数值中使用时,术语“约”意指该值可以从列举的值变动不多于1%。例如,如本文所用,表述“约100”包括99和101和之间的全部值(例如,99.1、99.2、99.3、99.4等)。
如本文所用,术语“治疗”指给予患者内用或外用治疗剂,包含本发明所提供的AAV病毒衣壳及其组成的药物组合物。所述患者具有一种或多种疾病症状,而已知所述治疗剂对这些症状具有治疗作用。通常,以有效缓解一种或多种疾病症状的治疗剂的量(治疗有效量)给予患者。
如本文所用,术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。例如,“任选的AAV Rep蛋白的核苷酸编码序列”是指AAV Rep蛋白的核苷酸编码序列可以选自AAV2、AAV5、AAV8或AAV9等。
如本文所用,术语“含有”或“包括(包含)”可以使开放式、半封闭式和封闭式的。换言之,所述术语也包括“基本上由…构成”或“由…构成”。
如本文所用,术语“腺相关病毒(AAV)”是指一种单链DNA病毒,其基因组大小约为4.7kb且两侧有反向末端重复序列(Inverted terminal repeat、ITR)。其包括天然的AAV(1-11型AAV、禽AAV、牛AAV、犬AAV、马AAV和绵羊AAV),和已知的或后来发现或发明的其他人工改造的AAV。不同血清型AAV的基因组序列和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(R)库,例如GenBank(R)登录号NC002077、NC001401、NC001729、NC001863、NC001829、NC001862、NC000883、NC001701、NC001510、AF063497、U89790、AF043303、AF028705、AF028704、J02275、JO1901、J02275、XO1457、AF288061、AHO09962、AY028226、AY028223、NC001358、NC001540、AF513851、AF513852、AY530579、AY631965、AY631966;其发明内容整体并入本文。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;国际专利出版物WO00/28061、WO99/61601、WO98/11244;U.S.专利号No.6,156.303。。
“载体”是指包裹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到靶细胞中。载体的分类包括但不限于质粒,病毒载体,脂质体和其他基因传递载体。待递送的多核苷酸有时被称为“转基因”,包含但不限于可以增强、抑制、削弱、保护、触发或预防某些生物学和生理学的某些蛋白质或合成多肽的编码序列,或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,多肽或肽的多核苷酸),RNAi组分的编码序列(例如,shRNA、siRNA、反义寡核苷酸),或可选的标记。
“转导”、“转染”、“转化”或本文用到的术语指的是将外源多核苷酸传递导至宿主细胞,转录和翻译产生多肽产物的过程,包括利用重组病毒将外源多核苷酸引入宿主细胞。
“基因传递”指的是将外源多核苷酸引入细胞来进行基因传递,可包括靶向,结合,摄取,转运,复制子整合和表达。
“基因表达”或“表达”指的是基因转录,翻译和翻译后修饰产生基因的RNA或蛋白产物的过程。
“感染”指的是病毒和病毒颗粒包含的多核苷酸组分可以传递多核苷酸至细胞中并产生其RNA和蛋白质产物。这也可能意味这在宿主细胞中病毒的复制过程。
“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(DNA),核糖核苷酸(RNA),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指可互换的单链和双链分子。除非另有说明,本文描述的任意实施例里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。
“多肽”指的是至少20个通过肽键连接的氨基酸的聚合物。术语“多肽”和“蛋白质”在本文中同义地是指由多于20个氨基酸组成的聚合物。该术语还包括合成氨基酸聚合物。
术语“反向末端重复”包含任意AAV病毒末端重复或合成序列,其组成发卡结构并作为顺式结构介导病毒的复制,包装,和整合。本文的ITR包括但不限于1-11型AAV、禽AAV、牛AAV、犬AAV、马AAV和绵羊AAV的末端重复。另外,AAV末端重复不需要是天然的,只要可用于AAV的复制、包装,和整合即可。
术语“肿瘤”是指大量的多细胞生物以及由具有致瘤特性的细胞(称为“肿瘤细胞”)产生的血管。这些细胞通常具有过度活化的增殖和抑制的分化能力,从而导致其急剧过度生长并遍布全身(远处转移)。由这种现象引起的这类疾病称为“癌症”,例如表现为白血病、淋巴瘤、肝癌、肾癌、前列腺癌、乳腺癌、肾癌、肺癌、甲状腺癌、大肠癌、宫颈癌、黑色素瘤、胶质母细胞瘤或其他。
术语“镶嵌”AAV衣壳核酸编码序列或衣壳蛋白指通过DNA shuffling,易错PCR和点突变的方法人工设计和改造AAV衣壳序列。
“靶向”在本文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。本领域技术人员已知如果没有顺式和反式作用因子(例如诱导型启动子或其他调节性核酸序列),从病毒基因组转录异源核酸序列就不能开始。
保守氨基酸的取代是本领域已知的。在一些实施例中,潜在的取代氨基酸在以下组的一个或多个内:甘氨酸,丙氨酸;和缬氨酸,异亮氨酸,亮氨酸和脯氨酸;天冬氨酸,谷氨酸;天冬酰胺,谷氨酰胺;丝氨酸,苏氨酸赖氨酸,精氨酸和组氨酸;和/或苯丙氨酸,色氨酸和酪氨酸;蛋氨酸和半胱氨酸。此外,本发明还提供了允许来自不同基团的氨基酸取代的非保守的氨基酸取代。
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的不同于天然多核苷酸的合成产物。重组病毒是一个包含重组多核苷酸的病毒颗粒。
本领域技术人员将容易理解本文所述的所有参数,尺寸,材料和构造的含义。实际参数,尺寸,材料和/或配置取决于使用本发明说明的特定应用。本领域技术人员能够理解,实施例或权利要求仅是通过示例的方式给出的,并且在等效物或权利要求的范围内,本发明的实施例可涵盖的范围不限于具体描述和要求的范围。
本文的定义和使用的所有定义应被理解为超过词典定义或通过引用并入的文档中的定义。
本文所发明的所有参考文献,专利和专利申请都相对于其所引用的主题通过引用并入,在某些情况下可能包含整个文档。
应当理解,对于本文所述的包括一个以上步骤的任何方法,步骤的顺序不一定限于这些实施例中描述的顺序。
本发明AAV5衣壳突变体及用途
腺相关病毒(adeno-associated virus,AAV)是一类微小、无被膜及具有二十面体结构的无致病性病毒。腺相关病毒的颗粒直径约在20-26nm之间,含有约4.70kb的线状单链DNA基因组。AAV病毒衣壳一般由3种衣壳蛋白VP1、VP2和VP3组成。在正常细胞中AAV不能独立复制,只有在腺病毒或疱疹病毒等辅助病毒的帮助下才能进行复制并具有产毒性感染。
根据腺相关病毒临床试验的全球流行病学分析表明,40-80%的人群血清抗AAV中和抗体呈阳性,机体可能存在针对其衣壳的抗体,可以降低转导效率。这说明野生的AAV衣壳不能作为理想的基因治疗载体。为避免人类先前存在的免疫反应,科学家们致力于从其他脊椎动物物种中分离出AAV衣壳,或者在野生型AAV的结构基础上进行改造,从而减少中和抗体并且具有高转导效率实现靶向治疗。
如本文所用,术语“AAV5衣壳突变体”、“AAVc1”、“本发明新型AAV血清型”、“新型AAV衣壳”、“本发明新型AAV载体”、“改造的AAV衣壳”可互换使用,均是指本发明第一方面所述的具有肌肉细胞高特异性、高转导率,以及低免疫原性的AAV5衣壳突变体。
本发明提供了一种腺相关病毒5(AAV5)衣壳突变体,所述的AAV5衣壳突变体具有选自下组特性:(a)特异性靶向肌肉细胞;(b)肌肉细胞高转导率,所述的肌肉细胞高转导率是指:所述的AAV5衣壳突变体在肌肉细胞中的转导率是其他AAV衣壳的2-3倍(较佳地可达到3倍);和(c)低免疫原性,所述的低免疫原性是指:所述AAV5衣壳突变体在免疫应答中所产生的中和抗体滴度值≤1:4(较佳地≤1:2,更佳地≤1:1)。
在本发明的一个实施方式中,在野生型AAV5衣壳蛋白氨基酸序列(如SEQ ID NO:4所示)Q574后插入7氨基酸的寡肽Pro-Gly-Pro-Ser-Pro-Ala-Asp(即PGPSPAD,SEQ ID NO:3)而改造获得的新型AAV衣壳(即AAVc1,氨基酸序列如SEQ ID NO:2所示),由此衣壳蛋白包装病毒基因组形成的AAV病毒颗粒和AAV5,8,9相比具有较强的感染小鼠成肌细胞C2C12并递送基因的能力,并在小鼠体内也展示了优于野生型AAV9的肌肉组织亲和感染力。
野生型AAV5衣壳氨基酸序列(SEQ ID NO:4):
MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKST\RTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL
本发明优选的AAV5突变型衣壳氨基酸序列(SEQ ID NO:2):
MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQPGPSPADSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL
应理解,与本发明的序列(优选地,序列如SEQ ID NO:2所示)具有一定同源性(保守性)的AAV5突变型衣壳蛋白的氨基酸序列或其基因序列,也包括在本发明的范围内,只要本领域技术人员在阅读了本申请后根据本申请提供的信息可以方便地从其它物种(尤其是哺乳动物)中分离得到该序列。
编码本发明的AAV5衣壳突变体的多核苷酸可以是DNA形式或RNA形式。DNA形式包括:DNA、基因组DNA或人工合成的DNA,DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码融合蛋白的编码区序列可以与SEQ ID NO:1所示的编码区序列相同或者是简并的变异体。
ATGTCTTTTGTTGATCACCCTCCAGATTGGTTGGAAGAAGTTGGTGAAGGTCTTCGCGAGTTTTTGGGCCTTGAAGCGGGCCCACCGAAACCAAAACCCAATCAGCAGCATCAAGATCAAGCCCGTGGTCTTGTGCTGCCTGGTTATAACTATCTCGGACCCGGAAACGGTCTCGATCGAGGAGAGCCTGTCAACAGGGCAGACGAGGTCGCGCGAGAGCACGACATCTCGTACAACGAGCAGCTTGAGGCGGGAGACAACCCCTACCTCAAGTACAACCACGCGGACGCCGAGTTTCAGGAGAAGCTCGCCGACGACACATCCTTCGGGGGAAACCTCGGAAAGGCAGTCTTTCAGGCCAAGAAAAGGGTTCTCGAACCTTTTGGCCTGGTTGAAGAGGGTGCTAAGACGGCCCCTACCGGAAAGCGGATAGACGACCACTTTCCAAAAAGAAAGAAGGCTCGGACCGAAGAGGACTCCAAGCCTTCCACCTCGTCAGACGCCGAAGCTGGACCCAGCGGATCCCAGCAGCTGCAAATCCCAGCCCAACCAGCCTCAAGTTTGGGAGCTGATACAATGTCTGCGGGAGGTGGCGGCCCATTGGGCGACAATAACCAAGGTGCCGATGGAGTGGGCAATGCCTCGGGAGATTGGCATTGCGATTCCACGTGGATGGGGGACAGAGTCGTCACCAAGTCCACCCGAACCTGGGTGCTGCCCAGCTACAACAACCACCAGTACCGAGAGATCAAAAGCGGCTCCGTCGACGGAAGCAACGCCAACGCCTACTTTGGATACAGCACCCCCTGGGGGTACTTTGACTTTAACCGCTTCCACAGCCACTGGAGCCCCCGAGACTGGCAAAGACTCATCAACAACTACTGGGGCTTCAGACCCCGGTCCCTCAGAGTCAAAATCTTCAACATTCAAGTCAAAGAGGTCACGGTGCAGGACTCCACCACCACCATCGCCAACAACCTCACCTCCACCGTCCAAGTGTTTACGGACGACGACTACCAGCTGCCCTACGTCGTCGGCAACGGGACCGAGGGATGCCTGCCGGCCTTCCCTCCGCAGGTCTTTACGCTGCCGCAGTACGGTTACGCGACGCTGAACCGCGACAACACAGAAAATCCCACCGAGAGGAGCAGCTTCTTCTGCCTAGAGTACTTTCCCAGCAAGATGCTGAGAACGGGCAACAACTTTGAGTTTACCTACAACTTTGAGGAGGTGCCCTTCCACTCCAGCTTCGCTCCCAGTCAGAACCTCTTCAAGCTGGCCAACCCGCTGGTGGACCAGTACTTGTACCGCTTCGTGAGCACAAATAACACTGGCGGAGTCCAGTTCAACAAGAACCTGGCCGGGAGATACGCCAACACCTACAAAAACTGGTTCCCGGGGCCCATGGGCCGAACCCAGGGCTGGAACCTGGGCTCCGGGGTCAACCGCGCCAGTGTCAGCGCCTTCGCCACGACCAATAGGATGGAGCTCGAGGGCGCGAGTTACCAGGTGCCCCCGCAGCCGAACGGCATGACCAACAACCTCCAGGGCAGCAACACCTATGCCCTGGAGAACACTATGATCTTCAACAGCCAGCCGGCGAACCCGGGCACCACCGCCACGTACCTCGAGGGCAACATGCTCATCACCAGCGAGAGCGAGACGCAGCCGGTGAACCGCGTGGCGTACAACGTCGGCGGGCAGATGGCCACCAACAACCAGCCTGGTCCATCTCCAGCAGACAGCTCCACCACTGCCCCCGCGACCGGCACGTACAACCTCCAGGAAATCGTGCCCGGCAGCGTGTGGATGGAGAGGGACGTGTACCTCCAAGGACCCATCTGGGCCAAGATCCCAGAGACGGGGGCGCACTTTCACCCCTCTCCGGCCATGGGCGGATTCGGACTCAAACACCCACCGCCCATGATGCTCATCAAGAACACGCCTGTGCCCGGAAATATCACCAGCTTCTCGGACGTGCCCGTCAGCAGCTTCATCACCCAGTACAGCACCGGGCAGGTCACCGTGGAGATGGAGTGGGAGCTCAAGAAGGAAAACTCCAAGAGGTGGAACCCAGAGATCCAGTACACAAACAACTACAACGACCCCCAGTTTGTGGACTTTGCCCCGGACAGCACCGGGGAATACAGAACCACCAGACCTATCGGAACCCGATACCTTACCCGACCCCTTTAA(SEQ ID NO:1)
编码本发明AAV5衣壳突变体的多核苷酸包括:只编码本发明AAV5衣壳突变体的编码序列;本发明AAV5衣壳突变体的编码序列和各种附加编码序列;本发明AAV5衣壳突变体的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码本发明AAV5衣壳突变体的多核苷酸”可以是包括编码此本发明AAV5衣壳突变体的多核苷酸,也可以是还包括附加编码和/或非编码序列的多核苷酸。本发明还涉及上述多核苷酸的变异体,其编码与本发明有相同的氨基酸序列的多苷或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的,等位变异体是一个多核苷酸的替换形式,它可能是一个或多个核苷酸的取代、缺失或插入,但不会从实质上改变其编码的本发明AAV5衣壳突变体的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50%,较佳地至少70%,更佳地至少80%相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中,“严格条件”是指:(1)在较低离子强度和较高温度下的杂交和洗脱,如0.2×SSC,0.1%SDS,60℃;或(2)杂交时加有变性剂,如50%(v/v)甲酞胺、0.1%小牛血清/0.1%Ficoll,42℃等;或(3)仅在两条序列之间的相同性至少在90%以上,更好是95%以上时才发生杂交。
编码本发明的本发明AAV5衣壳突变体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法,可根据本发明所公开的有关核苷酸序列,尤其是开放阅读框序列来设计引物,并用市售的DNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板,扩增而得有关序列。当序列较长时,常常需要进行两次或多次PCR扩增,然后再将各次扩增出的片段按正确次序拼接在一起。一旦获得了有关的序列,就可以用重组法来大批量地获得有关序列。通常是将其克隆入载体,再转入细胞,然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外,还可用人工合成的方法来合成有关序列,尤其是片段长度较短时。通常,通过先合成多个小片段,然后再进行连接可获得序列很长的片段。目前,已经可以完全通过化学合成来得到编码本发明本发明AAV5衣壳突变体(或其片段,或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外,还可通过化学合成将突变引入本发明融合蛋白序列中。
本发明还包括与本发明的SEQ ID NO:2所示序列具有50%或以上(优选60%以上,70%以上,80%以上,更优选90%以上,更优选95%以上,最优选98%以上,如99%)同源性的具有相同或相似功能的多肽或蛋白。
所述“相同或相似功能”主要是指:“特异性靶向肌肉细胞、肌肉细胞高转导率,以及低免疫原性”。
本发明的本发明AAV5衣壳突变体蛋白可以是重组多肽、天然多肽、合成多肽。本发明的融合蛋白可以是天然纯化的产物,或是化学合成的产物,或使用重组技术从原核或真核宿主(例如,细菌、酵母、植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主,本发明的本发明AAV5衣壳突变体蛋白可以是糖基化的,或可以是非糖基化的。本发明的本发明AAV5衣壳突变体蛋白序列还可包括或不包括起始的甲硫氨酸残基。
本发明还包括具有本发明AAV5衣壳突变体的活性的其他多肽片段和类似物。如本文所用,术语“片段”和“类似物”是指基本上保持本发明的AAV5衣壳突变体蛋白相同的生物学功能或活性的多肽。
本发明的多肽片段、衍生物或类似物可以是:(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽,而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的;或(ii)在一个或多个氨基酸残基中具有取代基团的多肽;或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物,例如聚乙二醇)融合所形成的多肽;或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列,或融合蛋白)。根据本文的定义这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明中,所述的AAV5衣壳突变体蛋白变体是如SEQ ID NO:2所示的氨基酸序列,经过若干个(通常为1-10个,较佳地1-8个,更佳地1-4个,最佳地1-2个)取代、缺失或添加至少一个氨基酸所得的衍生序列,以及在C末端和/或N末端添加一个或数个(通常为10个以内,较佳地为5个以内,更佳地为3个以内)氨基酸。例如,在所述蛋白中,用性能相近或相似的氨基酸进行取代时,通常不会改变蛋白质的功能,在C末端和/或N末端添加一个或数个(如1-3个)氨基酸通常也不会改变蛋白质的功能。这些保守性变异最好根据表A进行替换而产生。
表A
本发明还包括所要求保护的蛋白的类似物。这些类似物与天然SEQ ID NO:2差别可以是氨基酸序列上的差异,也可以是不影响序列的修饰形式上的差异,或者兼而有之。这些蛋白的类似物包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到,如通过辐射或暴露于诱变剂而产生随机诱变,还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物,以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解,本发明的蛋白并不限于上述列举的代表性的蛋白。
修饰(通常不改变一级结构)形式包括:体内或体外蛋白的化学衍生形式如乙酸化或羧基化。修饰还包括糖基化,如那些在蛋白质合成和加工中进行糖基化修饰。这种修饰可以通过将蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸,磷酸丝氨酸,磷酸苏氨酸)的序列。
在本发明中,所述的肌肉细胞包括:具有慢肌纤维(I型)和/或快肌纤维(II型)的肌肉细胞。优选地,所述的肌肉细胞包括:小鼠胫前肌(Tibial anterior,TA)、比目鱼肌(Soleus,SO)、腓肠肌(Gastrocnemius,GA)、膈肌或肱二头肌等I型或II型肌肉。
本发明所提供的AAVc1,和野生血清型相比,还显示了在猴群中有较低的中和抗体水平,这反映了当新型AAVc1进入灵长类动物身体后受到免疫系统抑制失活的概率也小于野生型,潜在副作用和不良反应也较低,并可能适用于更广范围的动物宿主或病人。
在一些实施方式中,改造的AAV衣壳可以装载异源核酸用于基因递送到靶细胞中。因此,这些改造的血清型可用于在体外或体内将核酸递送至细胞并表达产物蛋白。
药物组合物及治疗方法
如本文所用,术语“药物组合物”是指包含本发明的AAV5衣壳突变体或表达所述AAV5衣壳突变体的宿主细胞和药学上可接受的载体的组合物,其功能为治疗骨骼肌相关性疾病。
在一个实施方式中,本发明药物组合物包含AAVc1作为衣壳的病毒颗粒和相应的药学上可接受的载体。
在特定的实施方式中,由改造的AAV衣壳递送的异源多核苷酸编码充当报告子的多肽,以指示成功被AAV感染的细胞。这些报道蛋白包括但不限于绿色荧光蛋白、β-半乳糖苷酶、碱性磷酸酶、荧光素酶和氯霉素乙酰转移酶。
在一些情况下,递送的核酸包括用于治疗性(例如,医学或兽医学)用途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。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(包括一些截短的形式,称为微型肌营养不良蛋白或微肌营养不良蛋白)、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。这些蛋白可用于治疗各种肌肉类型的疾病,例如Duchenne肌营养不良(DMD)、Emery-Dreifuss肌营养不良(EDMD)、肢带肌营养不良(LGMD)、重症肌无力、先天性肌无力综合症、肌萎缩性侧索硬化症(ALS)、I型和II型肌强直性营养不良。
本发明还可应用于将抗体或抗原基因通过肌肉注射的方式递送至肌肉细胞中,并表达分泌至血液中递送致全身,起到免疫防疫(疫苗)和单抗靶向特异分子、进行细胞治疗的作用。可被包装进病毒基因组的抗原抗体包括但不限于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、Blosozumab)和抗RANKL抗体(例如Denosumab)、抗补体成分C5抗体(例如Ravulizumab、Eculizumab)、抗PD-1抗体(例如Nivolumab、Pembrolizumab、Cemiplimab)和PD-L1抗体(例如Avelumab、Atezolizumab)、抗CTLA-4抗体(例如Ipilimumab)、抗CGRP抗体(例如Fremanezumab、Galcanezumab、Erenumab)、抗HER2抗体(例如Trastuzumab、Pertuzumab)和抗EGFR抗体(例如Cetuximab、Panitumumab、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、Basiliximab)、冠状病毒2019(COVID2019)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、多种肿瘤细胞免疫抗原(例如MAGE抗原、HER2、ErbB2、粘蛋白抗原和雌激素受体)。
本发明中揭示的由AAV血清型递送的重组病毒基因核酸序列可能包括RNAi组分(例如,siRNA、shRNA、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。RNAi相关功能、原理和操作方法是业内专业人员所熟知的。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表达量减少至少30%(即30-100%以及其中任一数值)。
如本文所用,术语“药学上可接受的载体”是指任何和所有的药物载体,例如溶剂、分散介质、包衣、抗细菌或抗真菌剂、等渗剂和吸收延迟剂等。这些载体可以帮助病毒颗粒的储存和对受试者的给药。药学上可接受的载体可以包括任何合适的组分,例如但不限于盐水。盐水的说明性例子包括但不限于缓冲盐水、生理盐水、磷酸盐缓冲液、柠檬酸盐缓冲液、乙酸盐缓冲液、碳酸氢盐缓冲液、蔗糖溶液、盐溶液和聚山梨醇酯溶液。
在某些实施方式中,所述药物组合物可进一步包含添加剂,包括但不限于稳定剂、防腐剂、有助于细胞摄取的转染促进剂。合适的稳定剂包括但不限于谷氨酸钠、甘氨酸、EDTA和白蛋白(例如人血清白蛋白)。合适的防腐剂包括但不限于2-苯氧基乙醇、苯甲酸钠、山梨酸钾、羟基苯甲酸甲酯、苯酚、硫柳汞和抗生素。合适的转染促进剂包括但不限于钙离子。
在具体的实施方式中,本发明进一步提供了预防和/或治疗骨骼肌相关疾病的方法,包括但不限于通过肌肉注射、腹腔注射、静脉注射等给予受试者有效剂量的AAV病毒颗粒。上述方法要基于骨骼肌相关疾病的发病机制选择,且可以互相组合使用。
本发明的药物组合物中还可以包括用于治疗骨骼肌相关疾病的其他药物,或与用于治疗骨骼肌相关疾病的其他药物联用。
在其他实施方式中,本发明还提供了一种免疫接种方法和一种靶向治疗肿瘤的方法,均包括向有所需要的对象施用有效量的含有本发明的AAV5衣壳突变体的AAV病毒颗粒或本发明的药物组合物。
如本文所用,术语“受试者”、“对象”可互换使用,包括任何人或非人哺乳动物,例如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、大鼠、小鼠等。
如本文所用,术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。其是指治疗剂治疗、缓解或预防目标疾病或状况的量,或是表现出可检测的治疗或预防效果的量。治疗效果也包括生理性症状的减少。对于某一对象的精确有效量取决于该对象的体型和健康状况、病症的性质和程度、以及选择给予的治疗剂和/或治疗剂的组合。因此,预先指定准确的有效量是没用的。然而,对于某给定的状况而言,可以用常规实验来确定该有效量。
本发明所述的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于:所述药物的药代动力学参数例如药物组织分布、生物利用率、代谢、半衰期等;患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。例如,由治疗状况的迫切要求,可每天给予若干次分开的剂量,或将剂量按比例地减少。
本发明的主要优点包括:
1)本发明改造的AAV5突变体AAVc1具有稳定的肌肉亲和力,且有较低的中和抗体水平,可以作为新的AAV血清型治疗骨骼肌相关疾病。
2)AAVc1感染小鼠肌肉细胞和组织的效率明显高于AAV5、AAV8和AAV9,在临床应用中时对于体内有较高野生型中和抗体滴度的患者可以选用AAVc1进行治疗,进而扩大了临床使用AAV治疗疾病的选择。
3)病毒AAVc1包装效率(实心率)和产量至少不低于甚至高与野生型(如AAV8、9)。
4)AAVc1还可以应用于需要通过肌肉递送并分泌至全身起效的其他药物的递送和相关疾病的治疗,如抗体药物和抗原疫苗的递送。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring HarborLaboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数是重量百分比和重量份数。
实施例1:肽文库的构建和肌肉高亲和性突变体的筛选
本发明通过基于单个血清型骨架的AAV肽展示衣壳改造方法,在AAV5 cap基因第573位氨基酸后随机插入7个核苷酸寡肽片段,经合理设计并合成构建了AAV5随机质粒文库。
文库的构建流程如图1所示。
病毒载体是三质粒共转染方法获得的。将AAV5包装质粒、Ad5辅助(helper)质粒、pAAV-CMV-GFP质粒在聚乙烯亚胺(PEI)条件下共转染至HEK293细胞。转染48小时后,收集细胞并通过离心沉淀,将细胞重悬于150mM NaCl、50mM Tris-HCl(pH 7.5)中。冷冻融化数次。在37℃下用Benzonase酶(50U/ml)处理30分钟,通过离心去除细胞碎片。将上清液加到碘克沙醇梯度溶液(15%、25%、40%、60%碘克沙醇溶于PBS中配置)中,然后在4℃下以65000rpm的转速进行2.5小时。然后从40%碘克沙醇相中收获病毒颗粒,初步形成感染性的嵌合AAV5文库。
为了筛选得到靶向肌肉细胞的AAV5突变体,将AAV5嵌合文库与Ad5(MOI=25pfu/细胞)共感染小鼠成肌C2C12细胞,48h后收取细胞裂解液,用于下一轮的感染。
如此反复感染C2C12细胞四至五次,将每个感染周期收集的病毒提取病毒DNA,并对cap基因随机插入区域进行测序,分析各突变体的出现频次。
分析所得的出现频次最高的突变体,则被证明在C2C12中有较高的转导效率。肌肉靶向型AAV5突变体的筛选流程如图2所示。
筛选和分析的结果表明,插入有寡肽片段PGPSPAD的AAV5突变体AAVc1具有较高的转导效率。
实施例2:各AAV病毒效价的测定和比较
AAV的效价影响着其感染细胞的能力。因此,在本实施例中,对四种血清型AAVc1、AAV5、AAV8和AAV9的包装效率进行荧光定量PCR和银染分析,并比较了四种血清型的实心率(AAV病毒滴度/AAV总蛋白量)。
荧光定量PCR分析的结果如图3所示。结果显示,AAVc1病毒滴度为1.28×1012vg/ml,AAV5、AAV8和AAV9的病毒滴度分别为1.09×1012vg/ml、1.54×1012vg/ml和8.95×1011vg/ml。
银染分析的结果如图4所示。结果显示,AAVc1总蛋白量为1.65×1012vp/ml,AAV5、AAV8和AAV9的病毒滴度分别为3.25×1012vp/ml、2.31×1012vp/ml和1.93×1012vp/ml。
病毒滴度与AAV总蛋白量的比值,即包装病毒的实心率(实心与空壳的比例)结果如图5所示。结果表明,AAVc1、AAV5、AAV8和AAV9的实心率为66.32%、66.06%、47.38%和38.74%。通过对比发现AAVc1与AAV5的实心率相差不大,但相对于AAV8、AAV9则有更高的包装效率,说明筛选所得AAVc1具有较高且稳定的病毒包装效率。
实施例3:各AAV病毒转染效率的比较
C2C12是创伤后重建肌肉组织的前体细胞,常被用作为在体外系统中研究肌肉的发育和分化。在本实施例中,将AAVc1包装GFP报告基因感染C2C12细胞,同时与其他三种血清型AAV5、AAV8和AAV9相比较其转染效率。
将上述四种血清型包装GFP的病毒以MOI=1×105vg/细胞感染C2C12细胞,于72h后观察绿色荧光GFP的表达。
四种血清型AAV5、AAV8、AAV9、AAVc1携带GFP报告基因感染C2C12细胞的荧光强度结果如图6所示。结果表明,相对于几种野生型的AAV血清型,AAVc1有更强的荧光表达。
同时,在本实施例中对感染C2C12细胞GFP阳性细胞进行了定量,结果如图7所示。结果表明,选取荧光图对其中的GFP阳性细胞以及对总细胞进行计数,AAVc1阳性细胞数显著高于其他三种野生型AAV。
在本领域中,已知AAV5感染肌肉效率低;而AAV8、9对全身器官和组织有普遍的感染效率。因此,相较之下,本发明新筛选得到的AAVc1作为改造的AAV5突变体,对细胞的感染能力居然显著高于AAV8、AAV9,这显示了对肌肉细胞感染效率方面的意外优异的改造效果。
实施例4:小鼠体内的肌肉感染效率验证实验
在本实施例中,在小鼠上进一步验证了AAVc1的高肌肉感染效率。
用AAVc1-GFP和AAV9-GFP以3×1011vg/腿的剂量肌肉注射WT小鼠(n=5),观察其在体内的感染效率。在病毒注射小鼠3周后进行解剖取小鼠腿部肌肉,分别取小鼠胫前肌(Tibial anterior,TA)、比目鱼肌(Soleus,SO)以及腓肠肌(Gastrocnemius,GA)进行冰冻切片和GFP的免疫荧光染色。
骨骼肌纤维主要分慢肌纤维(I型)和快肌纤维(II型)。其中,I型肌纤维收缩慢,靠线粒体氧化磷酸化供能;II型肌纤维收缩快,主要以糖酵解反应供能。而胫前肌以II型肌纤维为主,比目鱼肌以I肌型纤维为主,腓肠肌则是混合肌含两种肌纤维。因此,可观察GFP在不同类型肌肉中的表达。
GFP在不同类型肌肉中的表达结果如图8所示。结果显示,可以观察到小鼠三种腿部肌肉中的绿色荧光表达,AAVc1显著高于AAV9。
同时,本发明人量化了转导效率,对免疫荧光图中的GFP阳性细胞以及总细胞计数,将GFP阳性肌肉细胞数的比例以及荧光强度进行了对比,结果如图9和10所示。结果表明,AAVc1和AAV9在感染小鼠肌肉上有着显著性差异。同时,AAVc1对不同的肌肉类型均有较强的转导效率,进一步说明了其强大的肌肉转导效率。
实施例5:猴子体内的中和抗体滴度检测
虽然AAV对人体没有致病性,但人体内免疫应答产生的中和抗体与AAV结合,抑制了外源基因的表达。一般认为,中和抗体滴度值大于1:4为中和抗体阳性,小于等于1:4为阴性;只有当中和抗体滴度小于等于1:4时才能采用这一血清型进行治疗。
在本实施例中,将AAV9和AAVc1分别包装高斯荧光素酶报告基因Gluc(Gaussialuciferase)用于检测10只猴子血清的中和抗体滴度。
结果如表1所示。从结果中可以看出AAVc1的中和抗体滴度相对于AAV9整体偏小,部分猴子对AAV9显示有较高的中和抗体,而对AAVc1的中和抗体滴度很低。整体说明新型AAV血清型AAVc1相对于AAV9有较低的中和抗体滴度,应用到临床中时对于体内有较高AAV9中和抗体的患者可以选用AAVc1这一血清型进行治疗,进而扩大了临床使用AAV治疗疾病的选择。
表1:猴子血清中不同AAV血清型的中和抗体检测。
Monkey ID | Anti-AAV9 | Anti-AAVc1 |
11559 | 1:32 | 1:2 |
13061 | 1:8 | 1:1 |
13073 | 1:16 | 1:2 |
13175 | 1:16 | 1:8 |
13343 | 1:2 | <1:1 |
13653 | 1:8 | 1:2 |
13725 | 1:32 | 1:8 |
80043 | 1:1 | 1:1 |
100039 | 1:16 | 1:1 |
12347A | 1:1 | <1:1 |
讨论
目前,合理设计、定向改造和计算机辅助设计是进行AAV衣壳改造的三种主要方法。最先应用于改进AAV载体的方法是通过肽序列的工程设计改造衣壳,改善衣壳的定位能力。临床试验中使用的第一个工程载体衣壳AAV2.5就是经过合理设计改造的,具有良好的肌肉传导能力。但由于对AAV结合细胞表面的具体过程认识不足,合理设计改造衣壳有一定局限性。定向进化通过模拟自然进化,让AAV衣壳受到选择性压力,产生有利于目的基因表达的遗传变异,然而从定向进化产生的衣壳库中筛选优质新型载体的工作量过于庞大,效率低下。计算机方法是最近新兴起的衣壳改造途径,计算机设计利用DNA序列信息和AAV血清型之间的系统分析来构建AAV衣壳库,产生具有强转导能力的新型衣壳变体。
本发明的主要目的是根据发明人的研究成果,为克服目前AAV基因治疗缺乏组织高效性和低中和抗体血清型的问题,提供一种具有高效肌肉感染能力的新型AAV血清型。
本发明主要通过肽展示的衣壳改造方法在AAV5的cap序列氨基酸Q574位后插入一个含有7个氨基酸片段的寡肽Pro-Gly-Pro-Ser-Pro-Ala-Asp(PGPSPAD),筛选得到具有较强的肌肉感染效率以及低中和抗体的AAV5突变体AAVc1。
在实施例中,本发明涉及到包括AAV衣壳蛋白和病毒基因组核酸的重组AAV载体,应用中它们可以是含有天然病毒基因组或者是外源核酸的重组AAV载体。在一些实施例中,外源核酸编码一种或多种哺乳动物蛋白(包括但不限于自然蛋白,抗体,抗原,合成可溶性受体或其他多肽序列),或者是RNAi组分的序列(例如shRNA、siRNA、反义寡核苷酸)。
在实施例中,AAVc1感小鼠肌肉细胞和组织的效率明显高于AAV5、8、9,且在猴血清里中和抗体含量明显低于野生型AAV9。在实施例中,病毒基因组的蛋白或核酸产物会增强、削弱、保护、激活或预防哺乳动物中参与代谢调节和健康维持的一种或多种内源信号通路。
因此,本发明的新型AAV血清型在基因治疗领域具有巨大的应用潜能。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 华东理工大学
<120> 新型肌肉高效亲和腺相关病毒血清型及相关应用
<130> P2021-0661
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 2196
<212> DNA
<213> 人工序列(artificial sequence)
<220>
<223> AAVc1衣壳蛋白编码基因序列
<400> 1
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 agcctggtcc atctccagca 1740
gacagctcca 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
<210> 2
<211> 731
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> AAVc1衣壳蛋白氨基酸序列
<400> 2
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 Pro Gly
565 570 575
Pro Ser Pro Ala Asp 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> 3
<211> 7
<212> PRT
<213> 人工序列(artificial sequence)
<220>
<223> 7氨基酸多肽
<400> 3
Pro Gly Pro Ser Pro Ala Asp
1 5
<210> 4
<211> 724
<212> PRT
<213> 腺相关病毒5(adeno-associated virus 5)
<400> 4
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 Ser Ser
565 570 575
Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro
580 585 590
Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp
595 600 605
Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala Met
610 615 620
Gly Gly Phe Gly Leu Lys His Pro Pro Pro Met Met Leu Ile Lys Asn
625 630 635 640
Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val Ser
645 650 655
Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met Glu
660 665 670
Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln
675 680 685
Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro Asp
690 695 700
Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr Leu
705 710 715 720
Thr Arg Pro Leu
Claims (10)
1.一种腺相关病毒5(AAV5)衣壳突变体,其特征在于,所述的AAV5衣壳突变体具有选自下组特性:
(a)特异性靶向肌肉细胞;
(b)肌肉细胞高转导率,所述的肌肉细胞高转导率是指:所述的AAV5衣壳突变体在肌肉细胞中的转导率是其他AAV衣壳的2-3倍(较佳地可达到3倍);和
(c)低免疫原性,所述的低免疫原性是指:所述AAV5衣壳突变体在免疫应答中所产生的中和抗体滴度值≤1:4(较佳地≤1:2,更佳地≤1:1)。
2.如权利要求1所述的AAV5衣壳突变体,其特征在于,所述AAV5衣壳突变体具有选自下组的氨基酸序列:
(i)在对应于野生型AAV5衣壳蛋白的氨基酸序列的第560至585位(较佳地为第Q574位)后插入一段PGPSPAD(SEQ ID NO:3),从而获得的氨基酸序列;
(ii)在(i)的氨基酸序列的基础上,进行一个或多个氨基酸残基的替换、缺失、改变或插入,或在其N端或C端添加1至10个氨基酸残基(较佳地1至5个氨基酸残基,更佳地1至3个氨基酸残基),并且保留与(i)具有≥85%(优选地≥90%,更优选地≥95%,例如≥96%、≥97%、≥98%或≥99%)的序列同一性的氨基酸序列。
3.如权利要求1所述的AAV5衣壳突变体,其特征在于,所述AAV5衣壳突变体具有选自下组的氨基酸序列:
(i)如SEQ ID NO:2所示的氨基酸序列;和
(ii)在如SEQ ID NO:2所示序列的基础上,进行一个或多个氨基酸残基的替换、缺失、改变或插入,或在其N端或C端添加1至10个氨基酸残基(较佳地1至5个氨基酸残基,更佳地1至3个氨基酸残基),从而获得的氨基酸序列;并且所述获得的氨基酸序列与如SEQ ID NO:2所示序列具有≥85%(优选地≥90%,更优选地≥95%,例如≥96%、≥97%、≥98%或≥99%)的序列同一性。
4.一种AAV病毒颗粒,其特征在于,所述AAV病毒颗粒包括:
(i)如权利要求1所述的AAV5衣壳突变体;和
(ii)包装于所述AAV5衣壳突变体中的核酸,所述核酸包括重组病毒基因核酸序列。
5.一种用于生产如权利要求4所述的AAV病毒颗粒的基因工程细胞,其特征在于,所述基因工程细胞中含有:
(i)第一核酸构建物,所述的第一核酸构建物中含有重组病毒基因核酸序列;和
(ii)第二核酸构建物,所述的第二核酸构建物中含有AAV包装所需的rep和cap基因,其中cap基因编码如权利要求1所述的AAV5衣壳突变体。
6.如权利要求1所述的AAV5衣壳突变体或如权利要求4所述的AAV病毒颗粒在制备药物中的用途,其特征在于,所述的药物选自下组:
(i)用于治疗骨骼肌相关疾病的药物组合物;
(ii)其他用于全身性治疗的药物组合物;和/或
(iii)用于免疫防疫的疫苗组合物。
7.如权利要求6所述的用途,其特征在于,所述的骨骼肌相关疾病包括:Duchenne肌营养不良(DMD)、Emery-Dreifuss肌营养不良(EDMD)、肢带肌营养不良(LGMD)、重症肌无力、先天性肌无力综合症、肌萎缩性侧索硬化症(ALS)、I型和II型肌强直性营养不良等。
8.如权利要求6所述的用途,其特征在于,所述用于全身性治疗的药物组合物是指用于肿瘤靶向治疗的药物。
9.一种药物组合物,其特征在于,所述药物组合物包括以下组分:
(i)如权利要求4所述的AAV病毒颗粒;和
(ii)药学上可接受的载体。
10.如权利要求9所述的药物组合物,其特征在于,所述药物组合物的剂型包括但不限于:肌肉注射剂、腹腔注射剂、静脉注射剂,或其组合。
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