CN117089560A - 融合核酸、腺相关病毒载体及其用途和药物制剂 - Google Patents
融合核酸、腺相关病毒载体及其用途和药物制剂 Download PDFInfo
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Abstract
本发明涉及基因工程技术领域,尤其涉及一种表达抗VEGF和Ang 2抗体基因片段系统的构建和应用,用于治疗新生血管增生疾病。具有表达稳定,长期有效,组织损伤风险低等优点。
Description
优先权信息
本申请要求于2022年5月18日提交的中国申请号为的202210543294.7优先权。
技术领域
本发明涉及基因工程技术领域,尤其涉及一种表达抗VEGF和抗Ang 2抗体基因片段系统的构建和应用。具体地说,是以基因治疗载体传输大分子药物基因体内表达以直接发挥生物功能的方法。该方法免除了传统大分子药物生产纯化的复杂工艺,在体内持续稳定表达治疗剂量的生物活性大分子。
背景技术
年龄相关性黄斑变性(AMD)和糖尿病视网膜病变(DR)是致盲的两个主要病因。年龄相关性黄斑变性(AMD)是一种能够导致中心视力丧失的疾病,累及黄斑,黄斑位于视网膜的中心,是眼后部的感光组织,提供清晰的中心视觉,视网膜是一层薄薄的组织,排列在眼睛的后部并接收光线,视网膜将光或图像转换成电冲动,然后将这些冲动或神经信号传递到大脑形成视觉。AMD引起的损害显著地影响眼睛将这些信号转换成图像的能力,视网膜下形成黄色沉积物(drusen),导致视力扭曲和模糊,随着时间的推移,沉积物的大小和数量增加,这导致血管在视网膜下生长(血管生成),从而泄漏血液并损伤视网膜。外围视觉(侧视觉)可能不受影响,但在晚期AMD中,直接看到前方的能力丧失。年龄相关性黄斑变性(AMD)有干性和湿性两种类型。干性年龄相关性黄斑变性视网膜色素上皮层、布鲁赫膜(Bruch膜)、脉络膜毛细血管等各层逐渐萎缩变性,一旦干性年龄相关性黄斑变性进展到晚期,没有任何治疗可防止视力丧失,无治疗意义。
本发明所针对的AMD为湿性年龄相关性黄斑变性(wet age-related maculardegeneration,wAMD),原因是玻璃膜疣等引起的布鲁赫膜(Bruch membrane)损害,诱发脉络膜的毛细血管向外层长出新生血管(即choroidal neovascularization,CNV),新生血管伴有成纤维细胞增生,可破坏脉络膜毛细血管、布鲁赫膜、色素上皮细胞和光感受器细胞,引起严重的视力丧失。
目前湿性年龄相关性黄斑变性采取激光疗法、光动力疗法或者注射疗法。激光疗法用高能量的光束直接对准新生血管,破坏它们来防止进一步的视力丧失。激光治疗也能破坏周围一些健康组织,因此只有一小部分湿性AMD患者适合激光治疗。如果渗漏血管远离黄斑中心凹,则适合激光治疗。光动力疗法(photodynamic therapy,PDT)维替泊芬为第二代卟啉类光敏剂,可被光(波长689nm)照射激活,通过非热能的激光照射,产生活性氧而闭塞不正常血管,从而终止血管的渗漏,保存视力。注射维替泊芬到手臂静脉血管中,这种药“粘附”于新生血管表面,然后用光束照入患者眼睛的病变部位约90秒,激活新生血管中的药物。与普通激光治疗不同,这种药物不会破坏周围健康组织,但是因为这种药物能被光激活,因此治疗后五天内必须避免皮肤和眼睛暴露于太阳光和室内明亮光线下。注射治疗,即抗血管内皮细胞生长因子疗法。湿性AMD患者眼内的血管内皮细胞生长因子(VEGF)异常增高,促进了异常新生血管增长,药物治疗例如雷珠单抗(Ranibizuma)可以阻止这种生长因子的作用,该疗法有助于延缓AMD引起的视力丧失,某些情况下能提高视力。
但是激光治疗后新生血管复发的危险性很高,光动力疗法能延缓视力下降速率,但不能阻止视力丧失或恢复已经因晚期AMD而丧失的视力,通常都需要重复治疗。注射治疗目前也需要多次注射。
糖尿病视网膜病变(Diabetic Retinopathy,DR)是糖尿病性微血管病变中最重要的表现,是一种具有特异性改变的眼底病变,是糖尿病的严重并发症之一。临床上根据是否出现视网膜新生血管为标志,将没有视网膜新生血管形成的糖尿病性视网膜病变称为非增殖性糖尿病性视网膜病变(Non-proliferative Diabetic Retinopathy,NPDR)(或称单纯型或背景型),而将有视网膜新生血管形成的糖尿病性视网膜病变称为增殖性糖尿病性视网膜病变(Proliferative Diabetic Retinopathy,PDR)。非增殖期的糖尿病视网膜病变的眼底表现为:视网膜静脉扩张、微血管瘤、深层和浅层出血、硬性渗出、棉絮斑、视网膜水肿,长期的黄斑水肿形成黄斑囊样水肿,视力明显下降。增殖期糖尿病视网膜病变损害进一步加重,较大面积毛细血管闭塞缺血,则发生视网膜新生血管,进而新生血管由视网膜表面长入内界膜与玻璃体后界膜间,形成纤维血管膜。新生血管易破裂出血,大量玻璃体积血、机化,导致牵拉性视网膜脱离。缺血区的视网膜产生的血管生长因子,经玻璃体进入前房,致虹膜、房角新生血管形成,最终导致继发闭角型青光眼即新生血管性青光眼而失明。
糖尿病黄斑水肿(Diabetic Macular Edema,DME)属于糖尿病视网膜病变的一种,是指由糖尿病引起在黄斑中心凹一个视盘直径范围内,细胞外液积聚所致的视网膜增厚或硬性渗出沉积。在过去三十多年中,增值性糖尿病视网膜病变的早期筛查和预防使其发病率逐年下降,因此,现阶段DME成为DM患者最主要的致盲原因。DME最主要的结构改变是血-视网膜屏障的破坏,DM环境使一些紧密连接蛋白遭到破坏,最终导致血管渗漏以及其通透性改变。造成这一改变最主要的分子介质为视网膜合成的血管内皮生长因子(VEGF)。
在DR的治疗方法中,药物治疗如羟苯磺酸钙、递法明、弥可保等主要用于非增殖期糖尿病视网膜病变。
激光光凝是通过其损伤作用,对有病变组织进行破坏,使之从耗氧变为不耗氧,进而使健康的视网膜组织有更好的氧气供应,有助于新生血管的逐渐萎缩,但是光凝的目的是尽量保持视力,阻止病变恶化。它是通过破坏不正常的视网膜,阻止新生血管形成和液体渗漏,然而,疾病仍在发展,不正常的新生血管和渗漏仍可以继续,还需要再次治疗。
玻璃体切除手术是治疗增殖性糖尿病视网膜病变的非常有效的方法,使屈光间质清晰,移走积血及分解物质并将机化膜切断、吸出,消除纤维组织赖以生长的支架,松解对视网膜的牵拉,并注入液体及(或)气体,恢复正常的视网膜解剖关系,保持眼球完整,还可以同时进行眼内光凝。早期的手术成功率可达90%以上,部分患者可以恢复视力,控制疾病的发展。但是,一旦进入疾病的晚期,手术则难以成功。眼部新生血管增生是年龄相关性黄斑变病(AMD)和糖尿病视网膜病变(DR)主要的病理性标志,血管内皮生长因子(VEGF)在其发病机制中起着重要作用。目前临床治疗以抗VEGF抗体为治疗标准,阻止VEGF引起的新生血管增生,临床效果良好。
单克隆抗体(mAbs)彻底改变了生物学和医学领域。自1989年Ferrara教授等证实血管内皮因子具有促进血管内皮细胞增殖作用以来,VEGF家族细胞因子VEGF-A、B、C、D、E及PLGF陆续被发现,其中VEGF-A和受体结合触发系列级联反应,促进血管内皮细胞分裂增生,新生血管生成,并维持新生血管的存活,VEGF-A是炎症细胞趋化因子,增加血管渗透性,在手术剥离的wAMD新生血管膜样本上检测到VEGF-A的高表达。作为CNV形成和进展的主要因素,VEGF-A是wAMD和DR的主要治疗靶点。
血管生成素是Tie受体配体家族的一员。到目前为止,已发现四种血管生成素:血管生成素1、2、3和4(Ang1、Ang2、Ang 3和Ang 4)。血管生成素-2(angiopoietin-2,Ang-2)是血管生成素家族的重要成员之一,为内皮细胞分泌的糖蛋白。Ang2促进内皮细胞的生存、增殖和迁移,并促进出芽式血管生成,它诱导血管壁周细胞的损失,增加血管通透性。Ang2与其他生长因子(如VEGF)结合,会促进血管重构、血管生成和炎症。
目前临床上抗体药物眼部注射已成为治疗眼部新生血管增生疾病的主要药物。然而这种治疗方式需要长期重复注射,给病人带来了很多痛苦和不便。
因为临床建议的治疗剂量需要重复多次注射给药,具有波动的血浆浓度,难以维持稳定的血浆水平;并且由于反复注射,患者需承受多次注射带来的组织损伤和痛苦,同时带来的是临床治疗费用极高。这是蛋白质药物临床眼部注射应用的主要缺点。重组腺相关病毒载体(rAAV)引导的基因疗法可以克服这些缺点。
腺相关病毒(Adeno-associated virus,AAV)是一种微小的(25毫微米)复制缺陷型病毒,属微小病毒科(parvovirus),为无包膜的单链线状DNA病毒。重组腺相关病毒载体(rAAV)是用任何感兴趣的基因或DNA序列取代野生型病毒基因组而构建的,可以感染复制期细胞和静止期细胞,具有长效的转基因表达优越性,并且没有观察到病毒引起的病理毒理反应。因此rAAV是近几十年来基因治疗最常用的病毒载体。
发明内容
本发明构建高表达rAAV表达载体,引导抗VEGF和抗Ang2抗体基因片段在体内高表达;用AAV包装表达基因,输送到眼部玻璃腔表达,用于治疗与新生血管增生相关性的眼部黄斑病变疾病。这种基因药物给药方式,单次给药,引导治疗基因的长期表达,能克服反复注射引起的眼球损伤,造福患者,并且与目前重组蛋白疗法相比,医疗费用将明显降低。
术语
为了可以更容易地理解本公开,首先定义某些术语。如本申请中所使用的,除非本文另有明确规定,否则以下术语中的每一个应具有下面给出的含义。
术语“启动子”意指启动特定基因的转录所需的由哺乳动物细胞中的酶/蛋白质识别的DNA序列。例如,与RNA聚合酶和/或任何相关因子结合并在此处启动转录的核苷酸序列。本文描述了启动子的非限制性实例,例如但不限于CB7启动子、巨细胞病毒(CMV)启动子、劳氏肉瘤病毒(RSV)启动子、GFAP启动子(胶质原纤维酸性蛋白)、MBP启动子(髓磷脂碱性蛋白)、MMT启动子、EF-1α启动子、UB6启动子、鸡β-肌动蛋白启动子、CAG启动子、RPE65启动子和视蛋白启动子、肝特异性启动子,诸如TBG(甲状腺素结合球蛋白)启动子、APOA2启动子、SERPINA1(hAAT)启动子、或mIR122启动子、或肌肉特异性启动子,诸如人结蛋白启动子或Pitx3启动子、诱导型启动子,诸如缺氧诱导型启动子或药物诱导型启动子。在某些实施方案中,本文提供的病毒载体包含一个或多个控制转基因表达的启动子。在某些实施方案中,启动子是组成型启动子。在一些实施方案中,CB7启动子包括增强通过载体驱动的转基因表达的其他控制元件。在某些实施方案中,启动子包括TATA盒。在某些实施方案中,启动子是CBA启动子(chickenβ-actin),核酸序列见SEQ ID NO:1。
术语“增强子”是指可增加编码感兴趣的蛋白,例如,特异性结合到VEGF的抗体或其抗原结合抗体片段或可溶性VEGF受体的核酸的转录水平的核苷酸序列。增强子序列(长度为50-1500个碱基对)通常通过为转录相关蛋白(例如,转录因子)提供额外的结合位点来增加转录水平。在一些实施方案中,在内含子序列内发现增强子序列。与启动子序列不同,增强子序列可在离转录起始位点更大的距离处起作用(例如,与启动子相比)。增强子的非限制性实例包括RSV增强子、CMV增强子或SV40增强子。
术语“内含子”是指基因中无编码作用的片段或间插序列。例如,鸡β-肌动蛋白内含子、小鼠微小病毒(MVM)内含子、人因子IX内含子、β-珠蛋白剪切供体/免疫球蛋白重链剪切受体内含子、腺病毒剪切供体/免疫球蛋白剪切受体内含子、SV40晚期剪切供体/剪切受体(19S/16S)内含子和杂合(hybrid)腺病毒剪切供体/IgG剪切受体内含子和多腺苷酸化信号,诸如兔β-珠蛋白多腺苷酸化信号、人生长激素(hGH)多腺苷酸化信号、SV40晚期多腺苷酸化信号、合成的多聚腺苷酸(SPA)信号和牛生长激素(bGH)多腺苷酸化信号等。在某些实施方案中,所述内含子是合成内含子chicken-β-actin intron(SEQ ID NO:2),在某些实施方案中,所述内含子是合成内含子chimeric intron(SEQ ID NO:3)。
术语“信号肽”是指存在于新生分泌蛋白的N端,但不存在于天然存在的成熟蛋白中的序列。在信号肽被翻译后,“信号肽”被蛋白酶(例如,信号肽酶)裂解。一种方法是使用来自与被表达的蛋白同源的蛋白的信号肽。例如,人抗体信号肽可以用于在CHO或其他细胞中表达IgG。另一种方法是鉴定针对用于表达的特定宿主细胞优化的信号肽。信号肽可以在不同蛋白之间或甚至在不同生物的蛋白之间互换,通常细胞类型的最丰富分泌的蛋白的信号序列被用于蛋白表达。例如,发现血浆中最丰富的蛋白——人白蛋白——的信号肽显著增加在CHO细胞中的蛋白生产收率。在优选实施方案中,信号序列与重链和轻链序列两者融合。优选的序列是用于在眼部/CNS组织中表达的信号肽。信号肽的非限制性实例包括:信号肽Vh-Leader,氨基酸序列为SEQ ID NO:4或SEQ ID NO:5。
术语“连接肽”是指分隔重链和轻链编码的序列。例如,弗林蛋白酶-F2A连接肽(Fang等,2005,Nature Biotechnology 23:584-590,和Fang,2007,Mol Ther 15:1153-9,它们各自通过引用完整地结合于此)可以结合到表达盒中以分隔重链和轻链编码序列。使用的另外的连接肽包括但不限于:连接肽1(SEQ ID NO:8)、连接肽2(SEQ ID NO:9)、连接肽3(SEQ ID NO:16)、连接肽4(SEQ ID NO:17)、Furin-F2A连接肽(SEQ ID NO:12或SEQ IDNO:13)。
术语“poly(A)信号序列”或“多聚腺苷酸化信号序列”是指触发mRNA的核酸内切酶裂解并且在裂解的mRNA的3’端添加一系列腺苷的序列。在一些实施方案中,poly(A)信号序列位于编码抗体重链、抗体轻链或抗原结合抗体片段的核酸序列的3’处。Poly(A)尾巴及其结合的蛋白质有助于保护mRNA免受核酸外切酶的降解。多聚腺苷酸化对于转录终止、mRNA从细胞核中输出和翻译也很重要。DNA转录为RNA后,多聚腺苷酸化在细胞核中发生,但随后也可在细胞质中发生。转录终止后,通过与RNA聚合酶相关联的核酸内切酶复合物的作用使mRNA链裂解。存在可使用的若干种poly(A)信号序列,包括源自以下的那些序列:牛生长激素(bGH)、小鼠β-珠蛋白、小鼠α-珠蛋白、人胶原蛋白、多瘤病毒、单纯疱疹病毒胸苷激酶基因(HSV TK)、IgG重链基因多聚腺苷酸化信号、人生长激素(hGH)、由SV40 poly(A)位点组成的组,诸如SV40晚期和早期的poly(A)位点等。在某些实施方案中,所述poly(A)序列是人的生长激素PolyA(hGH poly(A)signal)(SEQ ID NO:20)。
本发明用于体内表达抗VEGF抗体基因,Anti-VEGF重链(Vh)氨基酸序列为SEQ IDNO:7,Anti-VEGF轻链(VL-opt)氨基酸序列为SEQ ID NO:11。
本发明用于体内表达抗VEGF抗体基因,Anti-VEGF重链(Vh)核酸序列为SEQ IDNO:6,Anti-VEGF轻链(VL-opt)核酸序列SEQ ID NO:10。
本发明用于体内表达抗Ang2抗体基因,Anti-Ang2重链(Vh)氨基酸序列为SEQ IDNO:15,Anti-Ang2轻链(VL)氨基酸序列为SEQ ID NO:19。
本发明用于体内表达抗Ang2抗体基因,Anti-Ang2重链(Vh)核酸序列为SEQ IDNO:14,Anti-Ang2轻链(VL)核酸序列为SEQ ID NO:18。
一方面,本发明提供一种重组核苷酸序列(抗体基因体内表达框)。
在某些实施方式中,表达框单元序列包括选自下组的核苷酸序列:
a)所述核苷酸序列包含SEQ ID NO:10(anti-VEGF轻链)和SEQ ID NO:6(anti-VEGF重链);
b)所述核苷酸序列包含与SEQ ID NO:10(轻链)有≥99%相同性的序列,以及与SEQ ID NO:6(重链)有≥99%相同性的序列;
c)所述核苷酸序列包含与SEQ ID NO:10(轻链)有≥95%相同性的序列,以及与SEQ ID NO:6(重链)有≥95%相同性的序列;
d)所述核苷酸序列包含与SEQ ID NO:10(轻链)有≥99%相同性的序列,以及与SEQ ID NO:6(重链)有≥95%相同性的序列;
e)所述核苷酸序列包含与SEQ ID NO:10(轻链)有≥90%、≥95%、≥96%、≥97%、≥98%、≥99%、≥99.5%或100%相同性的序列,以及与SEQ ID NO:6(重链)有≥90%、≥95%、≥96%、≥97%、≥98%、≥99%、≥99.5%或100%相同性的序列。
在某些实施方式中,表达框单元序列包括选自下组的核苷酸序列:
a)所述核苷酸序列包含SEQ ID NO:18(Ang2轻链)和SEQ ID NO:14(Ang2重链);
b)所述核苷酸序列包含与SEQ ID NO:18(轻链)有≥99%相同性的序列,以及与SEQ ID NO:14(重链)有≥99%相同性的序列;
c)所述核苷酸序列包含与SEQ ID NO:18(轻链)有≥95%相同性的序列,以及与SEQ ID NO:14(重链)有≥95%相同性的序列;
d)所述核苷酸序列包含与SEQ ID NO:18(轻链)有≥99%相同性的序列,以及与SEQ ID NO:14(重链)有≥95%相同性的序列;
e)所述核苷酸序列包含与SEQ ID NO:18(轻链)有≥90%、≥95%、≥96%、≥97%、≥98%、≥99%、≥99.5%或100%相同性的序列,以及与SEQ IDNO:14(重链)有≥90%、≥95%、≥96%、≥97%、≥98%、≥99%、≥99.5%或100%相同性的序列。
在某些实施方式中,所述表达框还包括选自下组的一个或多个核苷酸序列:
在某些实施方式中,启动子是CBA启动子(chickenβ-actin),核酸序列见SEQ IDNO:1。
在某些实施方式中,启动子前还有增强基因表达的CMV增强子。
在某些实施方式中,所述内含子是合成内含子chicken-β-actin intron(SEQ IDNO:2),在某些实施方案中,所述内含子是合成内含子chimeric intron(SEQ ID NO:3)。在某些实施方案中,所述内含子是合成内含子chicken-β-actin intron(SEQ ID NO:2)和chimeric intron(SEQ ID NO:3)。
在某些实施方式中,所述信号肽Vh-Leader,氨基酸序列为SEQ ID NO:4或SEQ IDNO:5。
在某些实施方式中,所述连接肽为连接肽1(SEQ ID NO:8)、连接肽2(SEQ ID NO:9)、连接肽3(SEQ ID NO:16)、连接肽4(SEQ ID NO:17)、Furin-F2A连接肽(SEQ ID NO:12或SEQ ID NO:13)。
在某些实施方案中,所述poly(A)序列是人的生长激素PolyA(hGH poly(A)signal)(SEQ ID NO:20)。
在某些实施方式中,所述表达框包含,CMV增强子、CBA启动子、合成内含子chicken-β-actin intron和chimeric intron、Vh-Leader、anti-VEGF重链、连接肽、anti-VEGF轻链、Furin-F2A、Vh-Leader、Ang-2重链、连接肽、Ang-2轻链、hGH polyA。在某些实施方式中,以上几个核苷酸序列的相邻两个序列之间有多余的核苷酸序列存在。
本发明的另一方面,提供了一种腺相关病毒表达载体,其特征在于,所述载体含有前述的编码抗VEGF抗体和抗Ang-2抗体的核苷酸序列。
本发明的另一方面,提供了一种腺相关病毒表达载体,其特征在于,所述载体编码抗VEGF抗体和抗Ang-2抗体,编码的抗VEGF抗体氨基酸轻链序列为SEQ ID NO:11,重链氨基酸序列为SEQ ID NO:7;编码的抗Ang-2抗体的氨基酸轻链序列为SEQ ID NO:19,重链序列为SEQ ID NO:15。
在某些实施方式中,所述载体为腺相关病毒衣壳蛋白改造过的有关合成载体,包括但不限于腺相关病毒血清型AAV2、AAV5、AAV8中的一种或多种。
优选地,所述腺相关病毒为AAV2。
本发明的另一方面,提供了一种药物制剂,其特征在于,含有前述编码anti-VEGF抗体和Ang2抗体的重组核酸或腺相关病毒载体。在某些实施方式中,所述药物制剂还包含药学上可接受的载体和/或赋形剂;
在某些实施方式中,所述药物制剂为液体制剂。
本发明的另一方面,提供了如本发明所述的重组核酸、腺相关病毒载体或药物制剂在治疗脉络膜新生血管性疾病中的用途。
在某些实施方式中,所述脉络膜新生血管性疾病为年龄相关性黄斑变性(AMD)或糖尿病视网膜病变(DR)。
在某些实施方式中,所述糖尿病视网膜病变为糖尿病黄斑水肿(DMO)。
在某些实施方式中,所述药物制剂在眼内注射;优选地在玻璃体腔注射。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他附图。
图1A显示的是pAAV-CAG-chimeric-aVEGF_sFv-aAng2_sFV病毒载体的质粒图谱;
图1B显示的是pAAV-CAG-chimeric-aVEGF_sFv-aAng2_sFV病毒载体的质粒图谱;
图2显示的是pAAV-CAG-chimeric-aVEGF_sFv-aAng2_sFv病毒载体质粒细胞转染和表达;
图3A显示的是pAAV-CAG-chimeric-aVEGF_sFv-aAng2_sFv病毒载体质粒在Expi293细胞的表达;
图3B显示的是BCA法测定anti-VEGF_sFv蛋白质浓度所做标准曲线;
图3C显示的是BCA法测定anti-Ang2_sFv蛋白质浓度所做标准曲线;
图4A-F显示的是ELISA验证载体表达的anti-VEGF_sFv和anti-Ang2_sFv的特征型结合和结合选择性;
图5显示的是三质粒系统分别生产AAV2-aVEGF_sFv-aAng2_sFv和AAV2-aAng2_sFv。
图6A显示PBS对照组与AAV2-aVEGF_sFv-aAng2_sFv实验组2周与1月FFA结果(箭头指示渗漏位置)。
图6B显示PBS对照组与AAV2-aVEGF_sFv-aAng2_sFv实验组2周与1月渗漏面积统计结果(箭头指示渗漏位置)。
具体实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚、明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用于解释本发明,并不用于限制本发明。
需要说明的是,本发明的说明书和权利要求书及上述附图中的术语“第一”、“第二”等是用于区别类似的对象,而不必于描述特定的顺序或先后次序。应该理解这样使用的数据在适当情况下可以互换,以便这里描述的本发明的实施例。此外,术语“包括”和“包含”以及他们的任何变形,意图在于覆盖不排他的包含,例如,包含了一系列步骤或单元的过程、方法、系统或设备不必限于清楚地列出的那些步骤或单元,而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其他步骤或单元。
需要说明的是,在不冲突的情况下,本发明中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
重组核酸分子表达载体
一方面,本发明提供一种重组核酸分子,所述重组核酸分子可以含有编码抗VEGF抗体的核苷酸序列和编码抗Ang2抗体的核苷酸序列,所述重组核酸分子可以表达所述包含SEQ ID NO:7和SEQ ID NO:11所示的氨基酸序列的抗体,以及包含SEQ ID NO:15和SEQ IDNO:19所示的氨基酸序列的抗体。
另一方面,本发明提供一种腺相关病毒载体,所述载体含有前述的编码抗VEGF抗体基因片段的核苷酸序列和编码抗Ang2抗体的核苷酸序列。
所述载体为腺相关病毒衣壳蛋白改造过的有关合成载体,包括但不限于腺相关病毒血清型AAV2、AAV5、AAV8中的一种或多种。
药物制剂
本发明的另一方面,提供了一种药物制剂,其特征在于,含有前述编码anti-VEGF抗体和Ang2抗体的重组核酸或腺相关病毒载体。在某些实施方式中,所述药物制剂还包含药学上可接受的载体和/或赋形剂。
在某些实施方式中,所述药物制剂为液体制剂。
用途
本发明的另一方面,提供了如本发明所述的重组核酸、腺相关病毒载体或药物制剂在治疗脉络膜新生血管性疾病中的用途。
在某些实施方式中,所述脉络膜新生血管性疾病为年龄相关性黄斑变病(AMD)或糖尿病视网膜病变(DR)。
在某些实施方式中,所述药物制剂在眼内注射;优选地在玻璃体腔注射。
实施例
实施例1:病毒载体的构建
pAAV-CAG-chimeric-aVEGF_sFv-aAng2_sFv(sFv为单链Fv段)病毒载体构建(A)。载体基因组包含AAV反向末端重复序列ITR、CBA启动子(chickenβ-actin,核酸序列见SEQID NO:1)和单链抗体目的基因序列。其中在CBA启动子前加上增强基因表达的CMV增强子。目的基因表达框包含一段合成内含子(SEQ ID NO:2-3)、信号肽(SEQ ID NO:4-5)、重链(SEQ ID NO:6-7或SEQ ID NO:14-15)、轻链(SEQ ID NO:10-11或SEQ ID NO:18-19)、以连接肽(SEQ ID NO:8-9或SEQ ID NO:16-17)将轻链和重链连接,Furin-F2A连接肽(SEQ IDNO:12-13)分隔连接两个单链抗体,以及能够增加转录产物稳定性的人的生长激素poly(A)序列SEQ ID NO:20。
实施例2:病毒载体质粒细胞转染表达和表达验证
HEK293T细胞生长,消化收集细胞,接种到6孔板内,接种密度为1×106,分为空白组(PBS组)和转染组。取每个质粒2.5μg,聚乙烯亚胺(PEI)进行转染,空白组加入同样量的PBS。转染72小时后,收集细胞培养上清,使用经还原剂处理和未经还原剂处理的细胞上清液,每孔上30ul样品进行SDS-PAGE凝胶电泳分离和蛋白质印迹(Western blot)。1:1000孵育雷珠单抗(Ranibizumab,商品名Lucentis)的抗体(anti-Ranibizumab mAb;Gensceiptcat#A02035-40)后孵育羊抗鼠IgG二抗(Goat anti-mouse IgG(HRP)),如图2显示,样品顺序从左至右依次,各质粒表达anti-VEGF_sFv蛋白条带清晰(图示标注aVEGF_sFv),分子量与Ranibizumab_sFv(26KDa)一致。
实施例3:病毒载体在体外研究的表达和定量
Expi293F细胞生长密度达到3E6/mL以上,收集细胞,接种到20mL(100mL培养瓶内),接种密度为3×106cells/mL。取每个质粒20μg,PEI进行转染,质粒:PEI比例为1:3。转染72小时后,收集细胞培养上清,用Hitrap蛋白L亲和柱纯化单链可变片段(sFv)并用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离。如图3A显示,质粒表达anti-VEGF_sFv蛋白条带清晰(图3A左图),分子量与Ranibizumab_sFv一致;anti-Ang2_sFv条带清晰(图3A右图)。使用BCA法测定蛋白质浓度,根据所作标准曲线(图3B、图3C)与测得OD值计算蛋白浓度(表1、表2),由anti-VEGF_sFv样品1与样品2浓度平均值计算得anti-VEGF_sFv浓度约为1.2mg/4mL,anti-Ang2_sFv样品1与样品2浓度平均值计算得anti-Ang2_sFv浓度约为3.2mg/4mL。
表1anti-VEGF sFv蛋白浓度
表2anti-Ang2_sFv蛋白浓度
实施例4:ELISA验证载体表达的anti-VEGF_sFv和anti-Ang2_sFv的特征型结合和结合选择性
高吸附结合96孔板,不同列分别加样铺板:人VEGF165(VEGF-A),VEGF-B,VEGF-C,VEGF-D和人Ang2蛋白。各蛋白用碳酸盐缓冲液进行稀释至0.5μg/ml,100μl/孔加入包被板中(人VEGF165为1.31pmol/孔,人Ang2为0.88pmol/孔)4℃过夜包被。根据ELISA标准操作过程,每孔加入不同浓度(浓度如下表3所示)的anti-Ang2_sFv(50ng=2pmol)或anti-VEGF_sFv(50ng=1.92pmol)的孵育液,37℃进行孵育。Protein L-HRP作为二抗孵育(孵育anti-Ang2_sFv时比例为1:20000;孵育anti-VEGF_sFv时比例为1:10000),加入HRP底物显色后终止反应,检测anti-VEGF_sFv与人VEGF-A、VEGF-B,VEGF-C,and VEGF-D的结合;anti-Ang2_sFv与人Ang2的结合。结合活性结果见图4A、4B。如图4A所示,anti-Ang2_sFv与重组人血管生成素2(hrAng2)结合(图4A),anti-VEGF_sFv与重组人VEGF-165结合(图4B),两种sFv抗体的结合反应是剂量反应性的,anti-Ang2_sFv与重组人血管生成素2(hrAng2)的结合活性高于anti-VEGF_sFv与人VEGF165的结合活性。如图4C-F所示,anti-VEGF_sFv与重组人VEGF-A结合(图4C),但不与重组人VEGF-B(图4D),VEGF-C(图4E)和VEGF-D结合(图4F)。
表3 anti-Ang2_sFv与anti-VEGF_sFv浓度梯度(ng/ml)
实施例5:三质粒系统分别生产AAV2-aVEGF_sFv-aAng2_sFv和AAV2-aAng2_sFv
pAdHelper、pAAV-RC2、pAAV-CAG-chimeric-aVEGF_sFv-aAng2_sFv-hGH三质粒共转染293T细胞,收取细胞。用细胞裂解液重悬细胞,冻融1次。采用超速离心、POROSCaptureSelectAAV Resins、浓缩三步来分离、浓缩和纯化本申请所述AAV2-aVEGF_sFv-aAng2_sFv。制备SDS-PAGE分离胶和积层胶。电泳完毕后用考马斯亮蓝染色,常规脱色液脱色,作为病毒载体纯度鉴定。图5所示AAV2-GFP、AAV2-aAng2_sFv和AAV2-aVEGF_sFv-aAng2_sFv纯化病毒载体纯度鉴定:胶图显示AAV2三种衣壳蛋白的比例为VP1/VP2/VP3=1:1:10,符合AAV2衣壳蛋白比例特征,条带清晰。
实例6AAV2-aVEGF_sFv-aAng2_sFv兔RNV模型体内药效分析
荷兰兔玻璃体腔内(IVT)注射DL-α-氨基己二酸(AAA)构建兔视网膜新生血管(RNV)模型,导致兔眼部持续渗漏。定量血管渗漏面积,观察AAV2-aVEGF_sFv-aAng2_sFv改善血管渗漏的程度,用以研究AAV2-aVEGF_sFv-aAng2_sFv的生物效应。造模成功兔RNV模型且经荧光素眼底血管造影术(FFA)检测后(D0)分为实验组和对照组(n=3),实验组玻璃体腔注射50μL的AAV2-aVEGF_sFv-aAng2_sFv药物剂量为1E10vg/eye,对照组玻璃体腔注射50μL的PBS。注射药物或PBS后7天内两组均每天滴加消炎眼药水,每天观察兔眼状态。
注射药物或PBS 2周(2W)、1月(1M)后实验组与对照组荷兰兔经静脉注射10%荧光素钠进行FFA观察采图,使用imageJ统计给药前后血管渗漏面积并进行统计学分析。图6A所示为对照组与实验组2周与1月FFA结果,实验组与给药前相比2周时渗漏面积平均减少90%,1月时渗漏面积平均减少93%,而对照组与给药前相比渗漏面积仅减少4%,1月时渗漏面积减少6%。使用GraphPad prism进行t检验统计分析显示(图6B)2周时两组间渗漏面积变化有显著性差异(p<0.01),1月时两组间渗漏面积变化也有统计学差异(p<0.001)。证明AAV2-aVEGF_sFv-aAng2_sFv可以有效抑制血管渗漏,可用于治疗脉络膜新生血管性疾病,例如年龄相关性黄斑变性(AMD)或糖尿病视网膜病变(DR),其中糖尿病视网膜病变可以是糖尿病黄斑水肿(DMO)。
以上仅为本发明的较佳实施例而已,并不用于限制本发明,凡是在本发明的精神和原则之内所作的任何修改、等同替换、变形和改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种融合核酸,其包含:
a)所述融合核酸的核苷酸序列包含SEQ ID NO:6、SEQ ID NO:10、SEQ ID NO:14、SEQID NO:18;
b)所述融合核酸的核苷酸序列包含与SEQ ID NO:6、SEQ ID NO:10、SEQ ID NO:14、SEQID NO:18有≥99%相似性的序列;
c)所述融合核酸的核苷酸序列包含与SEQ ID NO:6、SEQ ID NO:10、SEQ ID NO:14、SEQID NO:18有≥95%相似性的序列;
d)所述融合核酸的核苷酸序列包含与SEQ ID NO:6、SEQ ID NO:10、SEQ ID NO:14、SEQID NO:18有≥90%相似性的序列。
2.如权利要求1所述的融合核酸,其特征在于,还包含启动子、内含子、信号肽、连接肽、Furin-F2A和polyA序列。
3.如权利要求2所述的融合核酸,其特征在于,包含如SEQ ID NO:1所示的启动子序列、如SEQ ID NO:2和/或SEQ ID NO:3所示的内含子序列,如SEQ ID NO:4和/或SEQ ID NO:5所示的信号肽序列,如SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:16、SEQ ID NO:17所示的连接肽序列,如SEQ ID NO:12和SEQ ID NO:13所示的Furin-F2A序列,如SEQ ID NO:20所示的polyA序列。
4.一种融合核酸,其特征在于,包含如权利要求1-3任一项所述的核苷酸的互补链、mRNA序列。
5.一种融合核酸,其特征在于,其表达如SEQ ID NO:7、SEQ ID NO:11、SEQ ID NO:15、SEQ ID NO:19所示的氨基酸序列。
6.一种腺相关病毒载体,其特征在于,所述载体含有权利要1-5任一项所述的融合核酸。
7.一种腺相关病毒载体,其特征在于,所述载体编码如SEQ ID NO:7、SEQ ID NO:11、SEQ ID NO:15、SEQ ID NO:19所示的氨基酸序列。
8.如权利要求6或7所述的载体,其特征在于,所述载体血清型选自AAV2、AAV5、AAV8中的一种或多种。
9.一种药物制剂,其特征在于,所述药物制剂含有如权利要求6-8任一项所述的载体,以及药学上可接受的载体或赋形剂。
10.如权利要求6-8任一项所述的载体在制备药物中的用途,所述药物用于治疗脉络膜新生血管性疾病,所述脉络膜新生血管性疾病为年龄相关性黄斑变性或糖尿病视网膜变性,优选地,所述糖尿病视网膜变性为糖尿病黄斑水肿。
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