CN117797249A - 基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗 - Google Patents
基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗 Download PDFInfo
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Abstract
本发明公开了基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗。该疫苗分别表达PF4、KL1和Klotho蛋白和PF4‑KL1及PF4‑Klotho融合蛋白,其中PF4具有增智、强记忆及提高认知的功能,Klotho延长生命时限并提高认知,KL1为Klotho的细胞外可溶性片段,也完全具有延长生命时限和提高认知的功能,利用包含所需寡核苷酸的多重PCR片段扩增,并利用重叠片段组装生成每个疫苗序列的完整体。本发明对上述基因序列和mRNA表达载体进行了优化和修饰。本发明具有抗衰老增智强记忆提高认知和抗老年痴呆的功效,而且融合蛋白表达疫苗效果更好,打一针功效至少可持续一月,该疫苗将用于增智、强记忆、提高认知、抗老年痴呆和抗衰老预防和治疗的临床实践,让人们健康长寿,免于痴呆。
Description
技术领域
本发明涉及疫苗技术领域,具体为基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗。
背景技术
阿尔茨海默病(Alzheimer's disease,AD)是最常见的神经退行性疾病之一,也是全球老年患者痴呆的主要原因。AD患者常出现定向障碍、语言障碍、认知下降和不可逆的记忆丧失。据世界卫生组织报道,目前AD患者人数为5000万;预计到2050年将增加到1.52亿人,并且由于人类寿命的延长,最终将成为世界主要死亡原因。全球已经投入了大量的社会和经济资源来阐明AD发病的分子机制,但仍缺乏有效的预防和治疗策略。AD的日益流行,以及人类寿命的延长,给世界带来了巨大的经济和社会负担。解决这一危机的唯一明智方法是开发有效预防和治疗的诊疗手段,以便在痴呆发病之前解决或阻止AD的进展。
血小板是小的无核血细胞,将生物活性因子储存在专门的细胞质囊泡内,在运动、组织损伤或应激等环境刺激下,不同形式的血小板活化会导致内容物的环境依赖性和选择性释放。因此,各种形式的血小板活化会转化从止血到神经发生的基本生物学作用。同样,血小板功能障碍与炎症、出血和中枢神经系统疾病有关。
血小板可能是大脑健康的信使的观点得到了运动激活血小板和随后释放的血小板因子4(PF4)支持,以及海马神经发生的观察结果的支持。认知是大脑功能的一种高度重视和核心表现,但随着衰老和疾病而下降,机制尚不清楚。这是一个重要的问题,因为认知功能障碍是我们最大的生物医学挑战之一,没有有效的治疗方法。
血小板作为一种生物疗法引起了人们的关注,因为它们具有惊人的强效抗炎、神经营养和抗氧化作用。血小板以富血小板血浆或血小板裂解物的形式,广泛用于许多再生应用,包括手术后愈合,治疗肌肉骨骼损伤和骨关节炎和皮肤年轻化。血小板衍生因子的协同神经保护作用最近也在创伤性脑损伤的小鼠模型、肌萎缩侧索硬化症和帕金森病中得到证实。
因此,我们研究了血小板因子在认知底层的作用和认知本身改善上的功能。
血小板因子,被定义为血小板激活时从血小板颗粒和溶酶体释放的蛋白质。我们的研究结果提供了一个证据,证明血小板释放的第四因子(Platelet factor 4,PF4),增加成人神经发生和恢复认知功能,从而巩固它们在调节大脑功能中的重要作用。
Klotho基因在肾脏中表现突出,可调节肾功能、甲状旁腺和脉络丛。它在钙磷酸盐代谢、髓鞘再生、认知过程和炎症过程中起重要作用。在人类中,Klotho的血清浓度随着年龄的增长而下降。血清Klotho水平较低可导致动脉僵硬,导致血管功能障碍,这反过来又有助于预测早期阶段的动脉粥样硬化。研究表明,血清Klotho浓度的下降及其基因在胸主动脉中的表达与预测冠状动脉疾病的存在和严重程度有关。α-Klotho的血清浓度随年龄增长而降低,主要在40岁后,导致人类与年龄相关的疾病,包括癌症、高血压和肾脏疾病。Klotho还被发现表现出抗衰老特性,并在认知中发挥作用。它会导致海马体和皮层突触结构发生变化,导致认知能力下降减慢。
克洛索(KLOTHO)是一个长寿因子,能改善认知功能。它在从跨膜形式裂解后以激素形式循环并影响胰岛素、成纤维细胞生长因子(FGF),Wnt16和N-甲基-D-天冬氨酸受体(NMDAR)信号传导。KLOTHO的人类遗传变异增加了其系统水平并与增强的大脑连接和认知提高有关。在老龄化人口中,在小鼠的实验研究中,分泌形式的α-klotho(klotho,KL)的急性和全身升高增加了突触可塑性,增加了认知和神经弹性,生理水平的klotho全身治疗也增强了衰老的非人灵长类动物大脑的认知能力。在遗传、解剖和功能复杂性增加的背景下,由于外周注射的klotho不会进入大脑,转导其信号的外围信使仍有待识别。因此,我们研究了潜在的Klotho亚域的分子KL1,其大小是klotho(KL)全长的一半,足以增强认知能力。
我们的研究揭示了血小板通过PF4在增强年轻和衰老大脑认知方面的非常规作用。Klotho以类似于运动的方式激活血小板。我们的研究结果表明,血小板可以作为循环信使,通过释放PF4等因子来调节认知本身。我们的数据表明,血小板激活是必需的,PF4进入大脑,足以概括解释klotho介导的认知增强。PF4增强了年轻小鼠的认知能力,并参与了谷氨酸能信号传导的突触机制。在衰老小鼠中,PF4还改善了认知能力,并恢复了年龄诱导的与认知相关的因子的增加。增加血小板因子可以增强年轻大脑的认知能力,并抵消衰老大脑中的认知缺陷。
于是,有鉴于此,针对现有的结构及缺失予以研究改良,提出基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗。
发明内容
本发明的目的在于提供基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗,以解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗,其特征在于,分别表达PF4、KL1、Klotho蛋白和PF4-KL1及PF4-Klotho融合蛋白,利用包含所需寡核苷酸的多重PCR片段扩增,并利用重叠片段组装生成每个疫苗序列的完整体,并对其mRNA序列进行了密码子优化及假尿苷取代,以及UTR和Poly A的改构优化,并插入PUC57质粒中,并进行质粒扩增、纯化和酶切等,IVT完成后进行加帽,双酶加帽反应,牛痘加帽系统和MTE系统,以mRNA疫苗结构为主体的疫苗,包含一个体外转录mRNA,由抗原编码的开放阅读框、和5’和3’端未翻译区,及一个7甲基鸟苷5帽结构,合并到第一个核苷酸的序列,和3端聚尾部,同时进行密码子优化,假尿苷取代。5’UTR区域结构,来自高表达的人类基因α-球蛋白的5-UTR,以及一个优化的下游Kozak共识序列GCCACCAUG;3'端UTR结构:3'UTR,它是由AES基因序列和mtRNA1组合而成,选择用于增加蛋白质表达和mRNA稳定性。
进一步的,基于mRNA的疫苗是指以mRNA表达载体为基础的疫苗生产制作,包括对载体的任何修饰,如加帽、密码子优化、假尿苷取代、UTR和Poly A的修饰。
进一步的,还包括mRNA自扩增表达技术。
进一步的,表达载体技术还包括除mRNA载体以外的质粒载体、腺病毒载体等各种病毒载体,即包括以DNA和mRNA病毒为基础的表达载体。
进一步的,还包括没有载体的脂质体包裹或直接的基因转移技术。
进一步的,PUC57含有T7启动子、5'-UTR、3'-UTR和polyA尾。
进一步的,Poly-A尾巴是高效翻译的必要元素,poly-A序列约30-300个核甘酸单位。各抗原之间的链接为GSGGGG。
进一步的,用于LNP制备的脂质是可电离脂质十七烷-9-基8-[2-羟乙基-(6-氧-6-十一氧己基)氨基]辛酸酯,辅助脂质1,2-二硬脂酰-sng-甘油-3-磷酸胆碱,胆固醇和1,2-二肉豆醇-sng-3-磷酸乙醇胺-n-[甲氧基(聚乙二醇)-2000],包括但不限于上述脂质体。
进一步的,使用微流体装置将有机相中的脂质与含有mRNA的水相混合制备的。
进一步的,SM-102的组成脂质摩尔百分比为48%,DSPC为12%,胆固醇为38%,DMG-PEG为2%,在1:3的流动比下,溶液在微流控装置中组合,每个微流控芯片的总流速为12mL/min,将LNP-mRNA混合物透析并离心浓缩,配制后的mRNA定量,采用RiboGreen测定法,配制后从LNPs中分离mRNA并进行琼脂糖凝胶电泳。
本发明提供了基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗,具备以下有益效果:该治疗疫苗具有抗衰老增智强记忆提高认知和抗老年痴呆的功效,而且融合蛋白表达疫苗效果更好,打一针功效至少可持续一月,该疫苗将用于增智、强记忆、提高认知、抗老年痴呆和抗衰老的预防和治疗的临床实践,让人们健康长寿,免于痴呆。
附图说明
图1:老年小鼠、老年人及AD患者血液PF4和Klotho明显降低;
图2:基于mRNA的PF4-Klotho抗衰老及增智强记忆抗老年痴呆(AD)疫苗设计;
图3:基于mRNA的PF4-Klotho的体外表达;
图4:小鼠皮下给予PF4明显改善认知和记忆;
图5:PF4 mRNA疫苗明显促进海马神经发生;
图6:小鼠注射KL1 mRNA疫苗明显改善海马电生理反应;
图7:Klotho mRNA疫苗注射使血小板PF4活化水平增加1倍以上;
图8:小鼠注射PF4-Klotho mRNA疫苗明显改善认知和记忆;
图9:恒河猴注射PF4、PF4-KL1、PF4-Klotho mRNA疫苗明显改善认知和记忆;
图10:小鼠注射PF4、PF4-KL1及PF4-Klotho mRNA疫苗生命时限明显增加25-30%;
图11:Klotho及KL1的蛋白质编码及分子结构的修饰;
图12.PF4的氨基酸序列。
具体实施方式
请参阅图1至图12,本发明提供技术方案:基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗,针对所述mRNA抗衰老抗痴呆疫苗的设计、蛋白表达及细胞、动物活体实验作以详细介绍。
实施例1.
老年小鼠、老年人及阿尔茨海默病(Alzheimer's disease,AD)患者血液血小板第四因子(Platelet factor,PF4)和Klotho明显降低
方法
将小鼠饲养在无特定病原体的条件下,在12小时-12小时光照-黑暗循环下,湿度保持在30-70%,温度保持在20-26℃。所有动物处理和使用均按照动物实验的赫尔辛基宣言和国家有关的法律法规和指南进行。
动物及人血浆采集、血小板级分制备
小鼠在安乐死时,通过安乐死时从年轻(3个月大)和老年(20个月大)小鼠的心内取血收集小鼠血液。用EDTA抗凝,以1,000g离心用于血浆制备。对于蛋白质印迹和ELISA分析,将血浆等分并储存在-80℃直至使用。在全身给药之前,使用PBS中的3.5kDa D管透析器(EMD密理博)透析血浆以去除EDTA。对于血小板级分制备,将血浆以20,000g离心,弃去上清液并将沉淀的血小板组分重悬于等量体积的盐水中。老年小鼠在100天内通过静脉尾静脉注射8次用盐水,血浆或血小板部分(每次注射24μl)全身处理。同样,生理盐水或PF4(5μgml-1)通过静脉尾静脉注射在100天内8次全身施用小鼠(每次注射24μl)。将小鼠PF4和人血小板来源的PF4(包括AD患者)溶解在无菌超纯水中,浓度为100μgml_1。
蛋白质印迹分析和酶联免疫结合
对于蛋白质印迹分析,将样品与RIPA裂解缓冲液(Abcam,ab156034)与完全蛋白酶抑制剂(4693116001,Sigma-Aldrich)和磷酸酶抑制剂(赛默飞世尔科技,78420)联合使用。随后,将样品与4×NuPage LDS上样缓冲液(Invitrogen,NP0008)混合,上样到SDS聚丙烯酰胺凝胶(Invitrogen)上并转移到硝酸纤维素膜上。使用Piceau S溶液(Sigma-Aldrich,P7170)确认样品的相同负载,并使用ChemiDoc系统(Bio-Rad)对膜进行成像。将印迹膜封闭在含有吐温-5的Tris缓冲盐水中的20%牛奶中,并与抗GAPDH(6C5,1:5,000,Abcam,ab8245)山羊抗mPF4(1μgml_1,研发系统,AF595),小鼠抗hPF4(170138,0.5μg毫升_1,R&DSystems,MAB7952),小鼠抗血小板反应素-1(A6.1,1:200,Santa Cruz,sc-59887,C2519)或兔抗亲环蛋白A(1:200,ENZO生命科学,BML-SA296-0100)。
结果
通过蛋白质印迹分析,我们检测到年轻小鼠血小板分数中的PF4水平高于老年小鼠。年轻人的血浆血小板PF4含量高于老年人,AD患者的血浆血小板PF4更明显低于年轻人的水平。
结论
随着年龄增长,小鼠和人类血浆中的PF4降低,AD患者的PF4降低更明显。
实施例2.
基于mRNA的PF4-Klotho抗衰老、增智强记忆抗老年痴呆(AD)疫苗设计方法
该疫苗分别含编码PF4(含70个氨基酸)、Klotho(971个氨基酸),KL1(476个氨基酸)序列(具体见图11,12),综合设计共有5种疫苗,分别为mRNA PF4,KL1,Klotho,PF4-KL1及PF4-Klotho疫苗。mRNA经5'端加帽,密码子优化,假尿苷取代及poly(A)尾调节,以增强体外转录翻译效率和降低其反应原性。
我们利用包含每个序列所需的寡核苷酸进行多重PCR片段扩增,并利用重叠片段组装生成每个完整序列。并插入PUC57中,PUC57含有T7启动子、5'-UTR、3'-UTR和polyA尾(图1A)。并插入pTwist_CMV_BetaGlobin_WPRE_Neo慢病毒载体中,以便于收获含有疫苗序列的假病毒,其含有T7启动子、5'-UTR、3'-UTR和polyA尾。
mRNA经脂质体包裹,其成分包括:七烷-9-基8-((2-羟乙基)(6-氧氧基-6-(十一氧基)己基)氨基辛酸盐(SM-102)、2-(聚乙二醇2000)-n,n-二十六烷基乙酰胺(ALC-0159),1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)和胆固醇。
结果
如图2所示,A,为5种疫苗的具体设计和mRNA载体;B为包裹的脂质体及其成分。
实施例3.
基于mRNA的PF4-Klotho的体外表达
方法
将构建的PF4及KL1、Klotho疫苗载体PUC57转染293细胞进行瞬时表达,收获细胞,进行Western Blot分析,并与人血浆提取纯化的蛋白进行比较。慢病毒载体经293T细胞表达及收获假病毒后,保存于-80℃备用。
结果
如图3所示,A为重组的PF4蛋白于人体PF4比较;B为人Klotho蛋白western blot分析;C为重组蛋白的表达分析,包括5种疫苗:PF4、KL1、Klotho、PF4-KL1及PF4-Klotho蛋白分析。
结论
我们构建的疫苗载体表达成功,结果良好。
实施例4.
小鼠给予PF4 mRNA疫苗明显改善认知和记忆方法
所有小鼠均在同源C57BL/6J背景上,并保持12小时的光照/黑暗循环,湿度为30-70%,温度为20-26℃,并随意获取食物和水。标准住房组是每个笼子五只小鼠,除了莫里斯水迷宫研究期间的单个住房。所用小鼠的性别和年龄在图例中标明。在光循环期间进行了认知和行为研究。除非另有说明,否则所有血小板活化测定、ELISA、认知和行为研究以及突触可塑性实验在研究执行和分析期间均对基因型和治疗不知情。
酶联免疫吸附法
对于mPF4的测量,根据制造商的指示进行ELISA测定。简而言之,用ELISA缓冲液稀释每个血浆样品,并通过ELISA分析mPF4。为了测量HIS标记的小鼠PF4(GeneTex,GTX00334-pro),首先将HIS标记的蛋白质在PBS(pH 7.5)中稀释并以500μg/kg的剂量使用,在灌注和脑组织收集前10分钟进行。解剖海马和皮质组织,然后用RIPA缓冲液裂解以获得匀浆样品。为了确定匀浆中的HIS信号,根据制造商的说明进行HISELISA测定。
免疫组化
将小鼠全脑分离并在4%(w/v)多聚甲醛中后固定48小时,然后在PBS中保存在30%(w/v)蔗糖中。在冷冻滑动切片机上以40μm的厚度冠状切片整个大脑。切片储存在-20℃的冷冻保护介质中。用驴血清封闭自由浮动切片,并在4℃以下浓度与一抗孵育过夜,进行显微镜检查:兔抗HIS(1:200,Invitrogen MA5-33032)和荧光素标记的凝集素(1:200,实验室)。洗涤后,切片与驴抗兔Alexa Fluor Plus 555(1:200,赛默飞世尔,A32794)和300nM的4′,6-二脒基-2-苯基吲哚(DAPI)在室温下孵育2小时,用转盘共聚焦显微镜(尼康CSU-W1)和数字荧光显微镜上成像。
NOR实验
第一天(习惯化阶段),小鼠通过探索空旷的竞技场10分钟来进行开放场地测试。记录红外光束断裂,并使用MotorMonitor软件(Kinder Scientific)分析运动指标。在第二天(训练阶段),将两个相同的物体放入习惯的竞技场中,并允许小鼠探索5分钟。在第三天(测试阶段),用一个新物体替换一个物体,并允许小鼠探索5分钟。使用智能视频跟踪软件(Panlab;哈佛仪器)。为了控制任何固有的物体偏好,一半的小鼠暴露于物体A作为他们的新对象,另一半暴露于物体B。为了控制任何潜在的与对象无关的位置偏好,新对象相对于训练对象的位置也被平衡。为了确定新对象的时间百分比,我们计算(新对象的时间)/(训练对象的时间+新对象的时间)×100。在训练阶段没有探索这两个物体的小鼠被排除在分析之外。
Y迷宫
在训练阶段,将小鼠放入面向墙壁的起始臂中,并允许探索起始臂和训练臂5分钟,同时阻止进入第三臂(新型臂)。在每只小鼠之间清洁迷宫以消除气味线索,并且训练过的手臂在小鼠之间交替。训练后,小鼠被送回鼠笼。3分钟后,将鼠标返回到起始臂,并允许探索所有三个臂45分钟。使用智能视频跟踪软件(Panlab;哈佛仪器)。每个组中的条目百分比定义为每个组中的条目数除以任务第一分钟内所有组中的条目总数。歧视指数由(新臂-训练臂)/(新臂+训练臂)量化。在测试的第一分钟内未执行三个条目的小鼠被排除在外。
RAWM范式评估空间学习和记忆
使用RAWM范式评估空间学习和记忆,小鼠在整个训练和测试阶段被训练到恒定目标臂的位置。起始臂改变了每次试验。进入不正确的手臂被评分为错误,并且错误在训练块上平均计算(连续三次试验)。在训练期间(第1天),小鼠被训练12次试验(块1-4),试验在可见和隐藏的平台之间交替进行。休息一个小时后,仅使用隐藏平台对3次试验(第5块)进行学习测试。在测试期间(第2天),用隐藏的平台对小鼠进行了15次试验(块6-10)。评分时,调查人员对治疗不知情。
结果
如图4A,对照组(Veh)或HIS标记的mPF4注射(500μg/kg)后脑组织采集的实验范式_1,i.p.),年轻和衰老的小鼠(雄性,年龄4个月和18个月,每组n=3-8只小鼠)。图4B,定量年轻小鼠大脑和血浆中的HIS水平,外周HIS-mPF4注射后4分钟进行。图4C,定量老年小鼠大脑和血浆中的HIS水平,外周HIS-mPF4注射后4分钟进行。(脑,每组n=8只小鼠;血浆,每组n=3只小鼠)。图4D,外周HIS-mPF4处理后年轻小鼠的代表性冠状脑免疫组化染色图像。在大脑中检测到外围注射的PF4。比例尺,1,000μm。图4E,对老年小鼠进行NOR和RAWM测试范式。图4F,hPF4处理的老年小鼠相对于熟悉的物体对新物体更感兴趣。图4G,hPF4处理的老年小鼠表现出对平台位置的学习和记忆的改善,错误明显减少。
结论
施用hPF4的老年动物表现出对平台位置的学习和记忆的改善,和对新物体更多兴趣。
实施例5.
PF4 mRNA疫苗明显促进海马神经发生方法
疫苗注射
8周龄雌性C57BL/6J小鼠分为对照组和PF4mRNA疫苗注射组,对照组给予30μl生理盐水,实验组给予10μg/kg疫苗,肌肉注射。14天后进行组织灌注和取材。
神经前体细胞的免疫组化染色
BrdU标记,盖玻片用0.9%NaCl洗涤两次,然后用1M盐酸(HCl,默克密理博)在37℃孵育30分钟。然后用0.1M硼酸盐缓冲液洗涤盖玻片一次,用0.01M PBS洗涤三次。对于分化细胞的免疫染色,并用0.01M PBS洗涤盖玻片一次。然后在含有1.0%叠氮化钠,0.1%正常山羊血清和0.01%Triton X-10的0.1M PBS中进行封闭100小时。在含有1.5%叠氮化钠,0.03%山羊血清和0.01.1%Triton X-500的0.1M PBS中孵育一抗大鼠抗BrdU(1:2000,AbDSerotec,cat#OBT5,RRID AB_8)或兔抗GFAP(1:4308,Dako Agilent,cat#Z0,RRID AB_01)和小鼠抗β-III-微管蛋白(0:01;克隆3G0,Promega,cat#G1,RRID AB_100)的组合在4℃下过夜。盖玻片用0.01M PBS洗涤三次,然后用相应的二抗(所有1:1000;山羊抗兔AlexaFluor 488(赛默飞世尔科技,猫#A11008,RRID AB_143165)和山羊抗小鼠Alexa Fluor 568(Invitrogen,cat#A11031,RRID AB_144696))在含有0.01%叠氮化钠,0.01%正常山羊血清和1.3%Triton X-0的0.1M PBS中在黑暗中孵育1小时。然后用0.01M PBS洗涤盖玻片,用4′,6-二脒基-2-苯基吲哚(DAPI;在PBS中为1:5000,赛默飞世尔科技)孵育10分钟,再次用0.01M PBS洗涤,最后浸入dH中2O分钟并使用荧光封片剂(达科安捷伦)安装在载玻片上。
增殖和分化细胞的定量
在5个随机视野(FOV)上对增殖和分化细胞进行定量,使每个条件总共10个FOV。在DAPI通道中确定FOV。图像以×200放大倍率采集,使用蔡司AxioImager Z120显微镜和蔡司ZEN软件(蓝色版)平均每个FOV捕获1个细胞。BrdU,β-III-微管蛋白,GFAP和DAPI细胞使用Adobe计数。通过量化BrdU细胞或β-III-微管蛋白和GFAP细胞的数量相对于细胞总数(DAPI细胞)来确定每个实验的增殖或分化细胞的比例,其中一个数据点是指10个FCldU和IdU的双重标签范式。
为了标记8周龄雌性C57BL/6J小鼠的所有增殖神经前体细胞,间隔5小时腹膜内注射2次42-氯-5'-脱氧尿苷(CldU;9.100μg/kg)。最后一次CldU注射后一小时,小鼠接受单次静脉注射4μlPF4(4μg/ml)。为了区分新募集增殖的细胞和PF4给药前已经增殖的细胞,腹膜内给予三剂2-碘-57'-脱氧尿苷(IdU;5.9mg/kg),间隔1小时。在最后一次IdU注射后1小时灌注小鼠。准备大脑进行组织学分析,并按照免疫组织化学部分所述进行CldU和IdU细胞的免疫组织化学染色。使用蔡司AxioImager Z100显微镜定量CldU和IdU细胞的总数。在DAPI通道中确定随机视场野,并根据其CldU和IdU标记对至少4个细胞进行表型分析。使用这种方法,在PF4处理之前已经增殖并在之后保持增殖的细胞(CldUIdU),在PF4处理后开始增殖的细胞(CldU++++++_IdU),以及在实验过程中失去增殖表型的细胞(CldU IdU++-)可以区分。取OV的平均值。每个条件总共分析~1200个细胞。
成熟神经元的神经树突分析
从雄性和雌性胚胎第17天C57BL/6J小鼠大脑中分离出原代海马神经元。原代海马神经元在体外13天用收获的带有荧光蛋白的PF4假病毒感染,感染后72小时。在蔡司LSM63共聚焦显微镜上用×510油浸物镜对固定神经元进行成像分析,同时与感染前的图像进行对比。以0.38μm的间隔收集一系列光学切片,并进行最大强度投影。使用ImageJ图像分析软件(v2.1.0/153c)对阈值图像进行Sholl分析。
结果
如图5显示,A中左图为对照组神经元树突生长情况,右图为mRNA疫苗处理组的神经树突生长,树突发生和生长明显多于对照组;B显示对照组神经前体细胞的染色,C显示PF4mRNA疫苗给药后的细胞增殖情况;D显示单个前体细胞增殖;E为PF4疫苗处理前的神经树突生长,F为PF4mRNA疫苗处理后的神经树突发生,证明跟踪细胞的总树突长度增加,生发的初级树突和长度明显增加。比例尺:4μm。
结论
全身性给予PF4mRNA疫苗增强脑内海马神经发生
实施例6.
小鼠注射KL1mRNA疫苗明显改善海马电生理反应方法
脑电生理学
从小鼠获得300μm厚的冠状脑切片。简而言之,在Schaffer侧支路径刺激后从CA1区域获得测量结果。用异氟醚麻醉小鼠,收集大脑并立即置于冰冷人工脑脊液中:124NaCl,2.8KCl,2MgSO4,1.25M NaHCO2,1M葡萄糖,2M氢氧化钠,1.3M抗坏血酸,并在振动切片机(徕卡)上切片。将切片在32℃下孵育30分钟,然后在室温下恢复1小时进行测试。将切片转移到具有循环,含氧(95%O2和5%一氧化碳)人工脑脊液环境,在30℃下,并在任何刺激前恢复10-15分钟。对于场电位记录,将急性海马切片放置在Med64-Quad II多电极阵列(AlphaMED Scientific)上。通过Quad II 2×8探针AL-MED-PG501A的平面电极,通过对齐电极和海马切片的辐射层区域,引出并记录fEPSP。在每次记录开始时执行输入输出曲线以确定适当的刺激强度。在30.0Hz下以最大强度的0-5%传递测试刺激,并且在LTP诱导之前建立5-20分钟的fEPSP稳定基线。LTP是使用θ脉冲群协议诱导的,该方案由每20秒发送的两个序列组成,每个序列包含10个5Hz的脉冲群,每个脉冲群包含100Hz的四个脉冲。将Ro 25(Tocris)2mg/ml溶解在的生理盐水中,并在fEPSP记录前1小时添加到急性海马切片的灌注液。使用Med64 Mobius软件(Alpha MED Scientific)进行记录和分析。
结果
如图6所示,我们评估了CA1 Schaffer侧支通路突触中急性海马切片中的LTP。用KL1 mRNA疫苗治疗后72小时测试fEPSP,证明该疫苗注射后增强正常成年大鼠大脑中的突触可塑性(图6A-E)。
结论
KL1 mRNA疫苗介导的认知刺激模拟表明,它们对海马体的代谢重塑可以使大脑改善认知。
实施例7.
Klotho mRNA疫苗给药使血小板PF4活化水平增加1倍以上
方法
通过流式细胞术活化和计数血小板
使用流式细胞术测量血小板活化状态。简而言之,通过心脏穿刺将全血收集到终浓度为0.38%柠檬酸钠溶液(pH 7)中,然后在室温下以200g离心10分钟。收集来自每只小鼠的等体积血浆并转移到带有Hanks平衡盐溶液(HBSS)(含乙二胺四乙酸,pH 6.4)的新管中,然后在室温下以1,200g离心20分钟。将血小板沉淀重悬于HBSS(pH 6.4)中,然后在室温下用CD61-PE(1:6,赛默飞世尔)和CD62P-Alexa 647(1:6,BD Bioscience)抗体染色30分钟。将用血小板标记物和活化标记物染色的血小板重悬于FACS缓冲液(PBS与1%牛血清白蛋白和1%叠氮化钠(pH 6.4))中提供足够的稀释度,以便在流经FACS机器时可以检测到非常小的血小板。活化的血小板在CD62阳性中被鉴定为CD61P阳性细胞。通过血小板浓度评估的血小板计数计算为每个样品CD61阳性血小板的总数。血小板计数归一化为FACS读取的体积。对于体外研究,从小鼠中分离的血小板重悬于HBSS(pH 6.4)中,然后在1℃下用载体,klotho或2mM ADP处理37分钟。通过流式细胞术分析CD61-PE和CD62P-Alexa 647的免疫染色血小板。
结果
在注射Klotho mRNA疫苗后72小时,我们测试了全身性klotho治疗是否可以激活血小板。从全血中分离血小板,并通过荧光激活细胞分选(FACS)进行。我们通过流式细胞术定量了血小板活化水平,表示为活化血小板(CD62P阳性)在总血小板群体(CD61阳性)中的百分比,血小板活化的静息水平来自从对照组小鼠分离的血小板。klotho疫苗治疗后使血小板活化的静息水平几乎翻了一番(图7A-C),而不改变血小板的数量。
结论
Klotho疫苗通过活化血小板来增加认知和提高记忆
实施例8.
小鼠注射PF4-Klotho mRNA疫苗明显改善认知和记忆
方法
小Y迷宫。
我们在小Y迷宫中测试了小鼠,该迷宫测量工作和空间记忆。老年(年龄,24个月)小鼠用载体或KL疫苗(10μg/kg,sc.c)处理,72小时后用空间线索探索小Y迷宫。简而言之,在适应前1小时将小鼠置于测试室进行驯化。对于每次测试,将鼠标放置在Y迷宫的三个相同臂之一内,并允许探索该装置6分钟。迷宫里覆盖着一块透明的塑料板,开始录音。将小鼠放回其主笼,并在试验之间用70%乙醇清洁装置。在观看视频时手动对自发的更改进行评分,并计算更改的百分比。
莫里斯水迷宫。
我们在莫里斯水迷宫中测试了一组单独的老年小鼠(24月大),该队列测量空间学习和记忆。水迷宫池(直径,122厘米)包含白色,不透明的水(21±1℃),一个小的方形的10cm2平台淹没并隐藏在地表以下2厘米处。测试室的三面墙上出现了明显的视觉提示。在隐蔽平台训练前,小鼠通过通道游动来安装隐蔽救援平台,进行两次预训练试验。在隐藏平台训练期间,平台位置保持不变,并且不同试验之间的下降位置不同。小鼠接受两次训练,每次两次试验,每天4天。每次试验允许的最长时间为60秒。对于探针试验,平台被移除,小鼠被允许搜索60秒。在探针测试之后,测试小鼠在用可见提示(放置在可见平台上的15厘米杆)标记时找到平台的能力。评分是根据标准说明使用EthoVision XT自动化软件(Noldus)进行的。
两审Y迷宫。
在两审Y迷宫中测试小鼠,该迷宫评估空间和工作记忆。简而言之,独特的视觉提示附在新颖而熟悉的手臂的末端,而起始臂则留空。在训练过程中,两个视觉提示臂中的一个被挡住了(新臂)。然后将小鼠置于起始臂中,并允许自由探索两个张开的臂(开始和熟悉)5分钟。新颖而熟悉的手臂名称在测试小鼠之间交替进行,以控制小鼠可能对一只手臂的任何先天偏好。训练后,小鼠被送回家笼。5小时后,在所有三个手臂畅通的情况下对小鼠进行测试。允许小鼠自由探索2分钟,并使用ANY-Maze系统(圣地亚哥仪器)自动测量持续时间以及在新颖和熟悉的手臂中行进的距离。
结果
如图7所示,正如预期的那样,mRNA PF4,PF4-KL1和PF4-Klotho疫苗增强了学习,这是通过减少延迟来衡量的,以找到隐藏的平台(图7AB)。此外,游泳速度或找到目标平台的能力改善(图5CD),表明疫苗结果对学习和记忆的特异性。在测试隐藏平台位置记忆的探针试验中,训练后1到4周,所有疫苗治疗组都表现出增强的记忆(图5EF)。因此,PF4和Klotho疫苗增强了学习和记忆。
结论
PF4及其Klotho mRNA疫苗增强了老年小鼠的学习和认知能力。
实施例9.
恒河猴注射PF4-Klotho mRNA疫苗明显改善认知和记忆
方法
老年恒河猴(每个测试组n=12-15只,平均年龄21.78岁,推定人类年龄当量平均值65岁)单次给mRNA疫苗10μg/kg或载体,使用空间延迟反应(SDR)任务对老年恒河猴进行认知测试。所有测试用均严格遵守动物实验赫尔辛基宣言和国家有关法律法规进行。
空间延迟响应任务(SDR)
NML和HML的SDR任务,简而言之,在NML任务的稳定性训练(65-75%正确)之后,猴子在一天中的大约同一时间接受测试,以获得非常可口的食物奖励。首先通过增加空间位置的数量或延长延迟(将0到1之间的可变乘数应用于一组2、3、4、6和9秒的基线延迟)来滴定任务难度,从而为每只猴子实现稳定的性能。一旦稳定,该猴子的孔数和延迟乘数在当前研究的测试期间保持固定。为了进行测试,猴子观察空间距离的井,而研究人员将首选的食物放在一个井中。覆盖井,降低猴子和井之间的不透明屏幕,在预定的延迟后,屏幕升高。如果选择井是正确的,猴子能够取回零食。根据孔的数量以及提示和任务之间的时间,行为被分类为HML(0-15孔;4-7-s延迟)或NML(0-32孔;3-5s延迟)。所有动物都对这项任务进行了广泛的训练和测试。动物在任何一次测试中进行20次试验,通常在任何一天进行一次试验。典型的测试过程持续不到10分钟。在几乎所有情况下,动物在HML会话之间至少进行了四个SDR会话的测试,以确保稳定性或检测性能的任何长期变化。在mRNA疫苗组,还测试了20μg/kg和30μg/kg剂量到的结果。
结果
如图9所示,AB为给药剂量和测试范式。C为各疫苗组间到比较。D为HML在4天与15-28天测试结果比较;E为NML在1-7天和8-14天的测试结果。F为PF4-Klotho不同剂量组间的结果测试。总的来说,我们的数据显示Klotho及其KL1疫苗(10微克/公斤)增强了衰老恒河猴的认知能力,这种效果在NML和HML记忆测量中都持续了至少2-4周。KL介导的认知增强同样在小鼠中持续至少2周,表明PF4及其Klotho mRNA疫苗对突触和大脑组织,有更持久和有益的影响。
结论
我们的数据显示,Klotho mRNA疫苗可以增强老年灵长动物的认知能力,这表明外周治疗或补充这种内源性激素可能对老年人具有治疗作用。
实施例10.
小鼠注射PF4、PF4-KL1及PF4-Klotho mRNA疫苗生命时限明显增加25-30%方法
我们取12月龄雌性C57BL/6J小鼠(约相当于人类寿命的50岁,小鼠的寿命通常为2年),分为对照组和PF4、PF4-KL1及PF4-Klotho mRNA疫苗治疗组进行研究,疫苗剂量为30μl,10μg/kg皮下注射,对照组给予生理盐水。每组n=30。治疗组每月进行一次注射,共注射10次,观察Klotho mRNA疫苗治疗后动物的生命跨度时限。
结果
PF4,PF4-KL1和PF4-Klotho mRNA疫苗治疗组的小鼠的寿命分别比对照延长19.8%,25.3%和30.8%
结论
PF4,PF4-KL1和PF4-Klotho mRNA疫苗治疗确实延长了小鼠的寿命,未来希望能够延长人类的寿命。
总之,经过上述各实施例的研究,我们的结果证明了PF4,PF4-KL1和PF4-KlothomRNA疫苗不但增强了小鼠和恒河猴的认知和记忆,也延长了小鼠的生命时限25-30%。未来希望该疫苗能够为人类的抗衰老、增智、强记忆和抗痴呆及延年益寿方面作出贡献。
本发明的实施例是为了示例和描述起见而给出的,而并不是无遗漏的或者将本发明限于所公开的形式。很多修改和变化对于本领域的普通技术人员而言是显而易见的。选择和描述实施例是为了更好说明本发明的原理和实际应用,并且使本领域的普通技术人员能够理解本发明从而设计适于特定用途的带有各种修改的各种实施例。
Claims (10)
1.基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗,其特征在于,分别表达PF4、KL1、Klotho蛋白和PF4-KL1及PF4-Klotho融合蛋白,利用包含所需寡核苷酸的多重PCR片段扩增,并利用重叠片段组装生成每个疫苗序列的完整体,并对其mRNA序列进行了密码子优化及假尿苷取代,以及UTR和Poly A的改构优化,并插入PUC57质粒中,并进行质粒扩增、纯化和酶切等,IVT完成后进行加帽,双酶加帽反应,牛痘加帽系统和MTE系统,以mRNA疫苗结构为主体的疫苗,包含一个体外转录mRNA,由抗原编码的开放阅读框、和5’和3’端未翻译区,及一个7甲基鸟苷5帽结构,合并到第一个核苷酸的序列,和3端聚A尾部,同时进行密码子优化,假尿苷取代5’UTR区域结构,来自高表达的人类基因α-球蛋白的5-UTR,以及一个优化的下游Kozak共识序列GCCACCAUG;3'端UTR结构:3'UTR,它是由AES基因序列和mtRNA1组合而成,选择用于增加蛋白质表达和mRNA稳定性。
2.根据权利要求1所述的基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗,其特征在于,基于mRNA的疫苗是指以mRNA表达载体为基础的疫苗生产制作,包括对载体的任何修饰,如加帽、密码子优化、假尿苷取代、UTR和Poly A的修饰。
3.根据权利要求2所述的基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗,其特征在于,还包括mRNA自扩增表达技术。
4.根据权利要求3所述的基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗,其特征在于,表达载体技术还包括除mRNA载体以外的质粒载体、腺病毒载体等各种病毒载体,即包括以DNA和mRNA病毒为基础的表达载体。
5.根据权利要求4所述的基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗,其特征在于,还包括没有载体的脂质体包裹或直接的基因转移技术。
6.根据权利要求5所述的基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗,其特征在于,PUC57含有T7启动子、5'-UTR、3'-UTR和polyA尾。
7.根据权利要求1所述的基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗,其特征在于,Poly-A尾巴是高效翻译的必要元素,poly-A序列约30-300个核甘酸单位。
8.根据权利要求7所述的基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗,其特征在于,用于LNP制备的脂质是可电离脂质十七烷-9-基8-[2-羟乙基-(6-氧-6-十一氧己基)氨基]辛酸酯,辅助脂质1,2-二硬脂酰-sng-甘油-3-磷酸胆碱,胆固醇和1,2-二肉豆醇-sng-3-磷酸乙醇胺-n-[甲氧基(聚乙二醇)-2000],包括但不限于上述脂质体。
9.根据权利要求8所述的基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗,其特征在于,使用微流体装置将有机相中的脂质与含有mRNA的水相混合制备。
10.根据权利要求9所述的基于mRNA的抗衰老抗老年痴呆预防及治疗疫苗,其特征在于,SM-102的组成脂质摩尔百分比为48%,DSPC为12%,胆固醇为38%,DMG-PEG为2%,在1:3的流动比下,溶液在微流控装置中组合,每个微流控芯片的总流速为12mL/min,将LNP-mRNA混合物透析并离心浓缩。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105188767A (zh) * | 2012-07-25 | 2015-12-23 | 布罗德研究所有限公司 | 可诱导的dna结合蛋白和基因组干扰工具及其应用 |
US20150366997A1 (en) * | 2012-12-07 | 2015-12-24 | Shire Human Genetics Therapies, Inc. | COMPOSITIONS AND METHODS FOR mRNA DELIVERY |
CN107405382A (zh) * | 2015-02-06 | 2017-11-28 | 加利福尼亚大学董事会 | 用于改善认知的方法和组合物 |
CN108289933A (zh) * | 2015-11-19 | 2018-07-17 | 巴塞罗那自治大学 | 作为认知和行为障碍药物的哺乳动物Klotho的分泌型剪接变体 |
US20190241633A1 (en) * | 2016-05-04 | 2019-08-08 | Curevac Ag | Rna encoding a therapeutic protein |
US20230123357A1 (en) * | 2020-03-04 | 2023-04-20 | The Regents Of The University Of California | Use of downstream factors in the klotho pathway to assess klotho activity |
US20230181691A1 (en) * | 2020-02-12 | 2023-06-15 | The Regents Of The University Of California | Platelet factors and cognitive improvement |
-
2023
- 2023-11-14 CN CN202311511073.2A patent/CN117797249A/zh active Pending
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105188767A (zh) * | 2012-07-25 | 2015-12-23 | 布罗德研究所有限公司 | 可诱导的dna结合蛋白和基因组干扰工具及其应用 |
US20150366997A1 (en) * | 2012-12-07 | 2015-12-24 | Shire Human Genetics Therapies, Inc. | COMPOSITIONS AND METHODS FOR mRNA DELIVERY |
CN107405382A (zh) * | 2015-02-06 | 2017-11-28 | 加利福尼亚大学董事会 | 用于改善认知的方法和组合物 |
CN108289933A (zh) * | 2015-11-19 | 2018-07-17 | 巴塞罗那自治大学 | 作为认知和行为障碍药物的哺乳动物Klotho的分泌型剪接变体 |
US20190241633A1 (en) * | 2016-05-04 | 2019-08-08 | Curevac Ag | Rna encoding a therapeutic protein |
US20230181691A1 (en) * | 2020-02-12 | 2023-06-15 | The Regents Of The University Of California | Platelet factors and cognitive improvement |
US20230123357A1 (en) * | 2020-03-04 | 2023-04-20 | The Regents Of The University Of California | Use of downstream factors in the klotho pathway to assess klotho activity |
Non-Patent Citations (1)
Title |
---|
DENA B DUBAL等: "Life extension factor klotho enhances cognition", 《CELL REPORTS》, vol. 7, no. 4, 22 May 2014 (2014-05-22), pages 1065 - 1076 * |
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