CN113041360A - 一种用于治疗年龄相关性黄斑变性的药物 - Google Patents
一种用于治疗年龄相关性黄斑变性的药物 Download PDFInfo
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Abstract
本发明属于生物技术领域，具体涉及一种用于治疗年龄相关性黄斑变性的药物，所述药物为由血管内皮生长因子抑制蛋白和补体抑制蛋白组成的融合蛋白，可用于治疗由血管内皮生长因子和/或补体激活而导致的疾病。该融合蛋白可以有多种方式进行给药，其具体形式包括但不限于，重组蛋白，通过纳米材料包裹技术直接递送编码该融合蛋白的基因，以及通过各种病毒载体直接递送编码该融合蛋白的基因。本发明的药物属于基因疗法，能稳定释放药物，不会在治疗周期内产生眼内药物浓度剧烈波动，仅需一次注射即可，减少了病人去医院的频率，并且可以预防湿性黄斑变性向干性转变，消除治疗隐患。
Description
技术领域
本发明属于生物技术领域，具体涉及一种用于治疗年龄相关性黄斑变性的药物。
背景技术
年龄相关性黄斑变性(age-related macular degeneration，AMD)是一种常见的老年病，表现为中央视觉模糊，是50岁以上人群失明的主要原因。根据病程，可分为早期，中期和晚期三种类型。而晚期又分为“干性”和“湿性”两种类型。干性年龄相关性黄斑变性(dryAMD)占AMD总数约90％，剩下的约10％为湿性年龄相关性黄斑变性(wetAMD)。两种类型的AMD都有十分复杂的发病原因，包括遗传因素。对于湿性AMD，一个比较明确的原因是血管内皮生长因子(VEGF)促进新的血管不断在色素上皮层下活跃地生成，从而引起一系列渗出、出血、瘢痕改变，从而导致黄斑变性。而对于干性AMD，一个比较明确的原因是免疫系统中的补体系统异常激活，攻击自身眼部的黄斑，从而造成其损坏。
由于VEGF在湿性AMD中的作用，治疗湿性AMD的一个常用方法是在眼内玻璃体腔中注射VEGF的拮抗蛋白或抗体，通过抑制新血管的产生而治疗疾病。已经批准用于治疗湿性AMD的VEGF拮抗蛋白或抗体包括：brolucizumab(抗VEGF单链抗体，商品名Beovu),aflibercept(VEGF受体融合蛋白，商品名Eylea和Zaltrap)和ranibizumab(抗VEGF抗体片段，商品名Lucentis)等。
尽管基于蛋白质的抗VEGF疗法疗效显著，但其在使用过程中的一个主要缺点是需要每4至8周进行一次玻璃体腔注射。治疗过程中频繁地对患者眼睛进行穿刺注射药物，极易对患者造成二次损伤，包括白内障，眼内出血，视网膜脱落和炎症等并发症。不仅将患者置于并发症的风险之中，还需要患者频繁地前往医院完成注射，造成了极大的不便和痛苦。并且湿性AMD的长期随访表明，患者的平均注射频率若低于推荐注射频率的一半，往往会导致疾病进展和视力下降。相反，接受定期或更频繁的抗VEGF治疗的患者的视力明显改善。除了这些依从性问题之外，每月的注射还会导致眼内药物的浓度处于一个持续变化的状态，可能刚完成注射时药物剂量过高，而月尾的时候眼内药物浓度又会过低。
对于干性AMD，目前尚无有效的方法。但考虑到补体激活在干性AMD病程进展中的作用，一些补体抑制剂有可能对治疗干性AMD有效。
发明内容
针对现有技术的缺陷，本发明提供了一种用于治疗年龄相关性黄斑变性的药物。本发明提供的药物属于基因疗法，能稳定释放药物，不会在治疗周期内产生眼内药物浓度剧烈波动，仅需一次注射即可，减少了病人去医院的频率，并且可以预防湿性黄斑变性向干性转变，消除治疗隐患。
为了达到上述目的，本发明采用的技术方案为：
一种用于治疗年龄相关性黄斑变性的药物，所述药物为由血管内皮生长因子抑制蛋白和补体抑制蛋白组成的融合蛋白。
优选地，所述血管内皮生长因子抑制蛋白的氨基酸序列如SEQ ID NO.1所示；所述补体抑制蛋白的氨基酸序列如SEQ ID NO.2所示。
>SEQ ID NO.1
MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
>SEQ ID NO.2
LQCYNCPNPTADCKTAVNCSSDFDACLITKAGLQVYNKCWKFEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQLEN；
优选地，所述融合蛋白由血管内皮生长因子抑制蛋白和补体抑制蛋白直接以任何顺序融合在一起，或通过四个以上柔性的氨基酸链接序列融合在一起。
优选地，所述融合蛋白通过基因工程表达融合蛋白，然后直接将重组蛋白注射进患者体内；或通过纳米材料包裹技术直接递送融合蛋白或编码该融合蛋白的基因进入患者体内，或通过各种病毒载体直接递送编码该融合蛋白的基因进入患者体内。
优选地，所述基因工程为通过细菌表达系统，酵母表达系统，昆虫杆状病毒或者哺乳动物细胞表达系统表达所述融合蛋白。
优选地，所述纳米材料包裹技术为通过纳米颗粒包裹融合蛋白或编码该融合蛋白的基因，帮助融合蛋白进入细胞或者直接在细胞内表达而发挥生物学功能。
优选地，所述病毒载体为腺相关病毒，腺病毒，慢病毒和痘苗病毒中的一种。
优选地，所述药物通过眼睛玻璃体内注射，或视网膜下注射，或脉络膜上注射方式给药。
本发明提供的融合蛋白可以同时抑制VEGF和补体，从而可以同时治疗由VEGF表达而引起的疾病，如湿性年龄相关性黄斑变性(wet AMD)，和由补体激活而引起的疾病，如干性年龄相关性黄斑变性(dry AMD)。本发明融合蛋白中的补体抑制蛋白部分可以抑制补体激活，故而此优选融合蛋白除了可以治疗wet AMD之外，也可以预防dry AMD的发生。
本发明利用基因药物进行治疗，仅需一次给药，终生有效。传统方法需要体外合成有治疗功能的蛋白质，多次给药注射入患者眼中。频繁的注射可能会增加并发症风险。本发明提供的治疗疗法通过将可以表达Aflibercept-CD59融合蛋白的AAV病毒注射到患者眼睛，使得患者眼内细胞可以自发并持续表达具有治疗作用的蛋白药物从而避免多次外部药物注射，且降低了成本。利用其中的蛋白药物CD59同时预防视网膜干性黄斑变性。相对于其它产品，具有对潜在的病情恶化的预防作用。
本发明采用专利申请号为202021161190.2的注射器针帽结合胰岛素注射器进行注射，与传统注射方式相比，改变了注射方式。脉络膜上腔作为眼睛巩膜下的一个潜在空间富含毛细血管且接近视网膜，将药物注射到脉络膜上腔可以在避免视网膜穿刺的情况下极大地提高药物在眼内的扩散。而且相对于传统的玻璃体腔注射需要刺入眼内4mm左右，脉络膜上腔注射只需要刺入眼0.75mm左右即可完成注射给药，极大地降低了注射导致的眼部并发症的形成。
本发明基于基因治疗递送抗VEGF蛋白Aflibercept和补体调节蛋白CD59可能提供了一个很有前途的替代蛋白治疗。这种方法可以使脉络膜新生血管处的外部视网膜和脉络膜产生稳定且持续的治疗水平药物，并减少频繁注射的需要；在脉络膜上腔注射方式仅仅刺入眼内0.75mm深，可以极大减少注射所引发的并发症。
现有的眼内注射方式有多种(可参见图2)：玻璃体腔注射需要将针头伸入眼睛内部进行给药，而视网膜下注射则需要将针头完全穿过眼睛，从而在眼的另一侧视网膜底部进行给药。这两种眼内注射或手术都可能导致眼内炎，且手术时间越长、越复杂，风险就越大。并且这类手术普遍都存在与针头相关的术后并发症，包括眼内感染，眼内炎症，结膜下出血，巩膜损伤和强烈的疼痛。
其中，视网膜下注射将光感受器与视网膜色素上皮分离开来，这会损害正常眼睛的光感受器，况且对于因遗传性视网膜变性而受损的眼睛来说，可能尤其有害。视网膜变性的眼睛通常也存在视网膜下纤维化，这增加了视网膜与色素上皮细胞之间的粘附，需要提高输液压力来产生视网膜下液泡。但是由于中央凹是黄斑最薄的部分，受压的视网膜下液可能通过中央凹逃逸，形成一个黄斑孔，这可能会降低视力。此外，黄斑孔的形成使药品逃逸到玻璃体腔内，造成药效的降低。
视网膜下载体注射后，药品的作用位置几乎完全发生在液泡区域(感光细胞和视网膜色素上皮细胞被含载体的液体分开的区域)。液泡的大小和位置很重要，但并不总是容易控制，因为最小阻力的路径决定了液泡扩散的方向，在手术时对视网膜的检查无法预测液泡的走向。有时，一个液泡从视网膜下注射部位对称地向外延伸，形成一个圆圈；有时，它向一个方向不对称地向视网膜周边扩散，未能触及目标视网膜后区。与x轴或y轴相比，液泡可能沿z轴延伸得更多，导致高液泡涉及相对较小的视网膜和视网膜色素上皮细胞区域。
这种不可预测性可能是眼内药物治疗的作用位置和数量变化的来源，导致在一些病人中可能出现很差的治疗疗效。并且，在不同位置进行多次视网膜下注射可能有助于将目标视网膜区域和视网膜色素上皮细胞暴露给病灶，从而增加并发症的风险。
随着现有技术的不断迭代，科学家发现眼睛内的脉络膜上腔是一个更为理想的给药位点，因为其富含血管，药物可以借由血液流动高效地扩散到整个眼内，并且不同于传统给药方式需要同时刺破巩膜，脉络膜和视网膜，在脉络膜上腔进行给药仅仅只需要刺穿眼睛表面的巩膜，从而极大的降低了注射并发症发作的可能，减少了注射对眼睛的伤害。
CD59又称膜攻击复合物抑制因子。补体激活后，会通过多个因子的级联反应，最终形成C5b6789n复合物，即膜攻击复合物MAC。插入细胞膜的MAC通过破坏局部磷脂双层而形成“渗漏斑”，或形成穿膜的亲水性孔道，最终导致细胞崩解。
本发明提供的药物属于一种基因治疗产品，可以在患者眼中表达抗血管内皮生长因子蛋白和CD59的融合蛋白，它既可以治疗湿性黄斑变性又可以预防其向干性黄斑变性发展。
本发明在进一步研究过程中发现，所述药物中的血管内皮生长因子抑制蛋白除了本申请所述的Aflibercept外，还可以为抗血管内皮生长因子抗体，或者是血管内皮生长因子的可溶性受体，或者是上述抑制蛋白的全部或者部分片段的各种组合，或者上述各种组合与其它功能性蛋白的组合体，比如与抗体重链恒定区的组合体；所述补体抑制蛋白除了CD59外，还可以为抑制补体激活通路的抗体，也可以是抑制补体激活通路的蛋白质或者短肽，包括但不限于Factor H，Factor I等。
与现有技术相比，本发明提供的药物具有如下优点：
(1)本发明提供的药物能稳定释放，不会在治疗周期内产生眼内药物浓度剧烈波动；
(2)本发明提供的药物注射一次即可，无需频繁注射，减少了病人去医院的频率，减轻病人心理压力，且注射方式对患者的损伤较小，相对于玻璃体腔注射疼痛更少且几乎没有并发症，且能更均匀高效的将药物分布在视网膜及脉络膜细胞中；
(3)本发明提供的药物治疗效果，既可以用于治疗湿性黄斑变性，又可以用于预防了湿性黄斑变性向干性转变，消除了治疗隐患。
附图说明
图1为优选融合蛋白Aflibercept-CD59示意图；
图2为不同眼内注射方式示意图；
图3为本发明实施例所述空表达载体结构示意图；
图4为酶切鉴定阳性克隆结果示意图；
图5为AAV8SYBR Green qPCR原始扩增曲线图；
图6为AAV8SYBR Green qPCR标准曲线图；
图7为AAV8纯度检测SDS-PAGE图；
图8为Aflibercept ELISA标准曲线图；
图9为CD59ELISA标准曲线图；
图10为绵羊红细胞溶血实验结果图；
图11为Aflibercept玻璃体表达量结果图；
图12为CD59玻璃体表达量结果图。
具体实施方式
下面结合具体实施例对本发明作进一步解释，但是应当注意的是，以下实施例仅用以解释本发明，而不能用来限制本发明，所有与本发明相同或相近的技术方案均在本发明的保护范围之内。本实施例中未注明具体技术或条件者，按照本领域常规技术方法和仪器说明书内容进行操作；所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
所述血清型载体pRepCapX和辅助载体pADHelper，来自广州派真生物技术有限公司；所述Aflibrecepte-CD59序列由生工生物工程(上海)股份有限公司合成；所述表达载体pAV-CAG-WPRE-beta-Globin-pA来自山东维真生物科技有限公司；所述质粒提取的试剂盒为QIAprep Spin Miniprep Kit和EndoFree Plasmid Kits，购自Qiagen公司；所述酶切体系中的10×Buffer，SfaAI，MluI，10×T4 Buffer，10U/μL的T4 DNA连接酶均可购自ThermoScientific公司；DMEM高糖培养基购于GIBICO公司；碘克沙醇购于SIGMA公司；鲎试剂、细菌内毒素工作标准品、细菌内毒素检查用水来自湛江安度斯生物有限公司。
实施例1一种用于治疗年龄相关性黄斑变性的基因治疗药物
所述药物由血管内皮生长因子(VEGF)抑制蛋白Aflibercept和补体抑制蛋白CD59直接连接成融合蛋白，所述融合蛋白的氨基酸序列如SEQ ID NO.3所示，编码所述融合蛋白的核苷酸序列如SEQ ID NO.4所示。
>SEQ ID NO.3
MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLQCYNCPNPTADCKTAVNCSSDFDACLITKAGLQVYNKCWKFEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQLEN；
>SEQ ID NO.4
序列4中加粗的序列分别为5’及3’端的酶切位点，倾斜序列分别为起始密码子及终止密码子，画横线的部分为添加的kozak区序列。
以通过各种病毒载体直接递送编码该融合蛋白的基因为例说明本发明所述药物的作用过程：
S1、先将SEQ ID NO.4所示的编码Aflibrecepte-CD59融合蛋白的基因序列克隆至pAV-CAG-WPRE-beta-Globin-pA(该载体的结构示意图如图3所示)，获得重组载体：
(1)用酶切Aflibercept-CD59基因片段，然后经胶回收，获得目的基因片段；酶切时，酶切体系共50μL：Aflibercept-CD59基因片段15μL，10×Buffer 5μL，SfaAI 1.5μL，MluI 1.5μL，ddH2O 27μL，37℃酶切2小时；
(2)用同种酶切载体pAV-CAG-WPRE-beta-Globin-pA，然后进行胶回收，获得酶切载体；酶切时，酶切体系共50μL：浓度为0.5μg/μL的pAV-CAG-aflibercept-WPRE-rabit-polyA质粒4μL，10×Buffer 5μL，SfaAI 1.5μL，MluI 1.5μL，ddH2O 38μL，37℃酶切2小时；
(3)将步骤(1)所得目的基因片段与步骤(2)所得酶切载体进行连接，获得连接产物；其中，连接体系共10μL：目的基因片段2～6μL，载体片段2～4μL，10×T4 Buffer 1μL，10U/μL的T4 DNA连接酶1μL，混匀后，于500rpm转速下离心30s，然后置于22℃下连接1h；
(4)将步骤(3)所得连接产物转化至大肠杆菌DH5α感受态细胞，涂布于相应抗性的LB平板上进行筛选，得成功转化的大肠杆菌菌落；其中，将连接产物转化至大肠杆菌DH5α感受态细胞的具体过程为：
a.从-80℃取出提前制备好的DH5α感受态置于冰浴中；
b.待DH5α感受态细胞融化后，取5μL连接产物于20μL DH5α感受态细胞中，充分混匀，冰浴中静置15分钟；
c.将离心管放入42℃水浴锅中40秒，期间不要摇动离心管，然后快速移至冰浴中，静置2分钟；
d.向离心管中加入200μL的无菌的LB培养基不加抗生素，混匀后置于摇床中37℃，220rpm，振摇1小时；使菌体复苏；
e.涂布到相应抗性的固体培养基平皿中，于37℃培养箱中培养过夜，即可。
(5)挑取步骤(4)所得成功转化的大肠杆菌单菌落后抽提纯化质粒，用SfaAI/MluI双酶切进行酶切鉴定阳性克隆，进一步经测序验证即可；所述质粒用QIAprep SpinMiniprep Kit试剂盒提取，鉴定结果如图4所示；
S2、将步骤S1所得重组表达载体连同AAV8血清型病毒外壳载体和辅助载体转染到HEK 293T细胞中，完成重组腺相关病毒AAV8的包装：具体转染过程为：
(1)准备HEK 293T细胞：提前一天分HEK 293T细胞，包装时细胞密度85％-90％且细胞分布均匀，状态良好；
(2)包装病毒转染前一到两个小时给细胞换液，换为无血清的DMEM培养基(1％HEPES和1％P/S)；
(3)质粒转染，将Aflibercept-CD59目的基因载体，血清型载体pRepCapX和辅助载体pADHelper与PEI混匀，室温静置30分钟；
(4)将上述静置后的液体加到HEK 293T细胞中并做好标记，加入后摇晃混匀。将细胞置于37℃，5％CO2培养箱中培养72h后收集病毒。
S3、将步骤S2所得重组腺相关病毒AAV8经碘克沙醇密度梯度离心进行纯化，并经SYBR Green qPCR测定病毒的滴度，然后检测病毒纯度和内毒素含量即可：
其中，病毒纯化具体过程为：
(1)将细胞吹起，与培养基一并收到50mL离心管中，于500rpm转速下离心15min，离心分离细胞沉淀及上清；
(2)将培养基上清转移到新管中，PEG8000沉淀过夜(每100mL加入2.33gNaCl+8.5gPEG8000)，第二天离心3500g、4℃离心30min，去掉上清，用PBS+0.001％PF68重悬；
(3)将细胞沉淀用PBS+0.001％PF68重悬(重悬目的是将细胞沉淀重悬起来，便于下一步裂解，让细胞内的毒素释放出来)，冻融一次(冻的温度是-80℃，融的温度是37℃)后，加入5M NaCl，涡旋混匀；
(4)将(2)的重悬液与(3)的重悬液混合，振荡混匀后超声至不粘稠，将超声后的液体3500g离心30min，收集上清。
(5)配置不同浓度的碘克沙醇，碘克沙醇浓度梯度为15％、25％、40％、60％。
(6)取一个超离管，逐层加入不同浓度的碘克沙醇。先加浓度高的再加浓度低的，加入不同浓度的碘克沙醇时要缓慢推入，避免浓度高的层与浓度低的层混在一起。
(7)将处理好的病毒液加入到最上层，48000rpm 4℃离心2.5h纯化病毒，离心完毕后，收集病毒。
(8)将收集的病毒置于超滤管中对病毒进行浓缩，将超滤管中剩下的液体反复吹打后吸至病毒储存管中，用病毒储存液(含0.001％F6的PBS)补充至500μL。
SYBR Green qPCR测定病毒的滴度具体过程为：
(1)DNaseⅠ消化病毒样品，去除游离DNA，按照表1所述配制体系
表1 DNaseI消化样品时的体系
取5μL样品加入95μLTris-HCl中稀释20倍，连续稀释2次，共计稀释400倍。取相应数目的PCR管，各管加18μL消化液(表1)，然后样本分别加入2μL稀释后样品、2μL Ref AAV和2μL标准品(2E+8，作为DNase消化对照)，相当于稀释10倍，37度孵育30min。消化后，取5μL样品加入95μLTris-HCl中，共计稀释80000倍。RefAAV消化结束后，取5μl加入95μLTris-HCl中稀释20倍，连续稀释2次，稀释400倍，RefAAV共计稀释4000倍。
(3)SYBR Green qPCR
标准品准备：取含2E+8拷贝/μL的AAV8标准品，按8μL+72μL Tris-HCl做6个连续稀释。则第一个梯度浓度为2E+8拷贝/μL，将其在软件中设置浓度为8E+14GC/mL，以反映样品的稀释梯度。后面系列的梯度依次为8E+13GC/mL，8E+12GC/mL，8E+11GC/mL，8E+10GC/mL，8E+9GC/mL。
| Reagent | Vol.per reaction |
| SYBR PCR试剂(2×) | 10μL |
| ROX(50×) | 0.4μL |
| FWD ITR(50M) | 0.1μL |
| REV ITR(50M) | 0.1μL |
| Nuclease-free water | 4.4μL |
| 样品DNA | 5μL |
其中引物序列，FWD ITR：GGAACCCCTAGTGATGGAGTT，REV ITR：CGGCCTCAGTGAGCGA。
按每个样品做3个重复孔计算，配制相应体积的混合物，每孔分18μL，再各自加入2μL样品。
SYBR Green qPCR条件：
预变性：95℃10min
循环：40cycle：95℃15sec；60℃30sec。
图5为原始扩增曲线(包含了对照和样本的扩增曲线)，图6为根据对照组的扩增结果得出的标准曲线。
病毒纯度检测的具体过程为：
1.衣壳蛋白纯度检测过程：
(1)取病毒液6μL，加入4×上样缓冲液2μL后95℃加热5min。
(2)用4-20％SDS-PAGE10孔梯度胶上样。
(3)电泳1小时，取胶考马斯亮蓝染色1.5h，去除染色液，用脱色液脱色4次，每20分钟换液一次。
(4)电脑成像；具体检测结果如图7所示
2.细菌内毒素检测过程：
参照《中国药典》鲎试剂凝胶法。
本发明通过AAV8导入融合蛋白通过专利号为202021161190.2的装置在脉络膜上注射。该方法简单，方便，可靠，而且可以把药物直接递送到视网膜的病变部位，具有很高的效率。
试验例Aflibercept-CD59生物学功能研究
1.试验样品：
(1)Aflibercept ELISA试剂盒，批号AA115-17MTP，土耳其ImmunoGuide公司；CD59ELISA试剂盒，批号111711825，美国Assaypro公司；4％绵羊红细胞，批号20201204，北京索莱宝科技有限公司；正常人血清，批号20200920，北京索莱宝科技有限公司；绵羊红细胞溶血素，批号20201112，北京索莱宝科技有限公司；含Mg++和EGTA的明胶弗洛拿缓冲液，批号880073，上海联迈生物工程有限公司；GVB缓冲液，批号C11550214，上海麦克林生化科技有限公司；EDTA，批号20200902，福州飞净生物科技有限公司。GVB-EDTA反应终止缓冲液，1mL 0.5M EDTA加入到49ml GVB缓冲液中，混匀备用；
(2)Aflibercept-CD59过表达载体转染的HEK 293T细胞上清；
(3)Eppendorf桌面台式离心机、涡旋震荡仪、酶标分析仪(DNM-9602，北京普朗新技术有限公司)。
2.试验方法：
2.1检测Aflibercept和CD59的表达：
细胞培养上清中Aflibercept和CD59的表达量检测，分别按照AfliberceptELISA试剂盒和CD59ELISA试剂盒的产品说明书进行。
2.2.绵羊红细胞溶血实验：
2.2.1取2mL 4％的绵羊红细胞于15mL离心管中，加入4ml GVB-Mg-EGTA缓冲液重悬细胞，500g，4℃，离心10min，去上清，重复洗涤细胞两次或洗涤至上清无色透明，用GVB-Mg-EGTA缓冲液重悬细胞并计数，调整细胞浓度为108个/mL；
2.2.2取100μL绵羊红细胞(1×107个)于EP管中，每管加入100μL细胞培养上清，对照组加入100μL GVB-Mg-EGTA缓冲液，37℃水浴中孵育30min；2.2.3每管加入0.125μL绵羊红细胞溶血素，300μL正常人血清；用热灭活的正常人血清作为阴性对照组，阳性对照组则是100μL ddH2O完全裂解的绵羊红细胞(1×107个)加溶血素(0.125μL)和正常人血清(300μL)，将上述混合液分别置于37℃水浴中孵育30min，期间每隔5min轻柔混匀，然后将反应液置于冰上，加入150μL预冷的GVB-EDTA缓冲液终止反应。
2.2.4 1000g，4℃，离心10min，取100μL上清转移到96孔酶标板中，用酶标仪检测OD405值。
2.3数据分析：绵羊红红细胞裂解率：绵羊红细胞在水中的裂解率定义为100％，裂解率换算公式：样本A405-阴性对照A405(缓冲液含EDTA)/(阳性对照A405(水裂解)-阴性对照A405(缓冲液含EDTA))。
3.试验结果：
3.1Aflibercept和CD59的表达：构建Aflibercept-CD59过表达载体，测序结果无误，转染HEK 293T细胞，收集细胞培养上清，结果如表2，表3，Aflibercept标准曲线结果如图8所示，CD59标准曲线如图9所示。
表2 Aflibercept浓度结果
| Plasmid | OD450 | Original Aflibercept concentration(ng/mL) |
| Aflibercept-CD59 | 2.131 | 35.169 |
表3 CD59浓度结果
由上表2、表3可知，用Aflibercept ELISA试剂盒检测细胞培养上清中Aflibercept的含量，Aflibercept的浓度为35.169ng/mL，CD59ELISA试剂盒检测细胞培养上清中CD59的浓度，Aflibercept-CD59(G08)组细胞培养上清中CD59浓度为416.33pg/mL，其中Aflibercept-CD59(G08)组细胞培养上清，分别用Aflibercept ELISA试剂盒和CD59ELISA试剂盒检测，结果存在一些差异，而Aflibercept和CD59为融合表达，该现象的出现可能归因于两种ELISA试剂盒的灵敏度和检出限存着差异，也可能由于Aflibercept和CD59的表达差异，导致细胞培养上清中Aflibercept和CD59的表达量并不完全一致。
3.2绵羊红细胞溶血试验：将绵羊红细胞与细胞培养上清37℃共孵育30分钟，然后加入溶血素和正常人血清再共孵育30min，GVB-EDTA缓冲液终止反应后，离心取上清，酶标仪检测OD405值，按照公式计算绵羊红细胞裂解率。3次实验结果的OD405值，计算标准差SD，以GVB组作为对照组，通过t-test分析得到p-value，试验结果如表4及图10所示：
表4 溶血试验结果图
由上表4及图10可知，阴性对照组是用灭活的正常人血清与绵羊红细胞孵育，溶血率设定为0，阳性对照组是ddH2O完全裂解绵羊红细胞，溶血率为100％，GVB缓冲液对照组溶血率为96.33％，Aflibercept-CD59细胞培养上清共孵育组，溶血率为67.37％，与GVB对照组比较，p值<0.01，呈极显著差异，实验结果初步显示，在正常人血清诱导的绵羊红细胞溶血实验中，Aflibercept-CD59可以抑制绵羊红细胞的溶血。
3.3玻璃体表达结果：
Aflibercept及CD59玻璃体表达结果见表4及图11、图12。
表4 Aflibercept及CD59玻璃体表达结果
用表达Aflibercept-CD59融合蛋白的AAV8，通过脉络膜腔注射，新西兰大白兔的眼球，每只眼睛注射100μL病毒滴度为1E+13GC/mL的AAV8，3周后，取100μL的兔眼球玻璃体，通过ELISA分别检测Aflibercept和CD59的表达。检测结果显示，左眼Aflibercept的表达量为51.19662ng/mL，CD59的表达为124.2173913pg/mL，右眼表达Aflibercept为52.09277ng/mL，CD59为95.95652174pg/mL，跟细胞转染上清中的表达类似，Aflibercept的表达量比CD59略高。
湿性黄斑变性，又称新生血管或渗出性年龄相关性黄斑病变，是晚期黄斑病变的“湿”形式，该病是由于脉络膜毛细血管中的异常血管生长(脉络膜新生血管)，通过Bruch膜引起视力丧失。本发明所述试验仅用以评估我公司研发的新型腺相关病毒基因疗法对湿性年龄相关黄斑变性的治疗效果，具体过程为，先构建Aflibercept-CD59的过表达载体，转染HEK 293T细胞，然后收集细胞培养上清，通过ELISA检测Aflibercept和CD59在细胞培养上清中的分泌表达，将培养上清与绵羊红细胞孵育，加入正常人血清诱导绵羊红细胞溶血反应，验证CD59在绵羊红细胞溶血过程中的调控作用。
最后应当说明的是，上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
序列表
<110> 深圳庭美奥生物技术有限公司
<120> 一种用于治疗年龄相关性黄斑变性的药物
<130> 2021.3.26
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
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<212> PRT
<213> 血管内皮生长因子抑制蛋白的氨基酸序列(Amino acid sequence ofvascular endothelial growth factor inhibitor protein)
<400> 1
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Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu
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Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile
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Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu
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Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe
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Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu
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Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr
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Phe Val Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys
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Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
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Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
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His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
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Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
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<210> 2
<211> 77
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<213> 补体抑制蛋白的氨基酸序列(Amino acid sequence of complementinhibitory protein)
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Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr Ala
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Lys Lys Asp Leu Cys Asn Phe Asn Glu Gln Leu Glu Asn
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<210> 3
<211> 535
<212> PRT
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Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Asp Thr Gly Arg Pro
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Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr
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Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys
65 70 75 80
Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr
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Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His
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Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile
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Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu
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Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr
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Phe Val Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys
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Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
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Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
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Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
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Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
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Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
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<210> 4
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gcgatcgcca ccatggtctc gtactgggac actggggtgc tattgtgtgc cctcttatca 60
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gttaaccgtg accttaagac acaatctgga tccgaaatga agaagttcct ctcaaccttg 600
acaatcgatg gcgtcactcg ctcagatcaa gggctttaca cgtgtgcagc aagttccgga 660
ctcatgacca agaagaattc cacatttgtt agagtgcacg aaaaggataa aacccatacc 720
tgtccaccct gccccgcacc ggagttgcta ggcggtccta gtgtatttct attcccgcct 780
aagccaaagg acacattgat gatatccaga acgcccgaag ttacttgcgt agtggtggac 840
gtctcccatg aagaccccga agttaagttc aattggtacg tggacggggt tgaagtccac 900
aacgcaaaga ctaaacctcg tgaggaacaa tacaacagta catatcgggt tgtttcagtc 960
cttacggtcc tacatcaaga ttggttgaac ggtaaggaat ataagtgcaa agtttctaac 1020
aaggcactac ccgcaccgat cgagaagaca atctctaaag ccaaaggtca gccgagagaa 1080
cctcaagttt acaccctccc tccatctaga gatgaactca ctaagaacca ggtctccctt 1140
acctgtttag ttaaggggtt ttacccgagt gacatagctg tcgagtggga gtctaatgga 1200
caacccgaga ataactacaa gactacacct ccagtcttgg actccgatgg ctcttttttc 1260
ttgtacagta agctaacggt ggataaatcg agatggcagc aagggaatgt gttttcatgc 1320
agtgtaatgc atgaagcgct tcataatcac tatacccaga aaagtctctc cctttctcct 1380
gggaaactgc agtgctacaa ctgtcctaac ccaactgctg actgcaaaac agccgtcaat 1440
tgttcatctg attttgatgc gtgtctcatt accaaagctg ggttacaagt gtataacaag 1500
tgttggaagt ttgagcattg caatttcaac gacgtcacaa cccgcttgag ggaaaatgag 1560
ctaacgtact actgctgcaa gaaggacctg tgtaacttta acgaacagct tgaaaattaa 1620
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ggaaccccta gtgatggagt t 21
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cggcctcagt gagcga 16
Claims (8)
1.一种用于治疗年龄相关性黄斑变性的药物，其特征在于，所述药物为由血管内皮生长因子抑制蛋白和补体抑制蛋白组成的融合蛋白。
2.如权利要求1所述的药物，其特征在于，所述血管内皮生长因子抑制蛋白的氨基酸序列如SEQ ID NO.1所示；所述补体抑制蛋白的氨基酸序列如SEQ ID NO.2所示。
3.如权利要求1所述的药物，其特征在于，所述融合蛋白由血管内皮生长因子抑制蛋白和补体抑制蛋白直接以任何顺序融合在一起，或通过四个以上柔性的氨基酸链接序列融合在一起。
4.如权利要求1所述的药物，其特征在于，所述融合蛋白为通过基因工程表达的重组融合蛋白，该重组蛋白可直接注射进患者体内；或通过纳米材料包裹技术直接递送重组融合蛋白或编码该融合蛋白的基因，或通过各种病毒载体直接递送编码该融合蛋白的基因。
5.如权利要求4所述的药物，其特征在于，所述基因工程为通过细菌表达系统，酵母表达系统，昆虫杆状病毒或者哺乳动物细胞表达系统表达所述融合蛋白。
6.如权利要求4所述的药物，其特征在于，所述纳米材料包裹技术为通过纳米颗粒包裹融合蛋白或编码该融合蛋白的基因，帮助融合蛋白进入细胞或者直接在细胞内表达而发挥生物学功能。
7.如权利要求4所述的药物，其特征在于，所述病毒载体为腺相关病毒，腺病毒，慢病毒和痘苗病毒中的一种。
8.如权利要求1所述的药物，其特征在于，所述药物通过眼睛玻璃体内注射，或视网膜下注射，或脉络膜上注射方式给药。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117467025A (zh) * | 2023-12-28 | 2024-01-30 | 上海鼎新基因科技有限公司 | 一种抗vegf和补体双功能融合蛋白及其应用 |
| CN117467025B (zh) * | 2023-12-28 | 2024-04-16 | 上海鼎新基因科技有限公司 | 一种抗vegf和补体双功能融合蛋白及其应用 |
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