CN105051190A - 信使rna的纯化方法 - Google Patents
信使rna的纯化方法 Download PDFInfo
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- CN105051190A CN105051190A CN201480013665.4A CN201480013665A CN105051190A CN 105051190 A CN105051190 A CN 105051190A CN 201480013665 A CN201480013665 A CN 201480013665A CN 105051190 A CN105051190 A CN 105051190A
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- mrna
- purifying
- rna
- external synthesis
- tangential flow
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Abstract
本发明尤其提供了纯化信使RNA(mRNA)的方法，所述方法包括使包含体外合成的mRNA的掺杂制剂经受变性条件，以及通过切向流过滤从来自于步骤(a)的所述掺杂制剂纯化所述mRNA的步骤，其中从步骤(b)纯化的所述mRNA基本上无提前中断的RNA序列和/或用于体外合成的酶试剂。
Description
相关申请的交叉引用
本申请案要求2013年3月14日提交的美国临时专利申请序列号61/784,996的权益，其整体通过引用并入本文。
发明背景
信使RNA疗法正成为日益重要的治疗各种疾病的方法。信使RNA疗法涉及向需要该疗法的患者施用信使RNA(mRNA)并在患者体内生成由mRNA编码的蛋白质。因此，重要的是确保生成高纯度且安全的mRNA产物。传统上，RNA纯化通常采用旋转柱并且涉及使用腐蚀性或易燃性溶剂，例如乙醇，这对于治疗剂施用和大规模生产而言不合需要。
发明概述
本文提供了基于切向流过滤(TFF)，纯化适合作为药物产品施用的mRNA的改进方法。在本发明之前，RNA纯化通常采用旋转柱并且涉及使用腐蚀性或易燃性溶剂，例如乙醇，这对于治疗剂施用和大规模生产而言不合需要。进一步地，先前技术方法通常不允许分离称为提前中断产物或“短聚物(shortmer)”的不完全转录产物，据报道这具高免疫刺激性并且其存在可大大改变mRNA作为活性药物成分(API)的毒性和耐受性。本发明部分基于发现切向流过滤对从mRNA生成混合物中去除反应物、酶、副产物，尤其是短聚物惊人地有效。如本文所述，切向流过滤，特别是与使用变性剂的预处理相结合，可有效地去除反应物、酶和包括提前中断RNA序列(即，短聚物)在内的副产物，同时仍保持mRNA的完整性。更令人惊讶的是，本发明人已经证明仅使用含水缓冲液作为溶剂，无需使用任何腐蚀性或易燃性溶剂，可成功地进行切向流过滤。因此，本发明提供了一种更有效、可靠且更安全的从大规模生产工艺治疗应用中纯化mRNA的方法。
一方面，本发明尤其提供了纯化信使RNA(mRNA)的方法，所述方法包括(a)使包含体外合成的mRNA的掺杂制剂经受变性条件，以及(b)通过切向流过滤从来自于步骤(a)的所述掺杂制剂纯化所述mRNA的步骤，其中从步骤(b)纯化的所述mRNA基本上无提前中断的RNA序列和/或用于体外合成的酶试剂。
在一些实施方案中，步骤(a)包括向掺杂制剂添加蛋白质变性剂。在一些实施方案中，步骤(a)包括在室温下用添加的蛋白质变性剂孵育掺杂制剂约1-10分钟(例如，约2-9、2-8、2-7、3-10、3-9、3-8、3-7、3-6、4-10、4-9、4-8、4-7、4-6分钟)。在一些实施方案中，步骤(a)包括在室温下用添加的蛋白质变性剂孵育掺杂制剂约1、2、3、4、5、6、7、8、9或10分钟。在一些实施方案中，步骤(a)包括在室温下用添加的蛋白质变性剂孵育掺杂制剂约5分钟。在一些实施方案中，适合的蛋白质变性剂选自脲、硫氰酸胍、KCl、十二烷基硫酸钠、十二烷基肌氨酸钠、其它洗涤剂及其组合。
在一些实施方案中，步骤(a)包括向掺杂制剂中添加脲以达到约1M或更高的所得脲浓度。在一些实施方案中，所得脲浓度为约2M或更高、3M或更高、4M或更高、5M或更高、6M或更高，7M或更高、8M或更高、9M或更高，或10M或更高。
在一些实施方案中，步骤(a)包括向掺杂制剂中添加硫氰酸胍以达到约1M或更高的所得硫氰酸胍浓度。在一些实施方案中，所得硫氰酸胍浓度为约2M或更高、3M或更高、4M或更高、5M或更高、6M或更高、7M或更高、8M或更高、9M或更高，或10M或更高。
在一些实施方案中，步骤(a)包括向掺杂制剂中添加KCl以达到约1M或更高的所得KCl浓度。在一些实施方案中，所得KCl浓度为约2M或更高、3M或更高、4M或更高，或5M或更高。
在一些实施方案中，仅使用水溶剂进行切向流过滤。在一些实施方案中，使用水作为溶剂进行切向流过滤。在一些实施方案中，以大约100-200mL/分钟(例如，大约100-180mL/分钟、100-160mL/分钟、100-140mL/分钟、110-190mL/分钟、110-170mL/分钟或110-150mL/分钟)的给料速度和/或大约10-50mL/分钟(例如，大约10-40mL/分钟、10-30mL/分钟、20-50mL/分钟或20-40mL/分钟)的流速进行切向流过滤。在一些实施方案中，以大约100、110、120、130、140、150、160、170、180、190或200mL/分钟的给料速度和/或大约10、20、30、40或50mL/分钟的流速进行切向流过滤。
在一些实施方案中，从步骤(b)纯化的mRNA含有少于约5％(例如，少于约4％、3％、2％或1％)的提前中断RNA序列和/或用于体外合成的酶试剂。在一些实施方案中，从步骤(b)纯化的mRNA含有少于约1％(例如，少于约0.9％、0.8％、0.7％、0.6％、0.5％、0.4％、0.3％、0.2％或0.1％)的提前中断RNA序列和/或用于体外合成的酶试剂。在一些实施方案中，从步骤(b)纯化的mRNA含有少于0.5％的提前中断RNA序列和/或用于体外合成的酶试剂。在一些实施方案中，从步骤(b)纯化的mRNA含有少于0.1％的提前中断RNA序列和/或用于体外合成的酶试剂。在一些实施方案中，如通过溴化乙锭和/或考马斯亮蓝染色(Coomassiestaining)测定，从步骤(b)纯化的mRNA含有不可检测的提前中断RNA序列和/或用于体外合成的酶试剂。
在一些实施方案中，提前中断RNA序列包含少于15个碱基(例如，少于14、13、12、11、10、9或8个碱基)。在一些实施方案中，提前中断RNA序列包含约8-12个碱基。
在一些实施方案中，用于体外合成的所述酶试剂包含T7RNA聚合酶、DNA酶I、焦磷酸酶和/或RNA酶抑制剂。在一些实施方案中，用于体外合成的所述酶试剂包含T7RNA聚合酶。
在一些实施方案中，切向流过滤在向体外合成的mRNA加帽和聚腺苷酸(poly-A)尾之前进行。在一些实施方案中，切向流过滤在向所述体外合成的mRNA加帽和聚腺苷酸尾之后进行。在一些实施方案中，切向流过滤在向所述体外合成的mRNA加帽和聚腺苷酸尾之前和之后进行。
在一些实施方案中，体外合成的mRNA长度大于约1kb、1.5kb、2kb、2.5kb、3kb、3.5kb、4kb、4.5kb或5kb。在一些实施方案中，体外合成的mRNA包含一个或多个修饰以增强稳定性。在一些实施方案中，所述一个或多个修饰选自经修饰的核苷酸、经修饰的糖磷酸骨架、5’和/或3’非翻译区。在一些实施方案中，体外合成的mRNA未经修饰。
在一些实施方案中，从步骤(b)纯化的mRNA具有高于约95％(例如，高于约96％、97％、98％、99％或更高)的完整性。在一些实施方案中，从步骤(b)纯化的mRNA具有高于98％的完整性。在一些实施方案中，从步骤(b)纯化的mRNA具有高于99％的完整性。在一些实施方案中，从步骤(b)纯化的mRNA具有高于大约100％的完整性。
本发明还提供了生产信使RNA(mRNA)的方法，包括体外合成mRNA，并且使用根据本文所述的方法纯化体外合成的mRNA的步骤。
本发明还提供了根据本文所述的方法纯化的信使RNA(mRNA)。
在本申请中使用的术语“约”和“大约”作为等效项使用。本申请中使用的任何数值，在有或无约/大约的情况下，意指涵盖相关领域中的普通技术人员理解的任何正常变动。
在随后的详细描述中，本发明的其它特征、目的和优点是显而易见。然而，应理解，详细描述虽然表明本发明的实施方案，但仅以示例说明而非限制的方式提供。基于详细描述，在本发明范围内的各种变化和修改对本领域技术人员而言将变得显而易见。
附图简述
以下图仅是出于示例说明目的而非限制。
图1显示了如凝胶电泳和考马斯亮蓝染色所示，连同各种对照一起，根据提供的包括暴露于脲的方法纯化的FFLmRNA样品体外转录的示例性蛋白质水平。
图2显示了如琼脂糖凝胶电泳和溴化乙锭染色所示，与根据传统方法纯化的mRNA相比，根据提供的方法纯化的体外转录样品的示例性萤火虫荧光素酶(FFL)mRNA水平。
图3显示了与根据传统方法凝胶电泳和考马斯亮蓝染色纯化的mRNA相比，根据提供的包括暴露和不暴露于5M脲的TFF的方法纯化的FFLmRNA样品体外转录的示例性蛋白质水平。
图4描绘了与由传统方法提供的纯化mRNA相比，从由提供的方法提供的翻译纯化FFLmRNA收集的示例性荧光数据。
图5显示与根据传统方法凝胶电泳和考马斯亮蓝染色纯化的mRNA相比，来自于根据提供的包括暴露于蛋白酶K和/或5M脲的方法纯化的因子IX(FIX)mRNA体外转录样品的示例性蛋白质水平。
图6显示了如琼脂糖凝胶电泳和溴化乙锭染色所示，根据提供的方法纯化的体外转录样品中的示例性FIXmRNA水平。
图7显示了与根据传统方法凝胶电泳和考马斯亮蓝染色纯化的mRNA相比，根据提供的包括暴露于2MKCl的方法纯化的囊性纤维化跨膜传导调节因子(CFTR)mRNA的体外转录样品中的示例性蛋白质水平。
图8显示了如琼脂糖凝胶电泳和溴化乙锭染色所示，根据提供的包括暴露于2MKCl的方法纯化的体外转录样品中的示例性CFTRmRNA水平。
图9显示了如琼脂糖凝胶电泳和溴化乙锭染色所示，与根据传统方法纯化的mRNA相比，根据提供的包括暴露于2MKCl的方法纯化的体外转录样品中的示例性CFTRmRNA水平。
定义
为了使本发明更易于理解，首先定义某些术语如下。对以下术语及其它术语的其它定义在整个说明书中进行阐述。
动物：如本文所用的，术语“动物”是指动物界的任何成员。在一些实施方案中，“动物”是指处于任何发育阶段的人类。在一些实施方案中，“动物”是指处于任何发育阶段的非人类动物。在某些实施方案中，非人类动物是哺乳动物(例如，啮齿动物、小鼠、大鼠、兔、猴、狗、猫、羊、牛、灵长类动物和/或猪)。在一些实施方案中，动物包括但不限于，哺乳动物、鸟类、爬行动物、两栖动物、鱼、昆虫和/或蠕虫。在一些实施方案中，动物可以是转基因动物、基因工程动物和/或克隆。
大约或约：如本文所用的，术语“大约”或“约”在应用于一个或多个相关的值时是指与所指的值相似的值。在某些实施方案中，除非另有说明或者从上下文显而易见有所不同(除了此类数值将超过可能值的100％的情况之外)，术语“大约”或“约”是指以任一方向在(大于或小于)所述参考值的25％、20％、19％、18％、17％、16％、15％、14％、13％、12％、11％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％或更小的范围内的值。
生物活性：如本文所用的，短语“生物活性”是指在生物体系中，特别是在生物体中具有活性的任何药物的特征。例如，在向生物体施用时对该生物体具有生物学效应的药物被视为具有生物活性。
表达：如本文所用的，核酸序列的“表达”是指将mRNA翻译成多肽(例如，抗体的重链或轻链)、将多个多肽(例如，抗体的重链或轻链)组装成完整蛋白(例如，抗体)和/或将多肽或完全组装的蛋白(例如，抗体)翻译后修饰。在本申请中，术语“表达”和“产生”以及语法等效项可互换使用。
功能性：如本文所用的，“功能性”生物分子是处于其呈现出它特征性的性质和/或活性的形式的生物分子。
改进、增高或降低：如本文所用的，术语“改进”、“增高”或“降低”或者语法等效项表明相对于基线测量值如在开始本文所述的治疗之前在相同个体中的测量值或者在没有本文所述的治疗下在对照受试者(或多个对照受试者)中的测量值的值。“对照受试者”是罹患与接受治疗的受试者相同的疾病形式的受试者，其与该接受治疗的受试者的年龄大约相同。
杂质：如本文所用的，术语“杂质”是指限定量的液体、气体或固体内，不同于靶材料或化合物的化学组成的物质。杂质也称为污染物。
体外：如本文所用的，术语“体外”是指发生在人工环境中，例如，在试管或反应容器中，在细胞培养物等中而不是在多细胞生物体内的事件。
体内：如本文所用的，术语“体内”是指发生在多细胞生物体如人和非人类动物内的事件。在基于细胞的系统的上下文中，该术语可以用来指发生在活细胞(与例如体外系统相对)内的事件。
分离的：如本文所用的，术语“分离的”是指物质和/或实体，其已经(1)与在最初产生(无论在自然界中和/或在试验环境中)时与它结合的组分中的至少一些分离，和/或(2)通过人工进行生产、制备和/或制造。分离的物质和/或实体可以从与它们最初结合的其它组分的约10％、约20％、约30％、约40％、约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或者大于约99％分离。在一些实施方案中，分离的药物是约80％、约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或者大于约99％纯度。在本文中使用时，若物质基本上不含有其它组分，则它是“纯的”。在本文中使用时，分离的物质和/或实体的百分比纯度的计算不应该包括赋形剂(例如，缓冲剂、溶剂、水等)。
信使RNA(mRNA)：如本文所用的，术语“信使RNA(mRNA)”是指编码至少一种多肽的多核苷酸。如本文所用的，mRNA涵盖经修饰的和未经修饰的RNA。mRNA可以包括一个或多个编码区和非编码区。
mRNA完整性：如本文所用的，术语“mRNA完整性”通常是指mRNA的质量。在一些实施方案中，mRNA完整性是指在纯化工艺(例如，切向流过滤)后未降解的mRNA的百分比。可使用本领域众所周知的方法，例如通过RNA琼脂糖凝胶电泳(例如，Ausubel等，JohnWeley&Sons,Inc.,1997,CurrentProtocolsinMolecularBiology)测定mRNA完整性。
核酸：如本文所用的，术语“核酸”广义上是指是多核苷酸链或者可以被包括在多核苷酸链中的任何化合物和/或物质。在一些实施方案中，核酸是多核苷酸链或者可以通过磷酸二酯键被包括在多核苷酸链中的化合物和/或物质。在一些实施方案中，“核酸”是指单个的核酸残基(例如，核苷酸和/或核苷)。在一些实施方案中，“核酸”是指包含单个的核酸残基的多核苷酸链。在一些实施方案中，“核酸”涵盖RNA以及单链和/或双链的DNA和/或cDNA。此外，术语“核酸”、“DNA”、“RNA”和/或类似的术语包括核酸类似物，即，具有除磷酸二酯骨架之外的类似物。例如，所谓的“肽核酸”是本领域已知的，并且在骨架中具有肽键而不是磷酸二酯键，被视为在本发明的范围内。术语“编码氨基酸序列的核苷酸序列”包括彼此的简并形式和/或编码相同氨基酸序列的所有核苷酸序列。编码蛋白和/或RNA的核苷酸序列可以包括内含子。核酸可以从天然来源进行纯化，利用重组表达系统产生，并任选地进行纯化，化学合成等。在适当的情况中，例如，在化学合成的分子的情况下，核酸可以包括核苷类似物如具有经化学修饰的碱基或糖的类似物，骨架修饰等。除非另外指明，核酸序列以5’至3’方向表示。在一些实施方案中，核酸是或者包括天然核苷(例如，腺苷、胸苷、鸟苷、胞苷、尿苷、脱氧腺苷、脱氧胸苷、脱氧鸟苷和脱氧胞苷)；核苷类似物(例如，2-氨基腺苷、2-硫代胸苷、肌苷、吡咯并嘧啶、3-甲基腺苷、5-甲基胞苷、C-5丙炔基-胞苷、C-5丙炔基-尿苷、2-氨基腺苷、C5-溴尿苷、C5-氟尿苷、C5-碘尿苷、C5-丙炔基-尿苷、C5-丙炔基-胞苷、C5-甲基胞苷、2-氨基腺苷、7-去氮杂腺苷、7-去氮杂鸟苷、8-氧代腺苷、8-氧代鸟苷、O(6)-甲基鸟苷和2-硫代胞苷)；化学修饰的碱基；生物修饰的碱基(例如甲基化的碱基)；嵌入碱基；经修饰的糖(例如，2’-氟核糖、核糖、2’-脱氧核糖、阿拉伯糖和己糖)；和/或经修饰的磷酸基(例如，硫代磷酸酯和5’-N-亚磷酰胺键)。在一些实施方案中，本发明具体地涉及“未经修饰的核酸”，意指尚未为了促进或实现递送而进行化学修饰的核酸(例如，多核苷酸和残基，包括核苷酸和/或核苷)。
患者：如本文所用的，术语“患者”或“受试者”是指为了例如试验、诊断、预防、美容和/或治疗目的可以向其施用提供的组合物的任何生物体。通常患者包括动物(例如，哺乳动物如小鼠、大鼠、兔、非人灵长类动物和/或人)。在一些实施方案中，患者是人。人包括产前和产后的形式。
药学上可接受的：如本文所用的，术语“药学上可接受的”是指物质在合理的医学判断的范围内适合用于与人类和动物的组织接触而没有过度的毒性、刺激、变态反应或其它问题或并发症，与合理的利益/风险比相匹配。
提前中断的RNA序列：如本文所用的，术语“提前中断的RNA序列”是指mRNA合成反应(例如，体外合成反应)的不完全产物。出于各种原因，RNA聚合酶并不总是完成DNA模板的转录，即RNA合成提前终止。RNA合成提前终止的可能原因包括DNA模板的质量、模板中存在的特定聚合酶的聚合酶终止序列、降解缓冲液、温度、核糖核苷酸的损耗及mRNA二级结构。提前中断的RNA序列可为小于所需转录产物预期长度的任何长度。例如，提前中断的mRNA序列可小于1000个碱基、小于500个碱基、小于100个碱基、小于50个碱基、小于40个碱基、小于30个碱基、小于20个碱基、小于15个碱基、小于10个碱基或更少。
盐：如本文所用的，术语“盐”是指由或可能由酸和碱之间的中和反应产生的离子化合物。
受试者：如本文所用的，术语“受试者”是指人或者任何非人动物(例如，小鼠、大鼠、兔、狗、猫、牛、猪、羊、马或灵长动物)。人包括产前和产后的形式。在许多实施方案中，受试者是人类。受试者可以是患者，其是指向医疗提供方寻求疾病的诊断或治疗的人。本文中使用的术语“受试者”与“个体”或“患者”可互换地使用。受试者可以罹患或者易患疾病或病症但是可能或可能不呈现出该疾病或病症的症状。
基本上：如本文所用的，术语“基本上”是指展现出完全或接近完全范围或程度的相关特征或性质的定性状态。任何生物学领域普通技术人员可理解生物和化学现象很少，难得彻底完成和/或持续完成或者获得或避免绝对结果。因此，术语“基本上”在本文中用来表现固有存在于许多生物和化学现象中的完全性的潜在缺乏。
基本上无：如本文所用的，术语“基本上无”是指相对少量或没有要去除的物质(例如，提前中断的RNA序列)存在的状态。例如，“基本上无提前中断的RNA序列”意指提前中断的RNA序列以低于杂质的大约5％、4％、3％、2％、1.0％、0.9％、0.8％、0.7％、0.6％、0.5％、0.4％、0.3％、0.2％、0.1％或更少(w/w)的水平存在。可选地，“基本上无提前中断的RNA序列”意指提前中断的RNA序列以低于约100ng、90ng、80ng、70ng、60ng、50ng、40ng、30ng、20ng、10ηg、1ηg、500ρg、100ρg、50ρg、10ρg或更少的水平存在。
详述
本发明尤其提供了基于切向流过滤从掺杂制剂(例如，体外合成反应混合物)纯化mRNA的改进方法。在一些实施方案中，根据本发明的发明方法包括(a)使包含体外合成的mRNA的掺杂制剂经受变性条件，以及(b)通过切向流过滤从来自于步骤(a)的掺杂制剂纯化mRNA的步骤，其中从步骤(b)纯化的mRNA基本上无提前中断的RNA序列和/或用于体外合成的酶试剂。
在以下章节中详细描述本发明的各方面。章节的使用并非意在限制本发明。每个章节可以应用于本发明的任何方面。在本申请中，除非另外说明，“或”的使用意指“和/或”。
mRNA的合成
mRNA通常被认为是将信息从DNA携带到核糖体的RNA类型。mRNA的存在通常非常短暂并且包括加工和翻译，接着是降解。通常，在真核生物中，mRNA加工包括在N(5’)末端加“帽”和在C(3’)末端加“尾”。典型的帽为7-甲基鸟苷帽，其为通过5’-5’-三磷酸键与第一个转录核苷酸连接的鸟苷。帽的存在在提供对大多数真核细胞内存在的核酸酶的抗性中很重要。尾通常为聚腺苷酸化事件，由此向mRNA分子的3’末端添加多聚腺苷酰基部分。这种“尾”的存在有助于保护mRNA免受核酸外切酶降解。信使RNA由核糖体翻译成一系列构成蛋白质的氨基酸。
根据本发明的mRNA可根据各种已知方法中的任一种合成。例如，根据本发明的mRNA可经由体外转录(IVT)合成。简言之，通常用含有启动子的线性或环状DNA模板、核糖核苷三磷酸池、可包括DTT和镁离子的缓冲体系和适当的RNA聚合酶(例如，T3、T7或SP6RNA聚合酶)、DNA酶I、焦磷酸酶和/或RNA酶抑制剂进行IVT。确切条件将根据特定应用变化。这些试剂的存在根据几个实施方案在最终产物中不合需要并且因此可称为杂质并且含有这些杂质中的一种或多种的制剂可称为掺杂制剂。
可在商业规模上纯化根据本发明的mRNA。在一些实施方案中，在每批为或大于0.1克、0.5克、1克、2克、3克、4克、5克、6克、7克、8克、9克、10克、20克、30克、40克、50克、60克、70克、80克、90克、100克、200克、300克、400克、500克、600克、700克、800克、900克或1,000克的规模上纯化mRNA。
根据各个实施方案，本发明可用于纯化各种长度的体外合成mRNA。在一些实施方案中，本发明可用于纯化长度大于约1kb、1.5kb、2kb、2.5kb、3kb、3.5kb、4kb、4.5kb、5kb、6kb、7kb、8kb、9kb、10kb、11kb、12kb、13kb、14kb或15kb的体外合成mRNA。在一些实施方案中，本发明可用于纯化含有通常增强稳定性的一个或多个修饰的mRNA。在一些实施方案中，一个或多个修饰选自经修饰的核苷酸、经修饰的糖磷酸骨架、5’和/或3’非翻译区。在一些实施方案中，本发明可用于纯化未经修饰的体外合成mRNA。
通常，mRNA经修饰以增强稳定性。mRNA的修饰可包括，例如RNA的核苷酸的修饰。因此根据本发明经修饰的mRNA可包括，例如骨架修饰、糖修饰或碱基修饰。在一些实施方案中，可由自然存在的核苷酸和/或核苷酸类似物(经修饰的核苷酸)，包括但不限于嘌呤(腺嘌呤(A)、鸟嘌呤(G))或嘧啶(胸腺嘧啶(T)、胞嘧啶(C)、尿嘧啶(U))，及嘌呤和嘧啶的经修饰核苷酸类似物或衍生物，例如1-甲基-腺嘌呤、2-甲基-腺嘌呤、2-甲基硫代-N-6-异戊烯-腺嘌呤、N6-甲基-腺嘌呤、N6-异戊烯-腺嘌呤、2-硫代-胞嘧啶、3-甲基-胞嘧啶、4-乙酰基-胞嘧啶、5-甲基-胞嘧啶、2,6-二氨基嘌呤、1-甲基-鸟嘌呤、2-甲基-鸟嘌呤、2,2-二甲基-鸟嘌呤、7-甲基-鸟嘌呤、肌苷、1-甲基-肌苷、假尿嘧啶(5-尿嘧啶)、二氢-尿嘧啶、2-硫代-尿嘧啶、4-硫代-尿嘧啶、5-羧甲基氨甲基-2-硫代-尿嘧啶、5-(羧基羟甲基)-尿嘧啶、5-氟-尿嘧啶、5-溴-尿嘧啶、5-羧甲基氨甲基-尿嘧啶、5-甲基-2-硫代-尿嘧啶、5-甲基-尿嘧啶、N-尿嘧啶-5-羟乙酸甲酯、5-甲基氨甲基-尿嘧啶、5-甲氧基氨甲基-2-硫代-尿嘧啶、5'-甲氧羰基甲基-尿嘧啶、5-甲氧基-尿嘧啶、尿嘧啶-5-羟乙酸甲酯、尿嘧啶-5-羟乙酸(v)、1-甲基-假尿嘧啶、Q核苷(queosine)、.β.-D-甘露糖基-Q核苷、怀丁氧苷(wybutoxosine)和氨基磷酸酯、硫代磷酸酯、肽核苷酸、甲基膦酸酯、7-去氮鸟苷、5-甲基胞嘧啶和肌苷合成编码抗体的mRNA(例如，编码重链和轻链的mRNA)。例如由美国专利第4,373,071号、美国专利第4,401,796号、美国专利第4,415,732号、美国专利第4,458,066号、美国专利第4,500,707号、美国专利第4,668,777号、美国专利第4,973,679号、美国专利第5,047,524号、美国专利第5,132,418号、美国专利第5,153,319号、美国专利第5,262,530和5,700,642号，本领域中的技术人员已知此类类似物的制备，所述专利的公开内容通过引用全部包括在本文中。
通常，mRNA合成包括在N(5’)末端加“帽”和在C(3’)末端加“尾”。帽的存在在提供对大多数真核细胞内存在的核酸酶的抗性中很重要。“尾”的存在有助于保护mRNA免受核酸外切酶降解。
因此，在一些实施方案中，mRNA包括5’帽结构。通常如下添加5’帽：首先，RNA末端磷酸酶从5’核苷酸去除一个末端磷酸基团，留下两个末端磷酸；然后经由鸟苷酸转移酶向末端磷酸添加三磷酸鸟苷(GTP)，生成5’5’5三磷酸键；然后通过甲基转移酶甲基化鸟嘌呤的7-氮。帽结构的实例包括但不限于m7G(5')ppp(5'(A,G(5')ppp(5')A和G(5')ppp(5')G。
虽然由体外转录反应提供的mRNA可能在一些实施方案中满足需要，但是其它mRNA来源被视为在本发明的范围之内，包括由细菌、真菌、植物和/或动物生成的野生型mRNA。
在一些实施方案中，mRNA包括5’和/或3’非翻译区。在一些实施方案中，5’非翻译区包括影响mRNA的稳定性或翻译的一个或多个元件，例如铁反应元件。在一些实施方案中，5’非翻译区长度可介于约50和500个核苷酸之间。
在一些实施方案中，3’非翻译区包括聚腺苷酸化信号序列、影响mRNA在细胞内定位的稳定性的蛋白质的结合位点或miRNA的一个或多个结合位点中的一个或多个。在一些实施方案中，3’非翻译区的长度可介于约50和500个核苷酸之间或更长。
本发明可用于纯化编码各种蛋白质的mRNA。在实施例部分详细描述了纯化编码萤火虫荧光素酶、因子IX和CFTR的mRNA的非限制性实例。
变性条件和变性剂
通常，通过破坏分子间力暂时或永久性地改变蛋白质或核酸的构象称为变性。变性导致结构变化并常常导致活性丧失。因为分子的天然构象通常最具水溶性，破坏分子的二级和三级结构可引起溶解性变化并且可导致蛋白质或核酸从溶液中沉淀。令人惊讶地，如本文所用的，变性条件与切向流过滤(TFF)结合使用可利于mRNA纯化，同时仍维持mRNA的完整性。
如本文所用的，术语“变性条件”指可引起变性的任何化学或物理条件。示例性变性条件包括但不限于化学试剂、高温、极端pH等。
在一些实施方案中，通过向含有待纯化的mRNA的掺杂制剂中添加一种或多种变性剂获得变性条件。在一些实施方案中，适合本发明的变性剂为蛋白质和/或DNA变性剂。在一些实施方案中，变性剂可为：1)酶(例如丝氨酸蛋白酶或DNA酶)、2)酸、3)溶剂、4)交联剂、5)离液剂、6)还原剂和/或7)经由高盐浓度的高离子强度。在一些实施方案中，特定试剂可属于这些类别中的一种以上。
在一些实施方案中，一种或多种酶可用作变性剂以降解蛋白质和用于mRNA合成的DNA模板。在一些实施方案中，适合的酶包括但不限于丝氨酸蛋白酶例如胰凝乳蛋白酶和胰凝乳蛋白酶样丝氨酸蛋白酶、胰蛋白酶和胰蛋白酶样丝氨酸蛋白酶、弹性蛋白酶和弹性蛋白酶样丝氨酸蛋白酶、枯草杆菌蛋白酶和枯草杆菌蛋白酶样丝氨酸蛋白酶及其组合、脱氧核糖核酸酶(DNA酶)(例如脱氧核糖核酸酶I、II和/或IV)、限制酶(例如EcoRI、EcoRII、BamHI、HindIII、SpeI、SphI、StuI、XbaI)及其组合。
在一些实施方案中，酸可用作变性剂。在一些实施方案中，适合的酸为乙酸、甲酸、草酸、柠檬酸、苯甲酸、氯乙酸、二氯乙酸、三氯乙酸、抗坏血酸、磺基水杨酸及其组合。
在一些实施方案中，溶剂可用作变性剂。在一些实施方案中，溶剂可为异丙醇、丙酮、甲乙酮、甲基异丁酮、乙醇、甲醇、地那铵(denatonium)及其组合。
在一些实施方案中，离液剂可用作变性剂。离液剂是通过干扰非共价力例如氢键和范德华力(vanderWaalsforce)破坏大分子例如蛋白质和核酸的结构的物质。在一些实施方案中，离液剂可为脲、硫脲、盐酸胍、硫氰酸胍、异硫氰酸胍、乙酸锂、氯化镁、十二烷基硫酸钠、高氯酸锂及其组合。
在一些实施方案中，用脲处理含有待纯化的mRNA的掺杂制剂。在一些实施方案中，添加一定量的脲以致所得脲浓度为约1M或更高。在一些实施方案中，添加脲以致所得脲浓度为约2M或更高、3M或更高、4M或更高、5M或更高、6M或更高、7M或更高、8M或更高、9M或更高，或10M或更高。在一些实施方案中，用硫氰酸胍处理含有待纯化的mRNA的掺杂制剂。在一些实施方案中，添加一定量的硫氰酸胍以致所得硫氰酸胍浓度为约1M或更高。在一些实施方案中，添加硫氰酸胍以致所得硫氰酸胍浓度为约2M或更高、3M或更高、4M或更高、5M或更高、6M或更高、7M或更高、8M或更高、9M或更高，或10M或更高。
在一些实施方案中，还原剂可用作变性剂。还原剂是为另一物质给出电子，从而自身变氧化的化合物。在一些实施方案中，还原剂可为氢化铝锂、钠汞齐、二硼烷、硼氢化钠、亚硫酸盐、二异丁基氢化铝、亚磷酸盐、一氧化碳、2-巯基乙醇、二硫苏糖醇或三(2-羧乙基)膦及其组合。
在一些实施方案中，pH、热量和/或重金属(例如铅、汞或镉)中的一种或多种可用作变性剂。已知极端pH引起蛋白质变性。虽然蛋白质链的骨架为中性，但是构成蛋白质的氨基酸残基常常含有酸性和碱性基团。这些基团通常带电并且可与相反电荷的基团形成盐桥。因此，极端pH可改变在这些酸性和碱性基团上的电荷，破坏盐桥。
在一些实施方案中，pH较温和的变化也可影响蛋白质的活性和溶解性。与个别氨基酸一样，蛋白质具有负电荷的数量等于正电荷的数量时的等电点。这通常是水溶性最低的点。在等电pH下，在分子上不存在净电荷。单分子具有相互靠近、凝结和从溶液中沉淀出来的倾向。在高于或低于等电pH的pH下，分子分别具有净负电荷或正电荷。因此当蛋白质分子相互靠近时，其具有相同的总电荷并且相互排斥。
在一些实施方案中，热量可用作变性剂。热量可为蛋白质分子供给动能，使其原子更迅速地振动。在一些实施方案中，这将破坏相对较弱的力例如氢键和疏水性相互作用。热量也可用于灭菌以使细菌中的酶变性并因此破坏细菌中的酶。
在一些实施方案中，金属离子例如汞(II)、铅(II)和银的盐由于其与二硫基和酸性氨基酸的羧酸盐离子形成强健的能力，可用作变性剂。因此，其破坏二硫桥和盐键并且使蛋白质从溶液中作为不溶性金属-蛋白质盐沉淀出来。
在一些实施方案中，高浓度的盐(高盐度)也可用作变性剂。已知高浓度的盐使蛋白质和核酸从水溶液中沉淀。在一些实施方案中，盐的高浓度可介于1M和10M之间，包括1M和10M。在一些实施方案中，盐的高浓度可介于2M和9M之间，包括2M和9M。在一些实施方案中，盐的高浓度可介于2M和8M之间，包括2M和8M。在一些实施方案中，盐的高浓度可介于2M和5M之间，包括2M和5M。在一些实施方案中，盐的高浓度可高于1M浓度。在一些实施方案中，盐的高浓度可高于2M浓度。在一些实施方案中，盐的高浓度可高于3M浓度。在一些实施方案中，盐的高浓度可高于4M浓度。在一些实施方案中，盐的高浓度可高于5M浓度。在一些实施方案中，盐的高浓度可高于6M浓度。在一些实施方案中，盐的高浓度可高于7M浓度。在一些实施方案中，盐的高浓度可高于8M浓度。在一些实施方案中，单种盐用作变性剂。在一些实施方案中，多于一种盐用作变性剂。
在一些实施方案中，用作变性剂的盐可为钙盐、铁盐、镁盐、钾盐、钠盐或其组合。在一些实施方案中适合用作变性剂的示例性特定盐类包括但不限于氯化钾(KCl)、氯化钠(NaCl)、氯化锂(LiCl)、氯化钙(CaCl2)、溴化钾(KBr)、溴化钠(NaBr)、溴化锂(LiBr)。在一些实施方案中，掺杂制剂经受的变性剂为氯化钾(KCl)。在一些实施方案中，添加KCl以致所得KCl浓度为约1M或更高。在一些实施方案中，添加KCl以致所得KCl浓度为约2M或更高、3M或更高、4M或更高，或5M或更高。
在一些实施方案中，可能可取的是用一种或多种变性剂孵育掺杂制剂一段时间。在一些实施方案中，用变性剂孵育掺杂制剂少于1分钟。在一些实施方案中，用变性剂孵育掺杂制剂1分钟。在一些实施方案中，用变性剂孵育掺杂制剂2分钟。在一些实施方案中，用变性剂孵育掺杂制剂3分钟。在一些实施方案中，用变性剂孵育掺杂制剂4分钟。在一些实施方案中，用变性剂孵育掺杂制剂5分钟。在一些实施方案中，用变性剂孵育掺杂制剂10分钟。在一些实施方案中，用变性剂孵育掺杂制剂1小时。在一些实施方案中，用变性剂孵育掺杂制剂2小时。
在一些实施方案中，在室温(例如，约20-25℃)下用一种或多种变性剂孵育掺杂制剂。在一些实施方案中，在低于室温的温度下用一种或多种变性剂孵育掺杂制剂。在一些实施方案中，在高于室温的温度下用一种或多种变性剂孵育掺杂制剂。
纯化
在几个实施方案中，在暴露于变性条件之前和/或之后，用切向流过滤从掺杂制剂纯化mRNA。在一些实施方案中，切向流过滤在向体外合成的mRNA加帽和聚腺苷酸尾之前进行。在一些实施方案中，切向流过滤在向体外合成的mRNA加帽和聚腺苷酸尾之后进行。在一些实施方案中，切向流过滤在向体外合成的mRNA加帽和聚腺苷酸尾之前和之后进行。
传统膜过滤
通常，膜过滤涉及使用一张或多张插入的渗透膜从流体中分离出固体。膜过滤也可用于从气态样品中过滤颗粒。存在两种主要的膜过滤形式，仅仅由于溶解-扩散进行的被动过滤，和使用正压或负压(即真空)迫使液体或气体穿过所述膜的主动过滤。
传统膜过滤也称为“死端”过滤。以这种形式，将进料载至膜上并通过正压或负压推动。死端过滤往往便宜而简单，主要缺点是膜受非渗透性或缓慢渗透性溶质(也称为渗余物)污损或堵塞，和浓度极化。通常，膜往往在推动力增加时更迅速地堵塞或污损。当膜污损或堵塞时，过滤速率降低并且最终没有渗透物能够通过直至更换或清洁过滤器。浓度极化是其中非渗透性溶质在过滤器表面聚集并且最终形成进一步阻止渗透性溶质穿过所述膜的一类次生膜的现象。因此，死端过滤通常用于批式工艺中。
切向流过滤
切向流过滤(TFF)，也称为错流过滤，是其中待过滤材料切向穿过过滤器，而不是通过过滤器的一类过滤。在TFF中，不需要的渗透物通过过滤器，而所需渗余物经过过滤器并且在下游聚集。重要的是注意在TFF中渗余物中通常含有所需材料，这与通常在传统死端过滤中遇到的相反。
根据待过滤的材料，TFF通常用于微滤或超滤。通常将微滤定义为过滤器的孔径介于0.05μm和1.0μm之间，包括0.05μm和1.0μm的情况，而超滤通常涉及孔径小于0.05μm的过滤器。孔径还决定了特定过滤器的标称分子量极限(NMWL)，也称为截留分子量(MWCO)，微滤膜通常具有大于1,000千道尔顿(kDa)的NMWL并且超滤过滤器具有介于1kDa和1,000kDa之间的NMWL。
切向流过滤的主要优点是改为沿着过滤器的表面运送在传统“死端”过滤期间可在过滤器内聚集并阻塞过滤器的非渗透性颗粒(有时称为“滤饼”)。这种优点允许切向流过滤广泛用于需要连续操作的工业过程中，因为通常不需要取下并清洁过滤器而明显减少了停机时间。
切向流过滤可用于几个目的，尤其包括浓缩和渗滤。浓缩是由此从溶液中去除溶剂，而保留溶质分子的过程。为了有效地浓缩样品，使用具有大大低于待保留的溶质分子的分子量的NMWL或MWCO的膜。通常，技术人员可以选择具有低于靶分子的分子量3至6倍的NMWL或MWCO的过滤器。
渗滤是不需要的小颗粒由此通过过滤器，而在渗余物中保持需要的较大分子，不改变溶液中那些分子的浓度的分馏过程。渗滤常常用于从溶液中去除盐或反应缓冲液。渗滤可以是连续或不连续的。在连续渗滤中，以滤液产生的相同速率向样品进料中添加渗滤溶液。在不连续渗滤中，首先稀释溶液，然后浓缩回到起始浓度。可重复不连续渗滤，直至达到所需的溶质分子浓度。
在典型TFF过程中重要的至少三个过程变量：跨膜压差、进料速率和渗透物的流速。跨膜压差是推动流体，带着渗透性分子一起通过过滤器的力。在一些实施方案中，跨膜压差介于1和30磅/平方英寸(psi)之间，包括1和30磅/平方英寸。
进料速率(也称为错流速度)是溶液流过进料通道并穿过过滤器的速率。进料速率决定了清除可能以其它方式填塞或污损过滤器并从而限制滤液流速的分子的力。在一些实施方案中，进料速率介于50和500mL/分钟之间。在一些实施方案中，进料速率介于50和400mL/分钟之间。在一些实施方案中，进料速率介于50和300mL/分钟之间。在一些实施方案中，进料速率介于50和200mL/分钟之间。在一些实施方案中，进料速率介于75和200mL/分钟之间。在一些实施方案中，进料速率介于100和200mL/分钟之间。在一些实施方案中，进料速率介于125和175mL/分钟之间。在一些实施方案中，进料速率为130mL/分钟。在一些实施方案中，进料速率介于60mL/分钟和220mL/分钟之间。在一些实施方案中，进料速率为60mL/分钟或更高。在一些实施方案中，进料速率为100mL/分钟或更高。在一些实施方案中，进料速率为150mL/分钟或更高。在一些实施方案中，进料速率为200mL/分钟或更高。在一些实施方案中，进料速率为220mL/分钟或更高。
渗透物的流速是从体系中去除渗透物的速率。对于恒定进料速率而言，增加渗透物流速可增加穿过过滤器的压力，导致过滤速率提高，同时还可能增加过滤器堵塞或污损的风险。Michaels等，"TangentialFlowFiltration"inSeparationsTechnology,PharmaceuticalandBiotechnologyApplications(W.P.Olson编，InterpharmPress,Inc.,BuffaloGrove,Ill.1995)中描述了用于TFF的原理、理论和装置。对代表对TFF的改进的高性能切向流过滤(HP-TFF)的描述，还见美国专利第5,256,294和5,490,937号。在一些实施方案中，流速介于10和100mL/分钟之间。在一些实施方案中，流速介于10和90mL/分钟之间。在一些实施方案中，流速介于10和80mL/分钟之间。在一些实施方案中，流速介于10和70mL/分钟之间。在一些实施方案中，流速介于10和60mL/分钟之间。在一些实施方案中，流速介于10和50mL/分钟之间。在一些实施方案中，流速介于10和40mL/分钟之间。在一些实施方案中，流速介于20和40mL/分钟之间。在一些实施方案中，流速为30mL/分钟。
可使用本文所述的各种工艺变量的任何组合。在一些实施方案中，以大约100-200mL/分钟(例如，大约100-180mL/分钟、100-160mL/分钟、100-140mL/分钟、110-190mL/分钟、110-170mL/分钟或110-150mL/分钟)的进料速率和/或大约10-50mL/分钟(例如，大约10-40mL/分钟、10-30mL/分钟、20-50mL/分钟或20-40mL/分钟)的流速进行切向流过滤。在一些实施方案中，以大约100、110、120、130、140、150、160、170、180、190或200mL/分钟的进料速率和/或大约10、20、30、40或50mL/分钟的流速进行切向流过滤。
进一步地适应大(商业)规模纯化的流速将需要以大约10L-200L/分钟(例如，大约10-180L/分钟、100-160L/分钟、100-140L/分钟、110-190L/分钟、110-170L/分钟或110-150L/分钟)的进料速率和/或大约10-50L/分钟(例如，大约10-40L/分钟、10-30L/分钟、20-50L/分钟或20-40L/分钟)的流速进行切向流过滤。在一些实施方案中，以大约100、110、120、130、140、150、160、170、180、190或200L/分钟的进料速率和/或大约10、20、30、40或50L/分钟的流速进行切向流过滤。
如上所述，用于TFF的过滤器可具有各种孔径中的任一种，并且因此具有NMWL。在一些实施方案中，过滤器将具有介于100kDa和1,000kDa之间的NMWL。在一些实施方案中，过滤器将具有介于200kDa和700kDa之间的NMWL。在一些实施方案中，过滤器将具有介于200kDa和500kDa之间的NMWL。在一些实施方案中，过滤器将具有300kDa的NMWL。在一些实施方案中，过滤器将具有500kDa的NMWL。
在一些实施方案中，仅使用水溶剂进行根据本发明所述的切向流过滤。在一些实施方案中，使用水作为溶剂进行根据本发明所述的切向流过滤。
纯化mRNA的表征
在各个实施方案中，根据本发明纯化的mRNA基本上无来自于mRNA合成过程的杂质，包括但不限于提前中断的RNA序列、DNA模板和/或用于体外合成的酶试剂。
本发明提供的独特优势是能够去除或消除高度的提前中断RNA序列(也称为“短聚物”)。在一些实施方案中，根据本发明所述的方法去除多于约90％、95％、96％、97％、98％、99％或基本上所有的提前中断RNA序列。在一些实施方案中，根据本发明纯化的mRNA基本上无提前中断的RNA序列。在一些实施方案中，根据本发明纯化的mRNA含有少于约5％(例如，少于约4％、3％、2％或1％)的提前中断RNA序列。在一些实施方案中，根据本发明纯化的mRNA含有少于约1％(例如，少于约0.9％、0.8％、0.7％、0.6％、0.5％、0.4％、0.3％、0.2％或0.1％)的提前中断RNA序列。在一些实施方案中，如通过(例如)溴化乙锭和/或考马斯亮蓝染色测定，根据本发明纯化的mRNA含有不可检测的提前中断RNA序列。在一些实施方案中，提前中断的RNA序列包含少于15个碱基(例如，少于14、13、12、11、10、9或8个碱基)。在一些实施方案中，提前中断的RNA序列包含约8-12个碱基。
在一些实施方案中，根据本发明所述的方法去除或消除高度的用于体外合成的酶试剂，包括但不限于T7RNA聚合酶、DNA酶I、焦磷酸酶和/或RNA酶抑制剂。在一些实施方案中，本发明对去除T7RNA聚合酶特别有效。在一些实施方案中，根据本发明所述的方法去除多于约90％、95％、96％、97％、98％、99％或基本上所有用于体外合成的酶试剂(包括)。在一些实施方案中，根据本发明纯化的mRNA基本上无用于体外合成的酶试剂(包括)。在一些实施方案中，根据本发明纯化的mRNA含有少于约5％(例如，少于约4％、3％、2％或1％)的用于体外合成的酶试剂(包括)。在一些实施方案中，根据本发明纯化的mRNA含有少于约1％(例如，少于约0.9％、0.8％、0.7％、0.6％、0.5％、0.4％、0.3％、0.2％或0.1％)的用于体外合成的酶试剂(包括)。在一些实施方案中，如通过(例如)溴化乙锭和/或考马斯亮蓝染色测定，根据本发明纯化的mRNA含有不可检测的用于体外合成的酶试剂(包括)。
在各个实施方案中，根据本文所述的方法纯化的mRNA保持高度完整性。如本文所用的，术语“mRNA完整性”通常是指纯化后mRNA的质量。在一些实施方案中，mRNA完整性是指在切向流过滤后未降解的mRNA的百分比。可使用本领域众所周知的方法，例如通过RNA琼脂糖凝胶电泳(例如，Ausubel等，JohnWeley&Sons,Inc.,1997,CurrentProtocolsinMolecularBiology)测定mRNA完整性。在一些实施方案中，根据本发明纯化的mRNA具有高于约95％(例如，高于约96％、97％、98％、99％或更高)的完整性。在一些实施方案中，根据本发明纯化的mRNA具有高于98％的完整性。在一些实施方案中，根据本发明纯化的mRNA具有高于99％的完整性。在一些实施方案中，根据本发明纯化的mRNA具有高于大约100％的完整性。
实施例
实施例1.信使RNA(mRNA)的生成和纯化
mRNA的合成
在以下每个实施例中，在完全无RNA酶的条件下进行mRNA的合成。除非另外明确表明，要求所有管、小瓶、移液管枪头、移液管、缓冲液等均无核酸酶。
在下列实施例中，除非另有描述，由线性化DNA模板经由体外转录合成mRNA。为生成所需mRNA前体(IVT)构建体，用无RNA酶的水制备～100ug线性化DNA、rNTP(3.33mM)、DTT(10mM)、T7RNA聚合酶、RNA酶抑制剂、焦磷酸酶和反应缓冲液(10x，800mMHepes(pH8.0)、20mM亚精胺、250mMMgCl2，pH7.7)的混合物至2.24mL的最终体积。在37℃下孵育反应混合物介于20分钟-120分钟之间范围的时间。完成后，用DNA酶I再处理混合物15分钟并相应地猝灭。
5’帽和3’尾的添加
使来自前述IVT步骤(也可能来自初始TFF过滤)的纯化mRNA产物在65℃下变性10分钟。单独地，将成份的GTP(20mM)、S-腺苷甲硫氨酸、RNA酶抑制剂、2’-O-甲基转移酶和鸟苷酸转移酶与反应缓冲液(10x，500mMTris-HCl(pH8.0)、60mMKCl、12.5mMMgCl2)混合在一起至8.3mL的终浓度。变性后，在冰上冷却mRNA，然后添加到反应混合物中。在37℃下孵育合并的溶液20-90分钟范围的时间。完成后，添加等分试样的ATP(20mM)、聚腺苷酸聚合酶和加尾反应缓冲液(10x，500mMTris-HCl(pH8.0)、2.5MNaCl、100mMMgCl2)并进一步在37℃下孵育总反应混合物20-45分钟范围的时间。完成后，猝灭最终反应混合物并相应地纯化。
经由切向流过滤纯化
在以下实施例中，除非另有描述，切向流过滤(TFF)系统由滤膜和蠕动泵(MilliporeLabscaleTFF系统)组成，液体以～130mL/分钟的进料速率穿过膜切向循环，渗透物的流速为30mL/流速。采用的TFF膜为MidiKros500kDamPES115cm2(SpectrumLabs)。使用之前，用无核酸酶的水洗涤滤筒并用0.2NNaOH进一步清洗。最后用无核酸酶的水清洗系统直至渗透物和渗余物的pH达到pH～6。
实施例2.纯化mRNA的分析
测试纯化mRNA中酶的存在
除非另有描述，进行标准考马斯亮蓝染色蛋白质凝胶以确定在纯化之前和之后存在的任何残留试剂酶的存在。在一些情况下，也进行BCA测定。
经由琼脂糖凝胶电泳测定评估mRNA完整性
除非另有描述，经由凝胶电泳评估信使RNA大小和完整性。采用自流式1.0％琼脂糖凝胶或InvitrogenE-Gel预制1.2％琼脂糖凝胶。信使RNA按每孔1.0-1.5ug量上样。完成后，使用溴化乙锭使信使RNA条带可见。
体外mRNA完整性测定
除非另有描述，使用HEK293T细胞进行萤火虫荧光素酶mRNA的体外转染。在单独的孔内使用lipofectamine进行1微克各mRNA构建体的转染。在所选时间点(例如4小时、8小时等)收获细胞并分析各自的蛋白质产量。对于FFLmRNA而言，经由生物发光测定分析细胞裂解产物的荧光素酶产量。
生物发光分析
在包括对提供的RNA的荧光评估的实施例中，除非另有说明，使用Promega荧光素酶测定系统(Item#E1500)进行生物发光测定。通过向荧光素酶测定底物中添加10mL的荧光素酶测定缓冲液并经由涡旋混合制备荧光素酶测定试剂。向96孔板上装载大约20uL的匀浆样品，接着向每份样品装载20uL板对照。单独地，向96孔平底板的每个孔添加120uL荧光素酶测定试剂(如上所述制备)。然后使用MolecularDeviceFlexStation仪器将每张板插入适当腔室内并测量发光(按相对光度单位(RLU)测量)。
实施例3.萤火虫荧光素酶(FFL)信使RNA(mRNA)的生成和纯
化
该实施例说明，根据各个实施方案，切向流过滤(TFF)和变性剂的组合可根据提供的方法使用以产生高度纯化的mRNA产物。在该实施例中，脲用作蛋白质变性剂。
在该实施例中，经由上述体外法转录一批5毫克的萤火虫荧光素酶(FFL)RNA(SEQIDNO:1，如下)以生成无帽和聚腺苷酸尾的前述中间构建体。该反应保持2.24mL的总体积并且在完成后用等体积的10M脲猝灭，使最终脲浓度达到5M。在室温下孵育所得溶液5分钟并转移到切向流过滤(TFF)系统贮器内。用无核酸酶的水将样品稀释至200mL并用1200mL无核酸酶的水，通过每次200mL超滤而洗涤。在此之后，用200mL10mM柠檬酸钠(pH6.4)处理样品，接着用600ml无核酸酶的水洗涤。最后将样品浓缩至～2mL并且在260nm(λmax)下经由吸收作用测定终浓度。
密码子优化的萤火虫荧光素酶(FFL)mRNA(SEQIDNO:1)
X2AUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGCCAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGAGCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGUGUAY2
5’和3’UTR序列
X2＝
GGGAUCCUACC(SEQIDNO：2)
Y2＝
UUUGAAUU(SEQIDNO：3)
如上所述，在9mL的最终反应体积中大约5mg经TFF纯化的萤火虫荧光素酶RNA加帽并加尾。在室温下(RT)用5M脲处理一份该反应混合物(6.7ml)5分钟并使用TFF纯化。仅使用水经由TFF纯化大约1.5mg的帽/尾反应混合物并分离。单独地，使用QiagenRNeasy纯化试剂盒根据公开的方法纯化另一小份帽/尾反应混合物。等分三批分离的最终FFLmRNA并且如下所述转染至HEK293T细胞内。经由荧光检测(FFL活性)分析细胞裂解产物中FFL蛋白的存在。
在该实施例中，为了去除该实施例中的反应酶，使一份FFLmRNAIVT混合物经受10M脲，产生5M脲终浓度。在室温下孵育该溶液5分钟，然后如上所述经由TFF纯化。图1显示了考马斯亮蓝染色的蛋白质凝胶，其显示了在采用前述脲条件的TFF后分离的所得mRNA。完成后不存在可检测的酶。
生成加帽和加尾FFLmRNA产物后，采用TFF方法进一步纯化最终靶mRNA。单独地等分成份的相同帽/尾反应混合物并经由不用脲的TFF或经由离心柱法(QiagenRNeasy试剂盒)纯化以进行比较。使用凝胶电泳进行通过TFF或离心柱分离的最终mRNA的比较并且在图2中进行描绘。进一步地，经由蛋白质凝胶监测残留酶水平(图3)。在图2中，人们可清楚地看到以～1900nt迁移的各“IVT”FFLmRNA条带，加帽和加尾(C/T)的最终mRNA大约2100nt长。在泳道4确实观察到在帽/尾步骤之后通常使用离心柱分离观察到的“短聚物”条带。
显然当采用经TFF纯化的mRNA时，在帽/尾步骤之后不存在短聚物条带。虽然可使用任一纯化方法去除大量的酶试剂，但是短聚物杂质不可以。这证明本文所述的切向流过滤是在mRNA转录期间纯化提前中断序列成功且有效的方法。
为了确定提供的mRNA是否可以翻译成所需蛋白质，进行每种分离FFLmRNA构建体(TFF与离心柱)的比较。将以下所列3种构建体中的每一种转染到HEK293T细胞内并在暴露于萤光素后呈FFL发光形式经由FFL蛋白质活性评估相应的FFL蛋白质产量(同上)。
FFL构建体：
1.经由TFF(脲)纯化的FFLIVT和经由TFF(无脲)的C/T步骤
2.经由TFF(脲)纯化的FFLIVT和经由TFF(脲)的C/T步骤
3.经由离心柱纯化的FFLIVT和经由离心柱的C/T步骤
图4中描绘了由每一种生成的FFL蛋白质发光输出的比较。在所述条件下(暴露于5M脲)在整个切向流过滤过程中保持经TFF纯化的FFLmRNA的完整性。
实施例4.因子IX(FIX)mRNA的生成和纯化
该实施例进一步说明，根据各个实施方案，切向流过滤(TFF)和变性剂的组合可根据提供的方法使用以产生高度纯化的mRNA产物。在该实施例中，硫氰酸胍用作蛋白质变性剂。
在该实施例中，生成第二种mRNA并纯化，这次编码因子IX(SEQIDNO:4，如下)。最初，经由上述体外法转录一批5毫克的因子IX(FIX)RNA以生成无帽和聚腺苷酸尾的前述RNA。该反应保持2.24mL的总体积并且在完成后通过添加蛋白酶K(4mg/mlIVT反应)，在37℃下于反应混合物中孵育5分钟猝灭反应。完成后，添加6M硫氰酸胍(4.3mL，最终～4M)并且在室温下孵育所得溶液5分钟并转移到TFF系统贮器内。用无核酸酶的水将样品稀释至200mL并用1600mL无核酸酶的水，通过每次200mL超滤而洗涤。完成后，将样品浓缩至～2mL并且在260nm(λmax)下经由吸收作用测定终浓度。
人因子IX(FIX)mRNA(SEQIDNO:4)
X1AUGCAGCGCGUGAACAUGAUCAUGGCAGAAUCACCAGGCCUCAUCACCAUCUGCCUUUUAGGAUAUCUACUCAGUGCUGAAUGUACAGUUUUUCUUGAUCAUGAAAACGCCAACAAAAUUCUGAGGCGGAGAAGGAGGUAUAAUUCAGGUAAAUUGGAAGAGUUUGUUCAAGGGAACCUUGAGAGAGAAUGUAUGGAAGAAAAGUGUAGUUUUGAAGAAGCACGAGAAGUUUUUGAAAACACUGAAAGAACAACUGAAUUUUGGAAGCAGUAUGUUGAUGGAGAUCAGUGUGAGUCCAAUCCAUGUUUAAAUGGCGGCAGUUGCAAGGAUGACAUUAAUUCCUAUGAAUGUUGGUGUCCCUUUGGAUUUGAAGGAAAGAACUGUGAAUUAGAUGUAACAUGUAACAUUAAGAAUGGCAGAUGCGAGCAGUUUUGUAAAAAUAGUGCUGAUAACAAGGUGGUUUGCUCCUGUACUGAGGGAUAUCGACUUGCAGAAAACCAGAAGUCCUGUGAACCAGCAGUGCCAUUUCCAUGUGGAAGAGUUUCUGUUUCACAAACUUCUAAGCUCACCCGUGCUGAGGCUGUUUUUCCUGAUGUGGACUAUGUAAAUUCUACUGAAGCUGAAACCAUUUUGGAUAACAUCACUCAAAGCACCCAAUCAUUUAAUGACUUCACUCGGGUUGUUGGUGGAGAAGAUGCCAAACCAGGUCAAUUCCCUUGGCAGGUUGUUUUGAAUGGUAAAGUUGAUGCAUUCUGUGGAGGCUCUAUCGUUAAUGAAAAAUGGAUUGUAACUGCUGCCCACUGUGUUGAAACUGGUGUUAAAAUUACAGUUGUCGCAGGUGAACAUAAUAUUGAGGAGACAGAACAUACAGAGCAAAAGCGAAAUGUGAUUCGAAUUAUUCCUCACCACAACUACAAUGCAGCUAUUAAUAAGUACAACCAUGACAUUGCCCUUCUGGAACUGGACGAACCCUUAGUGCUAAACAGCUACGUUACACCUAUUUGCAUUGCUGACAAGGAAUACACGAACAUCUUCCUCAAAUUUGGAUCUGGCUAUGUAAGUGGCUGGGGAAGAGUCUUCCACAAAGGGAGAUCAGCUUUAGUUCUUCAGUACCUUAGAGUUCCACUUGUUGACCGAGCCACAUGUCUUCGAUCUACAAAGUUCACCAUCUAUAACAACAUGUUCUGUGCUGGCUUCCAUGAAGGAGGUAGAGAUUCAUGUCAAGGAGAUAGUGGGGGACCCCAUGUUACUGAAGUGGAAGGGACCAGUUUCUUAACUGGAAUUAUUAGCUGGGGUGAAGAGUGUGCAAUGAAAGGCAAAUAUGGAAUAUAUACCAAGGUAUCCCGGUAUGUCAACUGGAUUAAGGAAAAAACAAAGCUCACUUAAY1
5’和3’UTR序列
X1＝
GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG(SEQIDNO：5)
Y1＝
CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC(SEQIDNO：6)
以上方法也如上所述进行，在蛋白酶K步骤期间添加放线菌素D(10μg/mlIVT反应)。通过用蛋白酶K(有或无放线菌素D)猝灭IVT反应，也可成功地实现所有酶的去除(图5)。虽然蛋白酶K可利于去除，但是大规模生产mRNA药物将需要大规模制备这种酶，招致不必要的附加成本，并且因此在一些实施方案中可能不是所需方法。如图6中所示，如上所述生成的FIXmRNA(有或无放线菌素D)，以及使用5M脲纯化的FIXmRNA，不含可检测水平的短聚物，与实施例3中所述对于FFLmRNA的结果类似。
实施例5.囊性纤维化跨膜传导调节因子(CFTR)mRNA的生成和
纯化
该实施例进一步说明，根据各个实施方案，切向流过滤(TFF)和变性剂的组合可根据提供的方法使用以产生高度纯化的mRNA产物。在该实施例中，氯化钾用作蛋白质变性剂。
在该实施例中，生成第三种mRNA并纯化，这次编码囊性纤维化跨膜传导调节因子(CFTR,SEQIDNO:7，如下)。最初，经由上述体外法转录一批5毫克的CFTRRNA以生成无帽和聚腺苷酸尾的前述RNA。该反应保持2.24mL的总体积并且在完成后通过添加2MKCl(～200mL)猝灭。在室温下孵育所得溶液5分钟并转移到TFF系统贮器内。在无核酸酶的水中用2MKCL以200mL恒定体积渗滤样品3-4个渗滤体积。这次之后，用400mL无核酸酶的水，通过每次200mL超滤而洗涤所得溶液。在此之后，用200mL1mM柠檬酸钠(pH6.4)处理样品，接着用600ml无核酸酶的水洗涤。最后，将样品浓缩至～2mL并且在260nm(λmax)下经由吸收作用测定终浓度。
密码子优化的囊性纤维化跨膜传导调节因子(CFTR)mRNA(SEQIDNO:7)
X1AUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAAY1
5’和3’UTR序列
X1＝
GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG(SEQIDNO：5)
Y1＝
CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC(SEQIDNO：6)
在该实施例中，为了去除反应酶，使用2MKCl渗滤。如经由蛋白质凝胶电泳测定(图7)，暴露于大体积的2MKCl导致成功去除反应混合物中存在的所有酶(包括T7聚合酶)。如琼脂糖凝胶电泳所示，靶信使RNA在暴露于此类条件后保持完整(图8)。
进一步地，在CFTRIVT构建体加帽和加尾后，可使用2MKCl经由TFF成功地纯化最终CFTR转录产物(加帽和加尾)。将这种分离的最终产物与经由离心柱方法纯化的相同产物比较时，正如经由凝胶电泳所测定，观察到大为减少的“短聚物”条带(图9)。
等效项和范围
仅仅使用常规实验，本领域技术人员可认识到，或者能够确定，本文所述的本发明的具体实施方案的许多等效项。本发明的范围无意受限于以上描述，而是如以下权利要求书中所述：
Claims (31)
1.一种纯化信使RNA(mRNA)的方法，其包括
(a)使包含体外合成的mRNA的掺杂制剂经受变性条件；以及
(b)通过切向流过滤从来自于步骤(a)的所述掺杂制剂纯化所述mRNA；
其中从步骤(b)纯化的所述mRNA基本上无提前中断的RNA序列和/或用于体外合成的酶试剂。
2.根据权利要求1所述的方法，其中步骤(a)包括向所述掺杂制剂添加蛋白质变性剂。
2a.根据权利要求2所述的方法，其中步骤(a)包括在室温下用添加的所述蛋白质变性剂孵育所述掺杂制剂约5分钟。
3.根据权利要求2或2a所述的方法，其中所述蛋白质变性剂选自脲、硫氰酸胍、KCl、十二烷基硫酸钠、十二烷基肌氨酸钠及其组合。
4.根据前述权利要求中任一项所述的方法，其中步骤(a)包括向所述掺杂制剂中添加脲以达到约1M或更高的所得脲浓度。
5.根据权利要求4所述的方法，其中所述所得脲浓度为约2M或更高、3M或更高、4M或更高、5M或更高、6M或更高、7M或更高、8M或更高、9M或更高，或10M或更高。
6.根据前述权利要求中任一项所述的方法，其中步骤(a)包括向所述掺杂制剂中添加硫氰酸胍以达到约4M或更高的所得硫氰酸胍浓度。
7.根据权利要求6所述的方法，其中所述所得硫氰酸胍浓度为约4M或更高、5M或更高、6M或更高、7M或更高、8M或更高，9M或更高，或10M或更高。
8.根据前述权利要求中任一项所述的方法，其中步骤(a)包括向所述掺杂制剂中添加KCl以达到约1M或更高的所得KCl浓度。
9.根据权利要求8所述的方法，其中所述所得KCl浓度为约2M或更高、3M或更高、4M或更高，或5M或更高。
10.根据前述权利要求中任一项所述的方法，其中仅使用水溶剂进行所述切向流过滤。
11.根据权利要求10所述的方法，其中使用水作为溶剂进行所述切向流过滤。
12.根据前述权利要求中任一项所述的方法，其中从步骤(b)纯化的所述mRNA含有少于1％的提前中断RNA序列和/或用于体外合成的酶试剂。
13.根据前述权利要求中任一项所述的方法，其中从步骤(b)纯化的所述mRNA含有少于0.5％的提前中断RNA序列和/或用于体外合成的酶试剂。
14.根据前述权利要求中任一项所述的方法，其中从步骤(b)纯化的所述mRNA含有少于0.1％的提前中断RNA序列和/或用于体外合成的酶试剂。
15.根据前述权利要求中任一项所述的方法，其中如通过琼脂糖凝胶电泳或色谱法测定，从步骤(b)纯化的所述mRNA含有不可检测的提前中断RNA序列和/或用于体外合成的酶试剂。
16.根据前述权利要求中任一项所述的方法，其中所述提前中断RNA序列包含少于15个碱基。
17.根据前述权利要求中任一项所述的方法，其中所述提前中断RNA序列包含约8-12个碱基。
18.根据前述权利要求中任一项所述的方法，其中用于体外合成的所述酶试剂包含T7RNA聚合酶、DNA酶I、焦磷酸酶和/或RNA酶抑制剂。
19.根据前述权利要求中任一项所述的方法，其中用于体外合成的所述酶试剂包含T7RNA聚合酶。
20.根据前述权利要求中任一项所述的方法，其中所述切向流过滤在向体外合成的mRNA加帽和聚腺苷酸尾之前进行。
21.根据权利要求1-19中任一项所述的方法，其中所述切向流过滤在向所述体外合成的mRNA加帽和聚腺苷酸尾之后进行。
22.根据权利要求1-19中任一项所述的方法，其中所述切向流过滤在向所述体外合成的mRNA加帽和聚腺苷酸尾之前和之后进行。
23.根据前述权利要求中任一项所述的方法，其中所述体外合成的mRNA长度大于约1kb、1.5kb、2kb、2.5kb、3kb、3.5kb、4kb、4.5kb或5kb。
24.根据前述权利要求中任一项所述的方法，其中所述体外合成的mRNA包含一个或多个修饰以增强稳定性。
25.根据权利要求24所述的方法，其中所述一个或多个修饰选自经修饰的核苷酸、经修饰的糖磷酸骨架、5’和/或3’非翻译区。
26.根据权利要求1-23中任一项所述的方法，其中所述体外合成的mRNA未经修饰。
27.根据前述权利要求中任一项所述的方法，其中从步骤(b)纯化的所述mRNA具有高于95％的完整性。
28.根据前述权利要求中任一项所述的方法，其中从步骤(b)纯化的所述mRNA具有高于98％的完整性。
29.根据前述权利要求中任一项所述的方法，其中从步骤(b)纯化的所述mRNA具有高于99％的完整性。
30.一种生产信使RNA(mRNA)的方法，其包括：
体外合成mRNA；以及
使用根据前述权利要求中任一项所述的方法纯化所述体外合成的mRNA。
31.一种信使RNA(mRNA)，其使用根据前述权利要求中任一项所述的方法纯化。
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| PCT/US2014/028441 WO2014152966A1 (en) | 2013-03-14 | 2014-03-14 | Methods for purification of messenger rna |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112930396A (zh) * | 2018-08-24 | 2021-06-08 | 川斯勒佰尔公司 | 用于纯化信使rna的方法 |
| CN113166790A (zh) * | 2018-11-08 | 2021-07-23 | 川斯勒佰尔公司 | 信使rna纯化的方法和组合物 |
| CN114127283A (zh) * | 2019-05-15 | 2022-03-01 | 川斯勒佰尔公司 | 用于纯化信使rna的方法 |
Families Citing this family (74)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PL3338765T3 (pl) | 2009-12-01 | 2019-06-28 | Translate Bio, Inc. | Pochodna steroidowa dla dostarczania mrna w ludzkich chorobach genetycznych |
| CN111671918A (zh) | 2011-06-08 | 2020-09-18 | 川斯勒佰尔公司 | Mrna递送的脂质纳米颗粒组合物和方法 |
| EP3336082B1 (en) | 2011-06-08 | 2020-04-15 | Translate Bio, Inc. | Cleavable lipids |
| US20150267192A1 (en) | 2012-06-08 | 2015-09-24 | Shire Human Genetic Therapies, Inc. | Nuclease resistant polynucleotides and uses thereof |
| AU2014236305B2 (en) | 2013-03-14 | 2019-01-17 | Ethris Gmbh | CFTR mRNA compositions and related methods and uses |
| ES2708561T3 (es) * | 2013-03-14 | 2019-04-10 | Translate Bio Inc | Métodos para la purificación de ARN mensajero |
| EP2971033B8 (en) * | 2013-03-15 | 2019-07-10 | ModernaTX, Inc. | Manufacturing methods for production of rna transcripts |
| US10077439B2 (en) | 2013-03-15 | 2018-09-18 | Modernatx, Inc. | Removal of DNA fragments in mRNA production process |
| ES2923018T3 (es) | 2013-03-15 | 2022-09-22 | Glaxosmithkline Biologicals Sa | Métodos de purificación de ARN |
| JP6506749B2 (ja) | 2013-10-22 | 2019-04-24 | シャイアー ヒューマン ジェネティック セラピーズ インコーポレイテッド | フェニルケトン尿症のためのｍＲＮＡ療法 |
| CN106413811A (zh) | 2013-10-22 | 2017-02-15 | 夏尔人类遗传性治疗公司 | 精氨基琥珀酸合成酶缺乏症的mrna疗法 |
| ES2750661T3 (es) | 2014-04-25 | 2020-03-26 | Translate Bio Inc | Métodos para la purificación de ARN mensajero |
| WO2016180430A1 (en) | 2015-05-08 | 2016-11-17 | Curevac Ag | Method for producing rna |
| EP4108769B1 (en) * | 2015-05-29 | 2023-08-30 | CureVac Manufacturing GmbH | A method for producing and purifying rna, comprising at least one step of tangential flow filtration |
| WO2017031241A1 (en) * | 2015-08-17 | 2017-02-23 | Modernatx, Inc. | Rna mapping/fingerprinting |
| US20210206818A1 (en) | 2016-01-22 | 2021-07-08 | Modernatx, Inc. | Messenger ribonucleic acids for the production of intracellular binding polypeptides and methods of use thereof |
| EP3442590A2 (en) | 2016-04-13 | 2019-02-20 | Modernatx, Inc. | Lipid compositions and their uses for intratumoral polynucleotide delivery |
| HRP20230050T1 (hr) | 2016-05-18 | 2023-03-03 | Modernatx, Inc. | Polinukleotidi koji kodiraju interleukin-12 (il12) i njihova upotreba |
| WO2018041921A1 (en) | 2016-08-31 | 2018-03-08 | Curevac Ag | Mixing device for the production of a liquid nucleic acid composition |
| US11202793B2 (en) | 2016-09-14 | 2021-12-21 | Modernatx, Inc. | High purity RNA compositions and methods for preparation thereof |
| WO2018096179A1 (en) | 2016-11-28 | 2018-05-31 | Curevac Ag | Method for purifying rna |
| US11384352B2 (en) | 2016-12-13 | 2022-07-12 | Modernatx, Inc. | RNA affinity purification |
| WO2018157154A2 (en) | 2017-02-27 | 2018-08-30 | Translate Bio, Inc. | Novel codon-optimized cftr mrna |
| IL268857B2 (en) | 2017-02-27 | 2024-09-01 | Translate Bio Inc | Methods for the purification of messenger RNA |
| US11560562B2 (en) | 2017-02-27 | 2023-01-24 | Translate Bio, Inc. | Large-scale synthesis of messenger RNA |
| DK3585892T3 (da) | 2017-02-27 | 2022-08-22 | Translate Bio Inc | Fremgangsmåder til oprensning af messenger-rna |
| EP3624824B1 (en) | 2017-05-16 | 2024-07-10 | Translate Bio, Inc. | Codon-optimized mrna encoding cftr for use in treating cystic fibrosis |
| US20230064106A1 (en) | 2017-05-17 | 2023-03-02 | Curevac Real Estate Gmbh | Method for determining at least one quality parameter of an rna sample |
| WO2018231990A2 (en) | 2017-06-14 | 2018-12-20 | Modernatx, Inc. | Polynucleotides encoding methylmalonyl-coa mutase |
| US11786607B2 (en) | 2017-06-15 | 2023-10-17 | Modernatx, Inc. | RNA formulations |
| MA49922A (fr) | 2017-08-18 | 2021-06-02 | Modernatx Inc | Procédés pour analyse par clhp |
| US11866696B2 (en) | 2017-08-18 | 2024-01-09 | Modernatx, Inc. | Analytical HPLC methods |
| WO2019046809A1 (en) | 2017-08-31 | 2019-03-07 | Modernatx, Inc. | METHODS OF MANUFACTURING LIPID NANOPARTICLES |
| EP3852728B1 (en) | 2018-09-20 | 2024-09-18 | ModernaTX, Inc. | Preparation of lipid nanoparticles and methods of administration thereof |
| EP3918065A4 (en) * | 2019-01-29 | 2022-10-26 | Flagship Pioneering Innovations V, Inc. | PROCEDURE FOR SEPARATION OF LONG POLYNUCLEOTIDS FROM A COMPOSITION |
| CN113396219A (zh) | 2019-02-11 | 2021-09-14 | 埃泽瑞斯公司 | 通过切向流过滤纯化mRNA |
| WO2020168466A1 (en) | 2019-02-19 | 2020-08-27 | Stemirna Therapeutics Co., Ltd. | Modified nucleoside and synthetic methods thereof |
| CN110133091B (zh) * | 2019-04-29 | 2020-08-28 | 中国科学院武汉物理与数学研究所 | 一种05sar-page的应用 |
| CA3146675A1 (en) | 2019-07-23 | 2021-01-28 | Translate Bio, Inc. | Stable compositions of mrna-loaded lipid nanoparticles and processes of making |
| AU2020329166A1 (en) | 2019-08-09 | 2022-03-03 | Nutcracker Therapeutics, Inc. | Microfluidic apparatus and methods of use thereof |
| EP4031662A1 (en) | 2019-09-20 | 2022-07-27 | Translate Bio, Inc. | Mrna encoding engineered cftr |
| EP4041400A1 (en) | 2019-10-09 | 2022-08-17 | Translate Bio, Inc. | Compositions, methods and uses of messenger rna |
| US20210145860A1 (en) | 2019-10-21 | 2021-05-20 | Translate Bio, Inc. | Compositions, methods and uses of messenger rna |
| KR20220142432A (ko) | 2019-12-20 | 2022-10-21 | 트랜슬레이트 바이오 인코포레이티드 | 메신저 rna의 직장 전달 |
| CA3165388A1 (en) | 2019-12-20 | 2021-06-24 | Translate Bio, Inc. | Improved process of preparing mrna-loaded lipid nanoparticles |
| US20240277830A1 (en) | 2020-02-04 | 2024-08-22 | CureVac SE | Coronavirus vaccine |
| US20210324369A1 (en) | 2020-02-10 | 2021-10-21 | Translate Bio, Inc. | Methods and compositions for messenger rna purification |
| CN115398546A (zh) | 2020-02-18 | 2022-11-25 | 翻译生物公司 | 用于信使rna体外转录的改进方法 |
| JP2023515485A (ja) * | 2020-02-21 | 2023-04-13 | バイオジェン・エムエイ・インコーポレイテッド | 限外濾過／透析濾過を用いたオリゴヌクレオチド組成物の調製方法 |
| WO2021173840A1 (en) | 2020-02-25 | 2021-09-02 | Translate Bio, Inc. | Improved processes of preparing mrna-loaded lipid nanoparticles |
| GB202307565D0 (en) | 2020-04-22 | 2023-07-05 | BioNTech SE | Coronavirus vaccine |
| CA3177940A1 (en) | 2020-05-07 | 2021-11-11 | Anusha DIAS | Optimized nucleotide sequences encoding sars-cov-2 antigens |
| EP4146342A1 (en) | 2020-05-07 | 2023-03-15 | Translate Bio, Inc. | Improved compositions for cftr mrna therapy |
| WO2021226463A1 (en) | 2020-05-07 | 2021-11-11 | Translate Bio, Inc. | Composition and methods for treatment of primary ciliary dyskinesia |
| AU2021273502A1 (en) | 2020-05-15 | 2023-02-02 | Translate Bio, Inc. | Lipid nanoparticle formulations for mRNA delivery |
| JP2023544197A (ja) | 2020-10-06 | 2023-10-20 | トランスレイト バイオ， インコーポレイテッド | 脂質ナノ粒子の改善された方法および製剤 |
| AU2021361986A1 (en) | 2020-10-12 | 2023-06-15 | Translate Bio, Inc. | Improved process of preparing mrna-loaded lipid nanoparticles |
| WO2022081548A1 (en) | 2020-10-12 | 2022-04-21 | Translate Bio, Inc. | Improved process of preparing ice-based lipid nanoparticles |
| WO2022099194A1 (en) | 2020-11-09 | 2022-05-12 | Translate Bio, Inc. | Improved compositions for delivery of codon-optimized mrna |
| CA3199895A1 (en) | 2020-11-25 | 2022-06-02 | Shrirang KARVE | Stable liquid lipid nanoparticle formulations |
| EP4277929A1 (en) | 2021-01-14 | 2023-11-22 | Translate Bio, Inc. | Methods and compositions for delivering mrna coded antibodies |
| EP4313115A1 (en) | 2021-03-25 | 2024-02-07 | Translate Bio, Inc. | Optimized nucleotide sequences encoding the extracellular domain of human ace2 protein or a portion thereof |
| AU2022261865A1 (en) | 2021-04-19 | 2023-11-30 | Translate Bio, Inc. | Improved compositions for delivery of mrna |
| EP4330427A1 (en) | 2021-04-29 | 2024-03-06 | Translate Bio, Inc. | Methods for measuring poly a tail length |
| US20240277872A1 (en) | 2021-05-12 | 2024-08-22 | The Broad Institute, Inc. | Modified mrna, modified non-coding rna, and uses thereof |
| CN117881395A (zh) | 2021-07-01 | 2024-04-12 | 翻译生物公司 | 用于递送mRNA的组合物 |
| EP4429713A1 (en) | 2021-11-10 | 2024-09-18 | Translate Bio, Inc. | Composition and methods for treatment of primary ciliary dyskinesia |
| JP2023181989A (ja) | 2022-06-13 | 2023-12-25 | 上海臻上医薬科技有限公司 | マイクロニードル注射製剤およびその使用 |
| US11878055B1 (en) | 2022-06-26 | 2024-01-23 | BioNTech SE | Coronavirus vaccine |
| WO2024112652A1 (en) | 2022-11-21 | 2024-05-30 | Translate Bio, Inc. | Compositions of dry powder formulations of messenger rna and methods of use thereof |
| WO2024126809A1 (en) | 2022-12-15 | 2024-06-20 | Sanofi | Mrna encoding influenza virus-like particle |
| WO2024133515A1 (en) | 2022-12-20 | 2024-06-27 | Sanofi | Rhinovirus mrna vaccine |
| WO2024133884A2 (en) | 2022-12-23 | 2024-06-27 | Sanofi | Optimized tailing of messenger rna |
| WO2024184489A1 (en) | 2023-03-07 | 2024-09-12 | Sanofi | Manufacture of messenger rna with kp34 polymerase |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998005673A1 (en) * | 1996-08-01 | 1998-02-12 | Megabios Corporation | Methods for purifying nucleic acids |
| EP0853123A1 (de) * | 1997-01-10 | 1998-07-15 | Roche Diagnostics GmbH | Reinigung von DNA durch Cross-Flow-Filtration |
| WO2001007599A1 (en) * | 1999-07-23 | 2001-02-01 | Genentech, Inc. | Method for rnase- and organic solvent-free plasmid dna purification using tangential flow filtration |
| WO2002042317A2 (en) * | 2000-11-21 | 2002-05-30 | Glaxo Group Limited | Method of separating extra-chromosomal dna from other cellular components |
| WO2012077080A1 (en) * | 2010-12-10 | 2012-06-14 | Tracy Thompson | Compositions for separation methods |
Family Cites Families (470)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2647121A (en) | 1951-02-02 | 1953-07-28 | Ruth P Jacoby | Diamine-bis-acetamides |
| US2819718A (en) | 1953-07-16 | 1958-01-14 | Isidore H Goldman | Drainage tube |
| US2717909A (en) | 1953-09-24 | 1955-09-13 | Monsanto Chemicals | Hydroxyethyl-keryl-alkylene-ammonium compounds |
| US2844629A (en) | 1956-04-25 | 1958-07-22 | American Home Prod | Fatty acid amides and derivatives thereof |
| US3096560A (en) | 1958-11-21 | 1963-07-09 | William J Liebig | Process for synthetic vascular implants |
| FR1378382A (fr) | 1962-12-01 | 1964-11-13 | Sandoz Sa | Amides de l'acide amino-propionique, utilisables en particulier pour le traitement des fibres textiles |
| GB1072118A (en) | 1962-12-01 | 1967-06-14 | Sandoz Ag | Amides of aminopropionic acid |
| JPS5141663B1 (zh) | 1966-03-12 | 1976-11-11 | ||
| JPS4822365B1 (zh) | 1968-10-25 | 1973-07-05 | ||
| NL143127B (nl) | 1969-02-04 | 1974-09-16 | Rhone Poulenc Sa | Versterkingsorgaan voor een defecte hartklep. |
| US3614955A (en) | 1970-02-09 | 1971-10-26 | Medtronic Inc | Standby defibrillator and method of operation |
| US3614954A (en) | 1970-02-09 | 1971-10-26 | Medtronic Inc | Electronic standby defibrillator |
| JPS5024216Y1 (zh) | 1970-12-29 | 1975-07-21 | ||
| JPS5024216B1 (zh) | 1970-12-29 | 1975-08-14 | ||
| US3945052A (en) | 1972-05-01 | 1976-03-23 | Meadox Medicals, Inc. | Synthetic vascular graft and method for manufacturing the same |
| US3805301A (en) | 1972-07-28 | 1974-04-23 | Meadox Medicals Inc | Tubular grafts having indicia thereon |
| JPS49127908A (zh) | 1973-04-20 | 1974-12-07 | ||
| JPS5624664B2 (zh) | 1973-06-28 | 1981-06-08 | ||
| US4013507A (en) | 1973-09-18 | 1977-03-22 | California Institute Of Technology | Ionene polymers for selectively inhibiting the vitro growth of malignant cells |
| JPS5123537A (ja) | 1974-04-26 | 1976-02-25 | Adeka Argus Chemical Co Ltd | Kasozaisoseibutsu |
| GB1527592A (en) | 1974-08-05 | 1978-10-04 | Ici Ltd | Wound dressing |
| US3995623A (en) | 1974-12-23 | 1976-12-07 | American Hospital Supply Corporation | Multipurpose flow-directed catheter |
| JPS5813576B2 (ja) | 1974-12-27 | 1983-03-14 | アデカ ア−ガスカガク カブシキガイシヤ | 安定化された合成高分子組成物 |
| DE2520814A1 (de) | 1975-05-09 | 1976-11-18 | Bayer Ag | Lichtstabilisierung von polyurethanen |
| US4281669A (en) | 1975-05-09 | 1981-08-04 | Macgregor David C | Pacemaker electrode with porous system |
| JPS5210847A (en) | 1975-07-16 | 1977-01-27 | Nippon Steel Corp | Pinch roll |
| US4096860A (en) | 1975-10-08 | 1978-06-27 | Mclaughlin William F | Dual flow encatheter |
| CA1069652A (en) | 1976-01-09 | 1980-01-15 | Alain F. Carpentier | Supported bioprosthetic heart valve with compliant orifice ring |
| US4134402A (en) | 1976-02-11 | 1979-01-16 | Mahurkar Sakharam D | Double lumen hemodialysis catheter |
| US4072146A (en) | 1976-09-08 | 1978-02-07 | Howes Randolph M | Venous catheter device |
| US4335723A (en) | 1976-11-26 | 1982-06-22 | The Kendall Company | Catheter having inflatable retention means |
| US4099528A (en) | 1977-02-17 | 1978-07-11 | Sorenson Research Co., Inc. | Double lumen cannula |
| US4140126A (en) | 1977-02-18 | 1979-02-20 | Choudhury M Hasan | Method for performing aneurysm repair |
| US4265745A (en) | 1977-05-25 | 1981-05-05 | Teijin Limited | Permselective membrane |
| US4182833A (en) | 1977-12-07 | 1980-01-08 | Celanese Polymer Specialties Company | Cationic epoxide-amine reaction products |
| US4180068A (en) | 1978-04-13 | 1979-12-25 | Motion Control, Incorporated | Bi-directional flow catheter with retractable trocar/valve structure |
| EP0005035B1 (en) | 1978-04-19 | 1981-09-23 | Imperial Chemical Industries Plc | A method of preparing a tubular product by electrostatic spinning |
| US4284459A (en) | 1978-07-03 | 1981-08-18 | The Kendall Company | Method for making a molded catheter |
| US4227533A (en) | 1978-11-03 | 1980-10-14 | Bristol-Myers Company | Flushable urinary catheter |
| US4375817A (en) | 1979-07-19 | 1983-03-08 | Medtronic, Inc. | Implantable cardioverter |
| US4500707A (en) | 1980-02-29 | 1985-02-19 | University Patents, Inc. | Nucleosides useful in the preparation of polynucleotides |
| US5132418A (en) | 1980-02-29 | 1992-07-21 | University Patents, Inc. | Process for preparing polynucleotides |
| US4458066A (en) | 1980-02-29 | 1984-07-03 | University Patents, Inc. | Process for preparing polynucleotides |
| DE3010841A1 (de) | 1980-03-21 | 1981-10-08 | Ulrich Dr.med. 6936 Haag Uthmann | Katheder |
| US4308085A (en) | 1980-07-28 | 1981-12-29 | Jenoptik Jena Gmbh | Process for the preparation of high molecular thermoplastic epoxide-amine-polyadducts |
| US4415732A (en) | 1981-03-27 | 1983-11-15 | University Patents, Inc. | Phosphoramidite compounds and processes |
| US4668777A (en) | 1981-03-27 | 1987-05-26 | University Patents, Inc. | Phosphoramidite nucleoside compounds |
| US4973679A (en) | 1981-03-27 | 1990-11-27 | University Patents, Inc. | Process for oligonucleo tide synthesis using phosphormidite intermediates |
| US4339369A (en) | 1981-04-23 | 1982-07-13 | Celanese Corporation | Cationic epoxide-amine reaction products |
| US4401796A (en) | 1981-04-30 | 1983-08-30 | City Of Hope Research Institute | Solid-phase synthesis of polynucleotides |
| US4373071A (en) | 1981-04-30 | 1983-02-08 | City Of Hope Research Institute | Solid-phase synthesis of polynucleotides |
| US4406656A (en) | 1981-06-01 | 1983-09-27 | Brack Gillium Hattler | Venous catheter having collapsible multi-lumens |
| US4475972A (en) | 1981-10-01 | 1984-10-09 | Ontario Research Foundation | Implantable material |
| US4401472A (en) | 1982-02-26 | 1983-08-30 | Martin Marietta Corporation | Hydraulic cement mixes and processes for improving hydraulic cement mixes |
| US4568329A (en) | 1982-03-08 | 1986-02-04 | Mahurkar Sakharam D | Double lumen catheter |
| US4546499A (en) | 1982-12-13 | 1985-10-15 | Possis Medical, Inc. | Method of supplying blood to blood receiving vessels |
| US4530113A (en) | 1983-05-20 | 1985-07-23 | Intervascular, Inc. | Vascular grafts with cross-weave patterns |
| US4550447A (en) | 1983-08-03 | 1985-11-05 | Shiley Incorporated | Vascular graft prosthesis |
| US4647416A (en) | 1983-08-03 | 1987-03-03 | Shiley Incorporated | Method of preparing a vascular graft prosthesis |
| US5104399A (en) | 1986-12-10 | 1992-04-14 | Endovascular Technologies, Inc. | Artificial graft and implantation method |
| US4571241A (en) | 1983-12-16 | 1986-02-18 | Christopher T Graham | Urinary catheter with collapsible urethral tube |
| US4710169A (en) | 1983-12-16 | 1987-12-01 | Christopher T Graham | Urinary catheter with collapsible urethral tube |
| US4737518A (en) | 1984-04-03 | 1988-04-12 | Takeda Chemical Industries, Ltd. | Lipid derivatives, their production and use |
| US4562596A (en) | 1984-04-25 | 1986-01-07 | Elliot Kornberg | Aortic graft, device and method for performing an intraluminal abdominal aortic aneurysm repair |
| US4782836A (en) | 1984-05-24 | 1988-11-08 | Intermedics, Inc. | Rate adaptive cardiac pacemaker responsive to patient activity and temperature |
| US4897355A (en) | 1985-01-07 | 1990-01-30 | Syntex (U.S.A.) Inc. | N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor |
| US4662382A (en) | 1985-01-16 | 1987-05-05 | Intermedics, Inc. | Pacemaker lead with enhanced sensitivity |
| US4762915A (en) | 1985-01-18 | 1988-08-09 | Liposome Technology, Inc. | Protein-liposome conjugates |
| US4860751A (en) | 1985-02-04 | 1989-08-29 | Cordis Corporation | Activity sensor for pacemaker control |
| US5223263A (en) | 1988-07-07 | 1993-06-29 | Vical, Inc. | Liponucleotide-containing liposomes |
| CA1320724C (en) | 1985-07-19 | 1993-07-27 | Koichi Kanehira | Terpene amino alcohols and medicinal uses thereof |
| US4701162A (en) | 1985-09-24 | 1987-10-20 | The Kendall Company | Foley catheter assembly |
| US4737323A (en) | 1986-02-13 | 1988-04-12 | Liposome Technology, Inc. | Liposome extrusion method |
| US5153319A (en) | 1986-03-31 | 1992-10-06 | University Patents, Inc. | Process for preparing polynucleotides |
| DE3616824A1 (de) | 1986-05-17 | 1987-11-19 | Schering Ag | Verwendung von haertbaren kunstharzmischungen fuer oberflaechenbeschichtungen und druckfarben und verfahren zu ihrer herstellung |
| DE3780374D1 (de) | 1986-07-31 | 1992-08-20 | Irnich Werner | Frequenzadaptierender herzschrittmacher. |
| US4960409A (en) | 1986-09-11 | 1990-10-02 | Catalano Marc L | Method of using bilumen peripheral venous catheter with adapter |
| JPH0829776B2 (ja) | 1986-10-29 | 1996-03-27 | 東燃化学株式会社 | 合成樹脂製容器及びその製造用金型 |
| US4720517A (en) | 1986-11-24 | 1988-01-19 | Ciba-Geigy Corporation | Compositions stabilized with N-hydroxyiminodiacetic and dipropionic acids and esters thereof |
| US4920016A (en) | 1986-12-24 | 1990-04-24 | Linear Technology, Inc. | Liposomes with enhanced circulation time |
| DE3728917A1 (de) | 1987-08-29 | 1989-03-09 | Roth Hermann J | Neue lipide mit unsymmetrisch substituierter disulfidbruecke |
| US4946683A (en) | 1987-11-18 | 1990-08-07 | Vestar, Inc. | Multiple step entrapment/loading procedure for preparing lipophilic drug-containing liposomes |
| US4843155A (en) * | 1987-11-19 | 1989-06-27 | Piotr Chomczynski | Product and process for isolating RNA |
| US5138067A (en) | 1987-12-17 | 1992-08-11 | Shionogi & Co. Ltd. | Lipid derivatives |
| US5047540A (en) | 1987-12-17 | 1991-09-10 | Shionogi & Co., Ltd. | Lipid derivatives |
| JPH01257491A (ja) * | 1988-04-08 | 1989-10-13 | Tosoh Corp | 不溶性融合異種蛋白質の処理方法 |
| US4892540A (en) | 1988-04-21 | 1990-01-09 | Sorin Biomedica S.P.A. | Two-leaflet prosthetic heart valve |
| US5176661A (en) | 1988-09-06 | 1993-01-05 | Advanced Cardiovascular Systems, Inc. | Composite vascular catheter |
| US5024671A (en) | 1988-09-19 | 1991-06-18 | Baxter International Inc. | Microporous vascular graft |
| US5200395A (en) | 1988-10-18 | 1993-04-06 | Ajinomoto Company, Inc. | Pharmaceutical composition of BUF-5 for treating anemia |
| CA2001401A1 (en) | 1988-10-25 | 1990-04-25 | Claude Piantadosi | Quaternary amine containing ether or ester lipid derivatives and therapeutic compositions |
| US5262530A (en) | 1988-12-21 | 1993-11-16 | Applied Biosystems, Inc. | Automated system for polynucleotide synthesis and purification |
| US5047524A (en) | 1988-12-21 | 1991-09-10 | Applied Biosystems, Inc. | Automated system for polynucleotide synthesis and purification |
| US6214804B1 (en) | 1989-03-21 | 2001-04-10 | Vical Incorporated | Induction of a protective immune response in a mammal by injecting a DNA sequence |
| US5703055A (en) | 1989-03-21 | 1997-12-30 | Wisconsin Alumni Research Foundation | Generation of antibodies through lipid mediated DNA delivery |
| FR2645866B1 (fr) | 1989-04-17 | 1991-07-05 | Centre Nat Rech Scient | Nouvelles lipopolyamines, leur preparation et leur emploi |
| US5194654A (en) | 1989-11-22 | 1993-03-16 | Vical, Inc. | Lipid derivatives of phosphonoacids for liposomal incorporation and method of use |
| US5670488A (en) * | 1992-12-03 | 1997-09-23 | Genzyme Corporation | Adenovirus vector for gene therapy |
| US20070053879A1 (en) * | 1990-03-05 | 2007-03-08 | Genzyme Corporation | Diagnostic and treatment methods involving the cystic fibrosis transmembrane regulator |
| US5279833A (en) | 1990-04-04 | 1994-01-18 | Yale University | Liposomal transfection of nucleic acids into animal cells |
| US5101824A (en) | 1990-04-16 | 1992-04-07 | Siemens-Pacesetter, Inc. | Rate-responsive pacemaker with circuitry for processing multiple sensor inputs |
| US5264618A (en) | 1990-04-19 | 1993-11-23 | Vical, Inc. | Cationic lipids for intracellular delivery of biologically active molecules |
| WO1992001425A1 (en) | 1990-07-26 | 1992-02-06 | Rodney James Lane | Self expanding vascular endoprosthesis for aneurysms |
| US5693338A (en) | 1994-09-29 | 1997-12-02 | Emisphere Technologies, Inc. | Diketopiperazine-based delivery systems |
| JPH0765267B2 (ja) | 1990-08-22 | 1995-07-12 | 花王株式会社 | 柔軟仕上剤 |
| US5256294A (en) | 1990-09-17 | 1993-10-26 | Genentech, Inc. | Tangential flow filtration process and apparatus |
| DE9117152U1 (de) | 1990-10-09 | 1996-07-11 | Cook Inc., Bloomington, Ind. | Stent |
| DE59008908D1 (de) | 1990-12-19 | 1995-05-18 | Osypka Peter | Herzschrittmacherleitung mit einem inneren Kanal und mit einem Elektrodenkopf. |
| US5116360A (en) | 1990-12-27 | 1992-05-26 | Corvita Corporation | Mesh composite graft |
| US5405363A (en) | 1991-03-15 | 1995-04-11 | Angelon Corporation | Implantable cardioverter defibrillator having a smaller displacement volume |
| US5330768A (en) | 1991-07-05 | 1994-07-19 | Massachusetts Institute Of Technology | Controlled drug delivery using polymer/pluronic blends |
| US5545449A (en) | 1991-10-02 | 1996-08-13 | Weyerhaeuser Company | Polyether-reinforced fiber-based materials |
| US5151105A (en) | 1991-10-07 | 1992-09-29 | Kwan Gett Clifford | Collapsible vessel sleeve implant |
| US5284491A (en) | 1992-02-27 | 1994-02-08 | Medtronic, Inc. | Cardiac pacemaker with hysteresis behavior |
| US5352461A (en) | 1992-03-11 | 1994-10-04 | Pharmaceutical Discovery Corporation | Self assembling diketopiperazine drug delivery system |
| SE9200951D0 (sv) | 1992-03-27 | 1992-03-27 | Kabi Pharmacia Ab | Pharmaceutical composition containing a defined lipid system |
| EP0635019B1 (en) | 1992-04-06 | 1999-05-26 | Biosite Diagnostics Inc. | Opiate derivatives and protein and polypeptide opiate derivative conjugates and labels |
| US6670178B1 (en) | 1992-07-10 | 2003-12-30 | Transkaryotic Therapies, Inc. | In Vivo production and delivery of insulinotropin for gene therapy |
| WO1994003598A1 (en) | 1992-08-04 | 1994-02-17 | The Green Cross Corporation | Antiallergic agent |
| US5334761A (en) | 1992-08-28 | 1994-08-02 | Life Technologies, Inc. | Cationic lipids |
| US5461223A (en) | 1992-10-09 | 1995-10-24 | Eastman Kodak Company | Bar code detecting circuitry |
| US5300022A (en) | 1992-11-12 | 1994-04-05 | Martin Klapper | Urinary catheter and bladder irrigation system |
| US5496362A (en) | 1992-11-24 | 1996-03-05 | Cardiac Pacemakers, Inc. | Implantable conformal coil patch electrode with multiple conductive elements for cardioversion and defibrillation |
| US5552155A (en) | 1992-12-04 | 1996-09-03 | The Liposome Company, Inc. | Fusogenic lipsomes and methods for making and using same |
| US5716395A (en) | 1992-12-11 | 1998-02-10 | W.L. Gore & Associates, Inc. | Prosthetic vascular graft |
| DE69424406T2 (de) | 1993-02-19 | 2000-10-26 | Nippon Shinyaku Co., Ltd. | Arzneistoffzusammensetzung, die ein nukleinsäurecopolymer enthält |
| US5395619A (en) | 1993-03-03 | 1995-03-07 | Liposome Technology, Inc. | Lipid-polymer conjugates and liposomes |
| US5697953A (en) | 1993-03-13 | 1997-12-16 | Angeion Corporation | Implantable cardioverter defibrillator having a smaller displacement volume |
| US5624976A (en) | 1994-03-25 | 1997-04-29 | Dentsply Gmbh | Dental filling composition and method |
| US5314430A (en) | 1993-06-24 | 1994-05-24 | Medtronic, Inc. | Atrial defibrillator employing transvenous and subcutaneous electrodes and method of use |
| DE4325848A1 (de) | 1993-07-31 | 1995-02-02 | Basf Ag | Verfahren zur Herstellung von N-(2-Hydroxyethyl)-piperazin |
| EP0875280B1 (en) | 1993-10-06 | 2001-08-22 | The Kansai Electric Power Co., Inc. | Method for removing carbon dioxide from combustion exhaust gas |
| US5609624A (en) | 1993-10-08 | 1997-03-11 | Impra, Inc. | Reinforced vascular graft and method of making same |
| SE9303481L (sv) | 1993-10-22 | 1995-04-23 | Berol Nobel Ab | Hygienkomposition |
| AU1091095A (en) | 1993-11-08 | 1995-05-29 | Harrison M. Lazarus | Intraluminal vascular graft and method |
| NZ277614A (en) | 1993-11-24 | 1998-05-27 | Megabios Corp | Piperazine-containing amphiphiles and their use in liposomes for delivering genetic material intracellularly |
| US5595756A (en) | 1993-12-22 | 1997-01-21 | Inex Pharmaceuticals Corporation | Liposomal compositions for enhanced retention of bioactive agents |
| US5464924A (en) | 1994-01-07 | 1995-11-07 | The Dow Chemical Company | Flexible poly(amino ethers) for barrier packaging |
| DE59511013D1 (de) * | 1994-02-11 | 2005-09-22 | Qiagen Gmbh | Verfahren zur Trennung von Doppelstrang/Einzelstrangnukleinsäurestrukturen |
| US5844107A (en) | 1994-03-23 | 1998-12-01 | Case Western Reserve University | Compacted nucleic acids and their delivery to cells |
| EP0758883B1 (en) | 1994-04-12 | 2003-03-19 | The Liposome Company, Inc. | Fusogenic liposomes and methods of making and using same |
| US5840576A (en) | 1994-07-20 | 1998-11-24 | Cytotherapeutics, Inc. | Methods and compositions of growth control for cells encapsulated within bioartificial organs |
| US5820873A (en) | 1994-09-30 | 1998-10-13 | The University Of British Columbia | Polyethylene glycol modified ceramide lipids and liposome uses thereof |
| US5885613A (en) | 1994-09-30 | 1999-03-23 | The University Of British Columbia | Bilayer stabilizing components and their use in forming programmable fusogenic liposomes |
| US5641665A (en) | 1994-11-28 | 1997-06-24 | Vical Incorporated | Plasmids suitable for IL-2 expression |
| US6071890A (en) | 1994-12-09 | 2000-06-06 | Genzyme Corporation | Organ-specific targeting of cationic amphiphile/DNA complexes for gene therapy |
| US5965434A (en) | 1994-12-29 | 1999-10-12 | Wolff; Jon A. | Amphipathic PH sensitive compounds and delivery systems for delivering biologically active compounds |
| US6485726B1 (en) | 1995-01-17 | 2002-11-26 | The Brigham And Women's Hospital, Inc. | Receptor specific transepithelial transport of therapeutics |
| US5830430A (en) | 1995-02-21 | 1998-11-03 | Imarx Pharmaceutical Corp. | Cationic lipids and the use thereof |
| JP2001523215A (ja) | 1995-04-17 | 2001-11-20 | イマークス ファーマシューティカル コーポレーション | ハイブリッド磁気共鳴造影剤 |
| US20010047091A1 (en) | 1998-09-09 | 2001-11-29 | Brian Miki | Cryptic regulatory elements obtained from plants |
| US5772694A (en) | 1995-05-16 | 1998-06-30 | Medical Carbon Research Institute L.L.C. | Prosthetic heart valve with improved blood flow |
| US5700642A (en) | 1995-05-22 | 1997-12-23 | Sri International | Oligonucleotide sizing using immobilized cleavable primers |
| US5783383A (en) | 1995-05-23 | 1998-07-21 | The Board Of Trustees Of The Leland Stanford Junior University | Method of detecting cytomegalovirus (CMV) |
| US7422902B1 (en) | 1995-06-07 | 2008-09-09 | The University Of British Columbia | Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer |
| US5705385A (en) | 1995-06-07 | 1998-01-06 | Inex Pharmaceuticals Corporation | Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer |
| US5609629A (en) | 1995-06-07 | 1997-03-11 | Med Institute, Inc. | Coated implantable medical device |
| US5981501A (en) | 1995-06-07 | 1999-11-09 | Inex Pharmaceuticals Corp. | Methods for encapsulating plasmids in lipid bilayers |
| CA2222328C (en) | 1995-06-07 | 2012-01-10 | Inex Pharmaceuticals Corporation | Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer |
| US5607385A (en) | 1995-08-17 | 1997-03-04 | Medtronic, Inc. | Device and algorithm for a combined cardiomyostimulator and a cardiac pacer-carioverter-defibrillator |
| US5744335A (en) | 1995-09-19 | 1998-04-28 | Mirus Corporation | Process of transfecting a cell with a polynucleotide mixed with an amphipathic compound and a DNA-binding protein |
| FR2740978B1 (fr) | 1995-11-10 | 1998-01-02 | Ela Medical Sa | Dispositif medical actif du type defibrillateur/cardioverteur implantable |
| US5874105A (en) | 1996-01-31 | 1999-02-23 | Collaborative Laboratories, Inc. | Lipid vesicles formed with alkylammonium fatty acid salts |
| DE69730218D1 (de) | 1996-04-11 | 2004-09-16 | Univ British Columbia | Fusogene liposomen |
| US5935936A (en) | 1996-06-03 | 1999-08-10 | Genzyme Corporation | Compositions comprising cationic amphiphiles and co-lipids for intracellular delivery of therapeutic molecules |
| US5913848A (en) | 1996-06-06 | 1999-06-22 | Luther Medical Products, Inc. | Hard tip over-the-needle catheter and method of manufacturing the same |
| US5677124A (en) | 1996-07-03 | 1997-10-14 | Ambion, Inc. | Ribonuclease resistant viral RNA standards |
| US5736573A (en) | 1996-07-31 | 1998-04-07 | Galat; Alexander | Lipid and water soluble derivatives of drugs |
| US7288266B2 (en) | 1996-08-19 | 2007-10-30 | United States Of America As Represented By The Secretary, Department Of Health And Human Services | Liposome complexes for increased systemic delivery |
| EP0941066B1 (en) | 1996-08-26 | 2003-10-29 | Transgene S.A. | Cationic lipid-nucleic acid complexes |
| CA2271388C (en) | 1996-09-13 | 2007-11-06 | The School Of Pharmacy, University Of London | Liposomes encapsulating polynucleotides operatively coding for immunogenic polypeptides |
| US5874268A (en) * | 1996-09-23 | 1999-02-23 | Duke University | Method of introducing exogenous compounds into cells by electroporation and apparatus for same |
| TW520297B (en) | 1996-10-11 | 2003-02-11 | Sequus Pharm Inc | Fusogenic liposome composition and method |
| JP4175667B2 (ja) | 1996-11-04 | 2008-11-05 | キアゲン ゲゼルシャフト ミット ベシュレンクテル ハフツング | トランスフェクションのためのカチオン試薬 |
| US6887665B2 (en) | 1996-11-14 | 2005-05-03 | Affymetrix, Inc. | Methods of array synthesis |
| US5985930A (en) | 1996-11-21 | 1999-11-16 | Pasinetti; Giulio M. | Treatment of neurodegenerative conditions with nimesulide |
| US6204297B1 (en) | 1996-11-26 | 2001-03-20 | Rhodia Inc. | Nonionic gemini surfactants |
| JPH10197978A (ja) | 1997-01-09 | 1998-07-31 | Mitsubishi Paper Mills Ltd | ハロゲン化銀写真感光材料 |
| FR2760193B1 (fr) | 1997-02-28 | 1999-05-28 | Transgene Sa | Lipides et complexes de lipides cationiques et de substances actives, notamment pour la transfection de cellules |
| US5837283A (en) | 1997-03-12 | 1998-11-17 | The Regents Of The University Of California | Cationic lipid compositions targeting angiogenic endothelial cells |
| US5945326A (en) | 1997-03-20 | 1999-08-31 | New England Biolabs, Inc. | Method for cloning and producing the Spel restriction endonuclease |
| EP1027033B1 (en) | 1997-05-14 | 2009-07-22 | The University Of British Columbia | High efficiency encapsulation of nucleic acids in lipid vesicles |
| US6835395B1 (en) | 1997-05-14 | 2004-12-28 | The University Of British Columbia | Composition containing small multilamellar oligodeoxynucleotide-containing lipid vesicles |
| US20030104044A1 (en) | 1997-05-14 | 2003-06-05 | Semple Sean C. | Compositions for stimulating cytokine secretion and inducing an immune response |
| WO1998055512A2 (en) * | 1997-06-02 | 1998-12-10 | Vlaams Interuniversitair Instituut Voor Biotechnologie | Smad-interacting polypeptides and their use |
| JPH115786A (ja) | 1997-06-13 | 1999-01-12 | Pola Chem Ind Inc | 新規アミノヒドロキシプロピルピペラジン誘導体 |
| US6067471A (en) | 1998-08-07 | 2000-05-23 | Cardiac Pacemakers, Inc. | Atrial and ventricular implantable cardioverter-defibrillator and lead system |
| JPH1180142A (ja) | 1997-09-05 | 1999-03-26 | Pola Chem Ind Inc | ジフェニルアルキル化合物の製造法 |
| EP2292771A3 (en) | 1997-09-19 | 2011-07-27 | Life Technologies Corporation | Sense mRNA therapy |
| US6165763A (en) | 1997-10-30 | 2000-12-26 | Smithkline Beecham Corporation | Ornithine carbamoyltransferase |
| US6096075A (en) | 1998-01-22 | 2000-08-01 | Medical Carbon Research Institute, Llc | Prosthetic heart valve |
| US6617171B2 (en) | 1998-02-27 | 2003-09-09 | The General Hospital Corporation | Methods for diagnosing and treating autoimmune disease |
| US6271209B1 (en) | 1998-04-03 | 2001-08-07 | Valentis, Inc. | Cationic lipid formulation delivering nucleic acid to peritoneal tumors |
| US6176877B1 (en) | 1998-04-20 | 2001-01-23 | St. Jude Medical, Inc. | Two piece prosthetic heart valve |
| US6986902B1 (en) | 1998-04-28 | 2006-01-17 | Inex Pharmaceuticals Corporation | Polyanionic polymers which enhance fusogenicity |
| DE19822602A1 (de) | 1998-05-20 | 1999-11-25 | Goldschmidt Ag Th | Verfahren zur Herstellung von Polyaminosäureestern durch Veresterung von sauren Polyaminosäuren oder Umesterung von Polyaminosäureestern |
| NO313244B1 (no) | 1998-07-08 | 2002-09-02 | Crew Dev Corp | Fremgangsmåte for isolering og produksjon av magnesitt eller magnesiumklorid |
| US6055454A (en) | 1998-07-27 | 2000-04-25 | Cardiac Pacemakers, Inc. | Cardiac pacemaker with automatic response optimization of a physiologic sensor based on a second sensor |
| AU771367B2 (en) | 1998-08-20 | 2004-03-18 | Cook Medical Technologies Llc | Coated implantable medical device |
| US6210892B1 (en) | 1998-10-07 | 2001-04-03 | Isis Pharmaceuticals, Inc. | Alteration of cellular behavior by antisense modulation of mRNA processing |
| WO2000030444A1 (en) | 1998-11-25 | 2000-06-02 | Vanderbilt University | Cationic liposomes for gene transfer |
| US6740643B2 (en) * | 1999-01-21 | 2004-05-25 | Mirus Corporation | Compositions and methods for drug delivery using amphiphile binding molecules |
| US6248725B1 (en) | 1999-02-23 | 2001-06-19 | Amgen, Inc. | Combinations and methods for promoting in vivo liver cell proliferation and enhancing in vivo liver-directed gene transduction |
| US6379698B1 (en) | 1999-04-06 | 2002-04-30 | Isis Pharmaceuticals, Inc. | Fusogenic lipids and vesicles |
| CA2370690A1 (en) | 1999-04-20 | 2000-10-26 | The University Of British Columbia | Cationic peg-lipids and methods of use |
| CA2369595C (en) | 1999-04-23 | 2010-10-05 | Alza Corporation | Conjugate having a cleavable linkage for use in a liposome |
| US6169923B1 (en) | 1999-04-23 | 2001-01-02 | Pacesetter, Inc. | Implantable cardioverter-defibrillator with automatic arrhythmia detection criteria adjustment |
| JP2003530070A (ja) | 1999-05-19 | 2003-10-14 | レキシジェン ファーマシューティカルズ コーポレイション | Ｆｃ融合タンパク質としてのインターフェロン−αタンパク質の発現および搬出 |
| US6696424B1 (en) | 1999-05-28 | 2004-02-24 | Vical Incorporated | Cytofectin dimers and methods of use thereof |
| US6346382B1 (en) | 1999-06-01 | 2002-02-12 | Vanderbilt University | Human carbamyl phosphate synthetase I polymorphism and diagnostic methods related thereto |
| WO2001005375A1 (en) | 1999-07-16 | 2001-01-25 | Purdue Research Foundation | Vinyl ether lipids with cleavable hydrophilic headgroups |
| US6358278B1 (en) | 1999-09-24 | 2002-03-19 | St. Jude Medical, Inc. | Heart valve prosthesis with rotatable cuff |
| US6371983B1 (en) | 1999-10-04 | 2002-04-16 | Ernest Lane | Bioprosthetic heart valve |
| US7060291B1 (en) | 1999-11-24 | 2006-06-13 | Transave, Inc. | Modular targeted liposomal delivery system |
| EP1242609A2 (en) | 1999-12-30 | 2002-09-25 | Novartis AG | Novel colloid synthetic vectors for gene therapy |
| EP1261379A2 (en) | 2000-02-17 | 2002-12-04 | Genzyme Corporation | Genetic modification of the lung as a portal for gene delivery |
| US6370434B1 (en) | 2000-02-28 | 2002-04-09 | Cardiac Pacemakers, Inc. | Cardiac lead and method for lead implantation |
| IL152527A0 (en) * | 2000-05-12 | 2003-05-29 | Univ Southern California | Retroviral vectors comprising an enhanced 3'transcription termination structure |
| US6565960B2 (en) | 2000-06-01 | 2003-05-20 | Shriners Hospital Of Children | Polymer composite compositions |
| IL138474A0 (en) | 2000-09-14 | 2001-10-31 | Epox Ltd | Highly branched water-soluble polyamine oligomers, process for their preparation and applications thereof |
| USRE43612E1 (en) | 2000-10-10 | 2012-08-28 | Massachusetts Institute Of Technology | Biodegradable poly(β-amino esters) and uses thereof |
| US7427394B2 (en) | 2000-10-10 | 2008-09-23 | Massachusetts Institute Of Technology | Biodegradable poly(β-amino esters) and uses thereof |
| US6998115B2 (en) | 2000-10-10 | 2006-02-14 | Massachusetts Institute Of Technology | Biodegradable poly(β-amino esters) and uses thereof |
| US7202226B2 (en) * | 2000-10-23 | 2007-04-10 | Detroit R & D | Augmentation of wound healing by elF-4E mRNA and EGF mRNA |
| CA2426244A1 (en) | 2000-10-25 | 2002-05-02 | The University Of British Columbia | Lipid formulations for target delivery |
| US20020094528A1 (en) | 2000-11-29 | 2002-07-18 | Salafsky Joshua S. | Method and apparatus using a surface-selective nonlinear optical technique for detection of probe-target interations |
| JP2002167368A (ja) | 2000-12-01 | 2002-06-11 | Nitto Denko Corp | アルキル置換デンドリマーおよびその製造法 |
| US20050004058A1 (en) | 2000-12-07 | 2005-01-06 | Patrick Benoit | Sequences upstream of the carp gene, vectors containing them and uses thereof |
| EP1366196A4 (en) | 2001-02-14 | 2004-07-07 | Baylor College Medicine | METHOD AND COMPOSITIONS FOR AMPLIFICATING RNA |
| DE10109897A1 (de) | 2001-02-21 | 2002-11-07 | Novosom Ag | Fakultativ kationische Liposomen und Verwendung dieser |
| US20020192721A1 (en) | 2001-03-28 | 2002-12-19 | Engeneos, Inc. | Modular molecular clasps and uses thereof |
| US7084303B2 (en) | 2001-04-23 | 2006-08-01 | Shin-Etsu Chemical Co., Ltd. | Tertiary amine compounds having an ester structure and processes for preparing same |
| US6585410B1 (en) | 2001-05-03 | 2003-07-01 | The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration | Radiant temperature nulling radiometer |
| EP2305699B1 (de) | 2001-06-05 | 2014-08-13 | CureVac GmbH | Stabilisierte mRNA mit erhöhtem G/C-Gehalt und optimierter Codon Usage für die Impfung gegen Schlafkrankheit, Leishmaniose und Toxoplasmose |
| EP2428571B1 (en) | 2001-09-28 | 2018-07-18 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | MicroRNA molecules |
| AU2002211719B2 (en) * | 2001-10-12 | 2007-06-14 | Qiagen North American Holdings, Inc. | Compositions and methods for using a solid support to purify RNA |
| AU2002340490A1 (en) | 2001-11-09 | 2003-05-19 | Bayer Healthcare Ag | Isotopically coded affinity markers 3 |
| DE10162480A1 (de) | 2001-12-19 | 2003-08-07 | Ingmar Hoerr | Die Applikation von mRNA für den Einsatz als Therapeutikum gegen Tumorerkrankungen |
| DE10207178A1 (de) | 2002-02-19 | 2003-09-04 | Novosom Ag | Komponenten für die Herstellung amphoterer Liposomen |
| AU2003220249A1 (en) * | 2002-03-15 | 2003-09-29 | Arcturus Bioscience, Inc. | Improved nucleic acid amplification |
| DE10214983A1 (de) | 2002-04-04 | 2004-04-08 | TransMIT Gesellschaft für Technologietransfer mbH | Vernebelbare Liposomen und ihre Verwendung zur pulmonalen Applikation von Wirkstoffen |
| GB0208390D0 (en) | 2002-04-11 | 2002-05-22 | Univ London | Adeno-associated virus producer system |
| US20030215395A1 (en) | 2002-05-14 | 2003-11-20 | Lei Yu | Controllably degradable polymeric biomolecule or drug carrier and method of synthesizing said carrier |
| US7601367B2 (en) | 2002-05-28 | 2009-10-13 | Mirus Bio Llc | Compositions and processes using siRNA, amphipathic compounds and polycations |
| DK1519714T3 (da) | 2002-06-28 | 2011-01-31 | Protiva Biotherapeutics Inc | Fremgangsmåde og apparat til fremstilling af liposomer |
| DE10229872A1 (de) | 2002-07-03 | 2004-01-29 | Curevac Gmbh | Immunstimulation durch chemisch modifizierte RNA |
| US20040028804A1 (en) | 2002-08-07 | 2004-02-12 | Anderson Daniel G. | Production of polymeric microarrays |
| CA2495535A1 (en) | 2002-08-22 | 2004-03-04 | Celltran Limited | Cell culture surface |
| US20060160759A1 (en) * | 2002-09-28 | 2006-07-20 | Jianzhu Chen | Influenza therapeutic |
| CA2504910A1 (en) | 2002-11-04 | 2004-05-21 | Ge Bayer Silicones Gmbh & Co. Kg | Linear polyamino and/or polyammonium polysiloxane copolymers i |
| AU2003281978A1 (en) | 2002-11-22 | 2004-06-18 | Boehringer Ingelheim International Gmbh | 2,5-diketopiperazines for the treatment of obesity |
| US7169892B2 (en) | 2003-01-10 | 2007-01-30 | Astellas Pharma Inc. | Lipid-peptide-polymer conjugates for long blood circulation and tumor specific drug delivery systems |
| EP1586983B1 (en) | 2003-01-20 | 2013-06-05 | Asahi Kasei EMD Corporation | Pointing device |
| NZ542665A (en) | 2003-03-05 | 2008-05-30 | Senesco Technologies Inc | Use of antisense oligonucleotides or siRNA to suppress expression of eIF-5A1 |
| US20040224912A1 (en) | 2003-05-07 | 2004-11-11 | Isis Pharmaceuticals Inc. | Modulation of PAI-1 mRNA-binding protein expression |
| US7619017B2 (en) | 2003-05-19 | 2009-11-17 | Wacker Chemical Corporation | Polymer emulsions resistant to biodeterioration |
| US7507859B2 (en) | 2003-06-16 | 2009-03-24 | Fifth Base Llc | Functional synthetic molecules and macromolecules for gene delivery |
| US20050059024A1 (en) | 2003-07-25 | 2005-03-17 | Ambion, Inc. | Methods and compositions for isolating small RNA molecules |
| EP1675943A4 (en) | 2003-09-15 | 2007-12-05 | Massachusetts Inst Technology | NANOLITRE SYNTHESIS OF BIOMATERIALS IN NETWORKS AND SCREENING THEREOF |
| US7803397B2 (en) | 2003-09-15 | 2010-09-28 | Protiva Biotherapeutics, Inc. | Polyethyleneglycol-modified lipid compounds and uses thereof |
| US20050069590A1 (en) | 2003-09-30 | 2005-03-31 | Buehler Gail K. | Stable suspensions for medicinal dosages |
| MXPA06003978A (es) | 2003-10-10 | 2006-06-27 | Powderject Vaccines Inc | Constructos de acidos nucleicos. |
| WO2005037226A2 (en) | 2003-10-17 | 2005-04-28 | Georgia Tech Research Corporation | Genetically engineered enteroendocrine cells for treating glucose-related metabolic disorders |
| AU2004287652A1 (en) | 2003-11-10 | 2005-05-19 | Nippon Kayaku Kabushiki Kaisha | Diimonium salt compound and use thereof |
| US8075780B2 (en) * | 2003-11-24 | 2011-12-13 | Millipore Corporation | Purification and concentration of synthetic biological molecules |
| EP1694692B1 (en) * | 2003-12-16 | 2010-04-28 | QIAGEN North American Holdings, Inc. | Formulations and methods for denaturing proteins |
| US7022214B2 (en) | 2004-01-21 | 2006-04-04 | Bio-Rad Laboratories, Inc. | Carrier ampholytes of high pH range |
| US7556684B2 (en) | 2004-02-26 | 2009-07-07 | Construction Research & Technology Gmbh | Amine containing strength improvement admixture |
| US20060228404A1 (en) | 2004-03-04 | 2006-10-12 | Anderson Daniel G | Compositions and methods for treatment of hypertrophic tissues |
| JP2006271201A (ja) * | 2004-03-26 | 2006-10-12 | Fuji Photo Film Co Ltd | Ｒｎａの選択的分離精製方法 |
| EP1727900A4 (en) | 2004-03-26 | 2008-05-28 | Fujifilm Corp | METHOD FOR SELECTIVELY SEPARATING AND CLEANING RNA, AND METHOD FOR SEPARATING AND CLEANING NUCLEIC ACID |
| ATE537263T1 (de) | 2004-06-07 | 2011-12-15 | Protiva Biotherapeutics Inc | Kationische lipide und verwendungsverfahren |
| AU2005252273B2 (en) | 2004-06-07 | 2011-04-28 | Arbutus Biopharma Corporation | Lipid encapsulated interfering RNA |
| US7670595B2 (en) | 2004-06-28 | 2010-03-02 | Merck Patent Gmbh | Fc-interferon-beta fusion proteins |
| GB0418172D0 (en) | 2004-08-13 | 2004-09-15 | Ic Vec Ltd | Vector |
| US7361465B2 (en) | 2004-09-07 | 2008-04-22 | Applera Corporation | Methods and compositions for tailing and amplifying RNA |
| DE102004043342A1 (de) | 2004-09-08 | 2006-03-09 | Bayer Materialscience Ag | Blockierte Polyurethan-Prepolymere als Klebstoffe |
| AU2005300638A1 (en) | 2004-11-05 | 2006-05-11 | Novosom Ag | Improvements in or relating to pharmaceutical compositions comprising an oligonucleotide as an active agent |
| CA2595594C (en) * | 2005-01-31 | 2012-05-01 | Merck & Co., Inc. | Upstream and a downstream purification process for large scale production of plasmid dna |
| GB0502482D0 (en) | 2005-02-07 | 2005-03-16 | Glaxo Group Ltd | Novel compounds |
| JP5042863B2 (ja) | 2005-02-14 | 2012-10-03 | サーナ・セラピューティクス・インコーポレイテッド | 生物学的に活性な分子をデリバリーするための脂質ナノ粒子系組成物および方法 |
| US7977452B2 (en) | 2005-03-28 | 2011-07-12 | Dendritic Nanotechnologies, Inc. | Janus dendrimers and dendrons |
| US9006487B2 (en) | 2005-06-15 | 2015-04-14 | Massachusetts Institute Of Technology | Amine-containing lipids and uses thereof |
| RU2008104411A (ru) * | 2005-08-05 | 2009-09-10 | Пфайзер Продактс Инк. (Us) | Автоматизированное серийное производство |
| SI3611266T1 (sl) | 2005-08-23 | 2023-02-28 | The Trustees Of The University Of Pennsylvania | Modificirani nukleozidi, ki vsebujejo RNA, in postopki za njihovo uporabo |
| US9012219B2 (en) | 2005-08-23 | 2015-04-21 | The Trustees Of The University Of Pennsylvania | RNA preparations comprising purified modified RNA for reprogramming cells |
| WO2007031091A2 (en) | 2005-09-15 | 2007-03-22 | Santaris Pharma A/S | Rna antagonist compounds for the modulation of p21 ras expression |
| US8101741B2 (en) | 2005-11-02 | 2012-01-24 | Protiva Biotherapeutics, Inc. | Modified siRNA molecules and uses thereof |
| US7238791B1 (en) | 2005-12-16 | 2007-07-03 | Roche Diagnostics Operations, Inc. | 6-monoacetylmorphine derivatives useful in immunoassay |
| WO2007073489A2 (en) | 2005-12-22 | 2007-06-28 | Trustees Of Boston University | Molecules for gene delivery and gene therapy, and methods of use thereof |
| CN100569877C (zh) | 2005-12-30 | 2009-12-16 | 财团法人工业技术研究院 | 含多uv交联反应基的分枝状结构化合物及其应用 |
| AU2007238624B2 (en) | 2006-04-14 | 2012-05-31 | Cellscript, Llc | Kits and methods for generating 5' capped RNA |
| US9085778B2 (en) | 2006-05-03 | 2015-07-21 | VL27, Inc. | Exosome transfer of nucleic acids to cells |
| US20070275923A1 (en) | 2006-05-25 | 2007-11-29 | Nastech Pharmaceutical Company Inc. | CATIONIC PEPTIDES FOR siRNA INTRACELLULAR DELIVERY |
| EP2032652A4 (en) | 2006-06-05 | 2011-08-17 | Massachusetts Inst Technology | NETWORKED DEVELOPABLE POLYMERS AND ITS USE |
| US20080102493A1 (en) | 2006-06-29 | 2008-05-01 | Millipore Corporation | Isolation of RNA and DNA from a biological sample |
| US20090186805A1 (en) | 2006-07-06 | 2009-07-23 | Aaron Thomas Tabor | Compositions and Methods for Genetic Modification of Cells Having Cosmetic Function to Enhance Cosmetic Appearance |
| ES2293834B1 (es) | 2006-07-20 | 2009-02-16 | Consejo Superior Investig. Cientificas | Compuesto con actividad inhibidora de las interacciones ubc13-uev, composiciones farmaceuticas que lo comprenden y sus aplicaciones terapeuticas. |
| WO2008011561A2 (en) | 2006-07-21 | 2008-01-24 | Massachusetts Institute Of Technology | End-modified poly(beta-amino esters) and uses thereof |
| KR101129509B1 (ko) | 2006-10-03 | 2012-04-13 | 알닐람 파마슈티칼스 인코포레이티드 | 지질 함유 조성물 |
| DE102006051516A1 (de) | 2006-10-31 | 2008-05-08 | Curevac Gmbh | (Basen-)modifizierte RNA zur Expressionssteigerung eines Proteins |
| WO2008140615A2 (en) | 2006-12-21 | 2008-11-20 | Novozymes, Inc. | Modified messenger rna stabilizing sequences for expressing genes in bacterial cells |
| DE102007001370A1 (de) | 2007-01-09 | 2008-07-10 | Curevac Gmbh | RNA-kodierte Antikörper |
| EP2125030A2 (en) | 2007-01-29 | 2009-12-02 | Medipol S.A. | Chitosan-based colloidal particles for rna delivery |
| US8859229B2 (en) | 2007-02-02 | 2014-10-14 | Yale University | Transient transfection with RNA |
| WO2008113364A2 (en) | 2007-03-20 | 2008-09-25 | Recepticon Aps | Amino derivatives to prevent nephrotoxicity and cancer |
| JP5186126B2 (ja) | 2007-03-29 | 2013-04-17 | 公益財団法人地球環境産業技術研究機構 | 新規トリアジン誘導体ならびにその製法およびそのガス分離膜としての用途 |
| PT2144609E (pt) | 2007-04-18 | 2015-02-06 | Cornerstone Pharmaceuticals Inc | Formulações farmacêuticas contendo derivados de ácido lipóico |
| WO2008137470A1 (en) | 2007-05-01 | 2008-11-13 | Pgr-Solutions | Multi-chain lipophilic polyamines |
| US20090163705A1 (en) | 2007-05-21 | 2009-06-25 | Alnylam Pharmaceuticals, Inc. | Cationic lipids |
| NZ584048A (en) | 2007-10-02 | 2012-08-31 | Marina Biotech Inc | Lipopeptides for delivery of nucleic acids |
| CA3043911A1 (en) | 2007-12-04 | 2009-07-02 | Arbutus Biopharma Corporation | Targeting lipids |
| ES2535419T3 (es) | 2007-12-27 | 2015-05-11 | Protiva Biotherapeutics Inc. | Silenciamiento de expresión de quinasa tipo polo usando ARN interferente |
| CA2710983A1 (en) | 2007-12-27 | 2009-10-01 | The Ohio State University Research Foundation | Lipid nanoparticle compositions and methods of making and using the same |
| AU2008347250A1 (en) | 2008-01-02 | 2009-07-16 | Tekmira Pharmaceuticals Corporation | Screening method for selected amino lipid-containing compositions |
| FR2926560A1 (fr) | 2008-01-21 | 2009-07-24 | Millipore Corp | Procede d'extraction et de purification d'acides nucleiques sur membrane |
| JP5788312B2 (ja) | 2008-04-11 | 2015-09-30 | アルニラム ファーマスーティカルズ インコーポレイテッドＡｌｎｙｌａｍ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． | 標的リガンドをエンドソーム分解性成分と組み合わせることによる核酸の部位特異的送達 |
| HUE034483T2 (en) | 2008-04-15 | 2018-02-28 | Protiva Biotherapeutics Inc | New lipid preparations for introducing a nucleic acid |
| US20090263407A1 (en) | 2008-04-16 | 2009-10-22 | Abbott Laboratories | Cationic Lipids and Uses Thereof |
| WO2009127230A1 (en) | 2008-04-16 | 2009-10-22 | Curevac Gmbh | MODIFIED (m)RNA FOR SUPPRESSING OR AVOIDING AN IMMUNOSTIMULATORY RESPONSE AND IMMUNOSUPPRESSIVE COMPOSITION |
| CA2726052A1 (en) | 2008-06-04 | 2009-12-10 | The Board Of Regents Of The University Of Texas System | Modulation of gene expression through endogenous small rna targeting of gene promoters |
| US20100012012A1 (en) | 2008-07-19 | 2010-01-21 | Mark Schaefbauer | Positioning apparatus |
| JP5024216B2 (ja) | 2008-07-23 | 2012-09-12 | トヨタ自動車株式会社 | 内燃機関の点火時期制御装置及び点火時期制御方法 |
| US20100035249A1 (en) | 2008-08-05 | 2010-02-11 | Kabushiki Kaisha Dnaform | Rna sequencing and analysis using solid support |
| DK2331954T3 (da) | 2008-08-27 | 2020-04-06 | Life Technologies Corp | Apparat og fremgangsmåde til processering af biologiske prøver |
| JP2010053108A (ja) * | 2008-08-29 | 2010-03-11 | Asahi Kasei Corp | 荷電を有する限外濾過膜を用いた生体成分の分離方法およびモジュール、装置 |
| EP2350043B9 (en) | 2008-10-09 | 2014-08-20 | TEKMIRA Pharmaceuticals Corporation | Improved amino lipids and methods for the delivery of nucleic acids |
| US20100112042A1 (en) | 2008-10-16 | 2010-05-06 | Mdrna, Inc. | Processes and Compositions for Liposomal and Efficient Delivery of Gene Silencing Therapeutics |
| US9080211B2 (en) | 2008-10-24 | 2015-07-14 | Epicentre Technologies Corporation | Transposon end compositions and methods for modifying nucleic acids |
| US20120009222A1 (en) | 2008-10-27 | 2012-01-12 | Massachusetts Institute Of Technology | Modulation of the immune response |
| WO2010053108A1 (ja) | 2008-11-05 | 2010-05-14 | 株式会社 日立メディコ | 位相シフト型インバータ回路、それを用いたｘ線高電圧装置、ｘ線ｃｔ装置、および、ｘ線撮影装置 |
| CA2742954C (en) | 2008-11-07 | 2018-07-10 | Massachusetts Institute Of Technology | Aminoalcohol lipidoids and uses thereof |
| SG10201901089TA (en) | 2008-11-10 | 2019-03-28 | Arbutus Biopharma Corp | Novel lipids and compositions for the delivery of therapeutics |
| TW201019969A (en) | 2008-11-17 | 2010-06-01 | Enzon Pharmaceuticals Inc | Branched cationic lipids for nucleic acids delivery system |
| JP2012511531A (ja) | 2008-12-09 | 2012-05-24 | エフ．ホフマン−ラ ロシュ アーゲー | 賦形剤不含抗体溶液を得るための方法 |
| US9023820B2 (en) | 2009-01-26 | 2015-05-05 | Protiva Biotherapeutics, Inc. | Compositions and methods for silencing apolipoprotein C-III expression |
| US20100222489A1 (en) | 2009-02-27 | 2010-09-02 | Jiang Dayue D | Copolymer composition, membrane article, and methods thereof |
| AU2010223967B2 (en) | 2009-03-12 | 2015-07-30 | Alnylam Pharmaceuticals, Inc. | Lipid formulated compositions and methods for inhibiting expression of Eg5 and VEGF genes |
| CN102334237B (zh) | 2009-04-02 | 2015-05-13 | 西蒙公司 | 电信接线板 |
| CN102497887A (zh) | 2009-04-17 | 2012-06-13 | Isis创新公司 | 递送遗传物质的组合物 |
| EP2421563B1 (en) * | 2009-04-22 | 2017-04-12 | Massachusetts Institute of Technology | Innate immune suppression enables repeated delivery of long rna molecules |
| SG10201402054UA (en) | 2009-05-05 | 2014-09-26 | Muthiah Manoharan | Lipid compositions |
| HUE056773T2 (hu) | 2009-06-10 | 2022-03-28 | Arbutus Biopharma Corp | Továbbfejlesztett lipid készítmény |
| US8273869B2 (en) | 2009-06-15 | 2012-09-25 | Alnylam Pharmaceuticals, Inc. | Lipid formulated dsRNA targeting the PCSK9 gene |
| US9051567B2 (en) | 2009-06-15 | 2015-06-09 | Tekmira Pharmaceuticals Corporation | Methods for increasing efficacy of lipid formulated siRNA |
| US9018187B2 (en) | 2009-07-01 | 2015-04-28 | Protiva Biotherapeutics, Inc. | Cationic lipids and methods for the delivery of therapeutic agents |
| US8569256B2 (en) | 2009-07-01 | 2013-10-29 | Protiva Biotherapeutics, Inc. | Cationic lipids and methods for the delivery of therapeutic agents |
| US8236943B2 (en) | 2009-07-01 | 2012-08-07 | Protiva Biotherapeutics, Inc. | Compositions and methods for silencing apolipoprotein B |
| WO2011011447A1 (en) | 2009-07-20 | 2011-01-27 | Protiva Biotherapeutics, Inc. | Compositions and methods for silencing ebola virus gene expression |
| ES2727711T3 (es) | 2009-07-30 | 2019-10-18 | Spiral Therapeutics Inc | Compuestos inhibidores de Apaf-1 |
| EP3581197A1 (de) | 2009-07-31 | 2019-12-18 | ethris GmbH | Rna mit einer kombination aus unmodifizierten und modifizierten nucleotiden zur proteinexpression |
| JP5769173B2 (ja) | 2009-08-19 | 2015-08-26 | 国立大学法人 奈良先端科学技術大学院大学 | 環境ストレス下の翻訳抑制を回避する５’ｕｔｒをコードする組換えｄｎａ分子 |
| DE102009043342A1 (de) | 2009-09-29 | 2011-03-31 | Bayer Technology Services Gmbh | Stoffe für selbstorganisierte Träger zur kontrollierten Freisetzung eines Wirkstoffs |
| JP5465331B2 (ja) * | 2009-10-15 | 2014-04-09 | クルセル ホランド ベー ヴェー | 高細胞密度の培養物からのアデノウイルスの精製方法 |
| PL3338765T3 (pl) | 2009-12-01 | 2019-06-28 | Translate Bio, Inc. | Pochodna steroidowa dla dostarczania mrna w ludzkich chorobach genetycznych |
| SI3112467T1 (en) | 2009-12-07 | 2018-06-29 | The Trustees Of The University Of Pennsylvania | RNA preparations comprising purified modified RNA for reprogramming cells |
| AU2010330814B2 (en) | 2009-12-18 | 2017-01-12 | Acuitas Therapeutics Inc. | Methods and compositions for delivery of nucleic acids |
| BR112012015755B1 (pt) | 2009-12-23 | 2021-06-22 | Novartis Ag | Lipídeo furtivo, e composição |
| US9315802B2 (en) | 2009-12-30 | 2016-04-19 | Quest Diagnostics Investments Incorporated | RNA isolation from soluble urine fractions |
| US20130123338A1 (en) | 2010-05-12 | 2013-05-16 | Protiva Biotherapeutics, Inc. | Novel cationic lipids and methods of use thereof |
| IL300109A (en) | 2010-06-03 | 2023-03-01 | Alnylam Pharmaceuticals Inc | Biodegradable lipids for the transfer of active substances |
| CN101863544B (zh) | 2010-06-29 | 2011-09-28 | 湖南科技大学 | 一种氰尿酸基重金属螯合絮凝剂及其制备方法 |
| WO2012000104A1 (en) | 2010-06-30 | 2012-01-05 | Protiva Biotherapeutics, Inc. | Non-liposomal systems for nucleic acid delivery |
| US20130323269A1 (en) | 2010-07-30 | 2013-12-05 | Muthiah Manoharan | Methods and compositions for delivery of active agents |
| WO2012019168A2 (en) | 2010-08-06 | 2012-02-09 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
| WO2012019630A1 (en) | 2010-08-13 | 2012-02-16 | Curevac Gmbh | Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded protein |
| US9193827B2 (en) | 2010-08-26 | 2015-11-24 | Massachusetts Institute Of Technology | Poly(beta-amino alcohols), their preparation, and uses thereof |
| WO2012026493A1 (ja) | 2010-08-26 | 2012-03-01 | アイシン精機株式会社 | 酵素結晶固定化電極及び酵素結晶固定化電極の製造方法、並びに酵素結晶固定化電極を備えるバイオ電池及びバイオセンサー |
| WO2012031294A2 (en) | 2010-09-03 | 2012-03-08 | University Of Maryland, College Park | Methods for determining protein ligand binding |
| TWI441591B (zh) * | 2010-09-07 | 2014-06-21 | Univ Nat Central | 使植物表現後天性高溫逆境耐受性狀的方法及其應用 |
| CA2821992A1 (en) | 2010-10-01 | 2012-04-05 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
| US8853377B2 (en) | 2010-11-30 | 2014-10-07 | Shire Human Genetic Therapies, Inc. | mRNA for use in treatment of human genetic diseases |
| PL2691443T3 (pl) | 2011-03-28 | 2021-08-30 | Massachusetts Institute Of Technology | Sprzężone lipomery i ich zastosowania |
| WO2012133737A1 (ja) | 2011-03-31 | 2012-10-04 | 公益財団法人地球環境産業技術研究機構 | 架橋性アミン化合物、該化合物を用いた高分子膜及びその製造方法 |
| DE12722942T1 (de) | 2011-03-31 | 2021-09-30 | Modernatx, Inc. | Freisetzung und formulierung von manipulierten nukleinsäuren |
| WO2012151503A2 (en) | 2011-05-04 | 2012-11-08 | The Broad Institute, Inc. | Multiplexed genetic reporter assays and compositions |
| AU2012255913A1 (en) | 2011-05-17 | 2013-11-21 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof for non-human vertebrates |
| EP2532649B1 (en) | 2011-06-07 | 2015-04-08 | Incella GmbH | Amino lipids, their synthesis and uses thereof |
| EP3336082B1 (en) | 2011-06-08 | 2020-04-15 | Translate Bio, Inc. | Cleavable lipids |
| CN111671918A (zh) | 2011-06-08 | 2020-09-18 | 川斯勒佰尔公司 | Mrna递送的脂质纳米颗粒组合物和方法 |
| WO2013003475A1 (en) | 2011-06-27 | 2013-01-03 | Cellscript, Inc. | Inhibition of innate immune response |
| US8980583B2 (en) * | 2011-06-30 | 2015-03-17 | Sigma-Aldrich Co. Llc | Cells deficient in CMP-N-acetylneuraminic acid hydroxylase and/or glycoprotein alpha-1,3-galactosyltransferase |
| US9464124B2 (en) | 2011-09-12 | 2016-10-11 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
| WO2013039857A1 (en) | 2011-09-12 | 2013-03-21 | modeRNA Therapeutics | Engineered nucleic acids and methods of use thereof |
| EP3384938A1 (en) | 2011-09-12 | 2018-10-10 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
| EP3492109B1 (en) * | 2011-10-03 | 2020-03-04 | ModernaTX, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
| EP2764007A4 (en) | 2011-10-05 | 2015-05-06 | Protiva Biotherapeutics Inc | COMPOSITIONS AND METHODS FOR DISABLING ALDEHYDE DEHYDROGENASE |
| CA3119789A1 (en) | 2011-10-27 | 2013-05-02 | Massachusetts Institute Of Technology | Amino acid derivatives functionalized on the n-terminal capable of forming drug encapsulating microspheres |
| WO2013090186A1 (en) | 2011-12-14 | 2013-06-20 | modeRNA Therapeutics | Modified nucleic acids, and acute care uses thereof |
| US20140378538A1 (en) | 2011-12-14 | 2014-12-25 | Moderma Therapeutics, Inc. | Methods of responding to a biothreat |
| JP2015501844A (ja) | 2011-12-16 | 2015-01-19 | モデルナ セラピューティクス インコーポレイテッドＭｏｄｅｒｎａ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． | 修飾ヌクレオシド、ヌクレオチドおよび核酸組成物 |
| AU2012358384A1 (en) | 2011-12-21 | 2014-07-31 | Moderna Therapeutics, Inc. | Methods of increasing the viability or longevity of an organ or organ explant |
| US20140371302A1 (en) | 2011-12-29 | 2014-12-18 | Modema Therapeutics, Inc. | Modified mrnas encoding cell-penetrating polypeptides |
| CA2862377A1 (en) | 2011-12-30 | 2013-07-04 | Cellscript, Llc | Making and using in vitro-synthesized ssrna for introducing into mammalian cells to induce a biological or biochemical effect |
| JP6275655B2 (ja) | 2012-02-24 | 2018-02-07 | プロティバ バイオセラピューティクス インコーポレイテッド | トリアルキルカチオン性脂質およびその使用方法 |
| CA2868030C (en) | 2012-03-29 | 2021-05-25 | Shire Human Genetic Therapies, Inc. | Lipid-derived neutral nanoparticles |
| AU2013237873B2 (en) | 2012-03-29 | 2017-12-14 | Translate Bio, Inc. | Ionizable cationic lipids |
| US9572897B2 (en) | 2012-04-02 | 2017-02-21 | Modernatx, Inc. | Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins |
| EP2833923A4 (en) | 2012-04-02 | 2016-02-24 | Moderna Therapeutics Inc | MODIFIED POLYNUCLEOTIDES FOR THE PRODUCTION OF PROTEINS |
| US9283287B2 (en) | 2012-04-02 | 2016-03-15 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of nuclear proteins |
| US20150050354A1 (en) | 2012-04-02 | 2015-02-19 | Moderna Therapeutics, Inc. | Modified polynucleotides for the treatment of otic diseases and conditions |
| US20140275229A1 (en) | 2012-04-02 | 2014-09-18 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding udp glucuronosyltransferase 1 family, polypeptide a1 |
| US9878056B2 (en) | 2012-04-02 | 2018-01-30 | Modernatx, Inc. | Modified polynucleotides for the production of cosmetic proteins and peptides |
| AU2013243949A1 (en) | 2012-04-02 | 2014-10-30 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of biologics and proteins associated with human disease |
| AU2013271392B2 (en) | 2012-06-08 | 2018-02-15 | Ethris Gmbh | Pulmonary delivery of mRNA to non-lung target cells |
| US20150267192A1 (en) | 2012-06-08 | 2015-09-24 | Shire Human Genetic Therapies, Inc. | Nuclease resistant polynucleotides and uses thereof |
| EP2674167A1 (en) | 2012-06-14 | 2013-12-18 | PLS-Design GmbH | Controlled activation of complement components for use as endogenous adjuvant |
| WO2014028487A1 (en) | 2012-08-13 | 2014-02-20 | Massachusetts Institute Of Technology | Amine-containing lipidoids and uses thereof |
| US20140093952A1 (en) | 2012-10-02 | 2014-04-03 | David Serway | Bioreactor Tangential Flow Perfusion Filter System |
| US20150307542A1 (en) | 2012-10-03 | 2015-10-29 | Moderna Therapeutics, Inc. | Modified nucleic acid molecules and uses thereof |
| NZ708523A (en) * | 2012-11-21 | 2018-09-28 | Serum Institute Of India Pvt Ltd | Production of high yields of bacterial polysaccharides |
| LT2922554T (lt) | 2012-11-26 | 2022-06-27 | Modernatx, Inc. | Terminaliai modifikuota rnr |
| WO2014089486A1 (en) | 2012-12-07 | 2014-06-12 | Shire Human Genetic Therapies, Inc. | Lipidic nanoparticles for mrna delivering |
| WO2014093574A1 (en) | 2012-12-13 | 2014-06-19 | Moderna Therapeutics, Inc. | Modified polynucleotides for altering cell phenotype |
| CN110106229B (zh) * | 2013-01-03 | 2023-03-28 | 外来体诊断公司 | 用于分离微囊泡的方法 |
| WO2014113089A2 (en) | 2013-01-17 | 2014-07-24 | Moderna Therapeutics, Inc. | Signal-sensor polynucleotides for the alteration of cellular phenotypes |
| EP2968397A4 (en) | 2013-03-12 | 2016-12-28 | Moderna Therapeutics Inc | DIAGNOSIS AND TREATMENT OF FIBROSIS |
| US20160024181A1 (en) | 2013-03-13 | 2016-01-28 | Moderna Therapeutics, Inc. | Long-lived polynucleotide molecules |
| EP2971010B1 (en) | 2013-03-14 | 2020-06-10 | ModernaTX, Inc. | Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions |
| EA201591293A1 (ru) | 2013-03-14 | 2016-02-29 | Шир Хьюман Дженетик Терапис, Инк. | Способы и композиции для доставки антител, кодируемых мрнк |
| AU2014236305B2 (en) | 2013-03-14 | 2019-01-17 | Ethris Gmbh | CFTR mRNA compositions and related methods and uses |
| WO2014152940A1 (en) | 2013-03-14 | 2014-09-25 | Shire Human Genetic Therapies, Inc. | Mrna therapeutic compositions and use to treat diseases and disorders |
| ES2708561T3 (es) * | 2013-03-14 | 2019-04-10 | Translate Bio Inc | Métodos para la purificación de ARN mensajero |
| JP2016514970A (ja) | 2013-03-14 | 2016-05-26 | シャイアー ヒューマン ジェネティック セラピーズ インコーポレイテッド | メッセンジャーｒｎａのキャップ効率の定量的評価 |
| ES2797974T3 (es) | 2013-03-14 | 2020-12-04 | Translate Bio Inc | Acidos ribonucleicos con nucleótidos modificados con 4-tio y procedimientos relacionados |
| EP2971098B1 (en) | 2013-03-14 | 2018-11-21 | Translate Bio, Inc. | Quantitative assessment for cap efficiency of messenger rna |
| US10077439B2 (en) | 2013-03-15 | 2018-09-18 | Modernatx, Inc. | Removal of DNA fragments in mRNA production process |
| ES2923018T3 (es) * | 2013-03-15 | 2022-09-22 | Glaxosmithkline Biologicals Sa | Métodos de purificación de ARN |
| EP3757570B1 (en) | 2013-03-15 | 2023-10-11 | Translate Bio, Inc. | Synergistic enhancement of the delivery of nucleic acids via blended formulations |
| US20160032273A1 (en) | 2013-03-15 | 2016-02-04 | Moderna Therapeutics, Inc. | Characterization of mrna molecules |
| WO2014144711A1 (en) | 2013-03-15 | 2014-09-18 | Moderna Therapeutics, Inc. | Analysis of mrna heterogeneity and stability |
| EP2983804A4 (en) | 2013-03-15 | 2017-03-01 | Moderna Therapeutics, Inc. | Ion exchange purification of mrna |
| US8980864B2 (en) | 2013-03-15 | 2015-03-17 | Moderna Therapeutics, Inc. | Compositions and methods of altering cholesterol levels |
| EP4279610A3 (en) | 2013-03-15 | 2024-01-03 | ModernaTX, Inc. | Ribonucleic acid purification |
| EP2971033B8 (en) * | 2013-03-15 | 2019-07-10 | ModernaTX, Inc. | Manufacturing methods for production of rna transcripts |
| WO2014179562A1 (en) | 2013-05-01 | 2014-11-06 | Massachusetts Institute Of Technology | 1,3,5-triazinane-2,4,6-trione derivatives and uses thereof |
| WO2014210356A1 (en) | 2013-06-26 | 2014-12-31 | Massachusetts Institute Of Technology | Multi-tailed lipids and uses thereof |
| JP7019233B2 (ja) | 2013-07-11 | 2022-02-15 | モデルナティエックス インコーポレイテッド | ＣＲＩＳＰＲ関連タンパク質をコードする合成ポリヌクレオチドおよび合成ｓｇＲＮＡを含む組成物ならびに使用方法 |
| CN110974981A (zh) | 2013-07-23 | 2020-04-10 | 野草莓树生物制药公司 | 用于递送信使rna的组合物和方法 |
| WO2015034925A1 (en) | 2013-09-03 | 2015-03-12 | Moderna Therapeutics, Inc. | Circular polynucleotides |
| EP3041934A1 (en) | 2013-09-03 | 2016-07-13 | Moderna Therapeutics, Inc. | Chimeric polynucleotides |
| WO2015048744A2 (en) | 2013-09-30 | 2015-04-02 | Moderna Therapeutics, Inc. | Polynucleotides encoding immune modulating polypeptides |
| US20160264614A1 (en) | 2013-10-02 | 2016-09-15 | Moderna Therapeutics, Inc. | Polynucleotide molecules and uses thereof |
| EP3052511A4 (en) | 2013-10-02 | 2017-05-31 | Moderna Therapeutics, Inc. | Polynucleotide molecules and uses thereof |
| US20160243221A1 (en) | 2013-10-18 | 2016-08-25 | Moderna Therapeutics, Inc. | Compositions and methods for tolerizing cellular systems |
| JP6506749B2 (ja) | 2013-10-22 | 2019-04-24 | シャイアー ヒューマン ジェネティック セラピーズ インコーポレイテッド | フェニルケトン尿症のためのｍＲＮＡ療法 |
| CN106413811A (zh) | 2013-10-22 | 2017-02-15 | 夏尔人类遗传性治疗公司 | 精氨基琥珀酸合成酶缺乏症的mrna疗法 |
| CN105658800A (zh) | 2013-10-22 | 2016-06-08 | 夏尔人类遗传性治疗公司 | Mrna的cns递送及其用途 |
| US20170173128A1 (en) | 2013-12-06 | 2017-06-22 | Moderna TX, Inc. | Targeted adaptive vaccines |
| US20150167017A1 (en) | 2013-12-13 | 2015-06-18 | Moderna Therapeutics, Inc. | Alternative nucleic acid molecules and uses thereof |
| US20170002060A1 (en) | 2014-01-08 | 2017-01-05 | Moderna Therapeutics, Inc. | Polynucleotides for the in vivo production of antibodies |
| SG10201912038TA (en) | 2014-04-23 | 2020-02-27 | Modernatx Inc | Nucleic acid vaccines |
| ES2750661T3 (es) * | 2014-04-25 | 2020-03-26 | Translate Bio Inc | Métodos para la purificación de ARN mensajero |
| JP6347163B2 (ja) | 2014-07-10 | 2018-06-27 | 株式会社リコー | 定着装置及び画像形成装置 |
| EP3201338B1 (en) | 2014-10-02 | 2021-11-03 | Arbutus Biopharma Corporation | Compositions and methods for silencing hepatitis b virus gene expression |
| WO2016071857A1 (en) | 2014-11-07 | 2016-05-12 | Protiva Biotherapeutics, Inc. | Compositions and methods for silencing ebola virus expression |
| WO2016077125A1 (en) | 2014-11-10 | 2016-05-19 | Moderna Therapeutics, Inc. | Alternative nucleic acid molecules containing reduced uracil content and uses thereof |
| US20170362627A1 (en) | 2014-11-10 | 2017-12-21 | Modernatx, Inc. | Multiparametric nucleic acid optimization |
| EP3247363A4 (en) | 2015-01-21 | 2018-10-03 | Moderna Therapeutics, Inc. | Lipid nanoparticle compositions |
| EP3247398A4 (en) | 2015-01-23 | 2018-09-26 | Moderna Therapeutics, Inc. | Lipid nanoparticle compositions |
| US20180245077A1 (en) | 2015-03-20 | 2018-08-30 | Protiva Biotherapeutics, Inc. | Compositions and methods for treating hypertriglyceridemia |
| WO2016164762A1 (en) | 2015-04-08 | 2016-10-13 | Moderna Therapeutics, Inc. | Polynucleotides encoding low density lipoprotein receptor egf-a and intracellular domain mutants and methods of using the same |
| WO2016183366A2 (en) | 2015-05-12 | 2016-11-17 | Protiva Biotherapeutics, Inc. | Compositions and methods for silencing expression of hepatitis d virus rna |
| EP4108769B1 (en) * | 2015-05-29 | 2023-08-30 | CureVac Manufacturing GmbH | A method for producing and purifying rna, comprising at least one step of tangential flow filtration |
| US10626393B2 (en) | 2015-06-04 | 2020-04-21 | Arbutus Biopharma Corporation | Delivering CRISPR therapeutics with lipid nanoparticles |
| US20180245074A1 (en) | 2015-06-04 | 2018-08-30 | Protiva Biotherapeutics, Inc. | Treating hepatitis b virus infection using crispr |
| EP3307305A4 (en) | 2015-06-10 | 2019-05-22 | Modernatx, Inc. | TARGETED ADAPTIVE VACCINES |
| WO2017149139A1 (en) | 2016-03-03 | 2017-09-08 | Curevac Ag | Rna analysis by total hydrolysis |
| IL268857B2 (en) * | 2017-02-27 | 2024-09-01 | Translate Bio Inc | Methods for the purification of messenger RNA |
| CN113396219A (zh) | 2019-02-11 | 2021-09-14 | 埃泽瑞斯公司 | 通过切向流过滤纯化mRNA |
-
2014
- 2014-03-14 ES ES14714150T patent/ES2708561T3/es active Active
- 2014-03-14 MX MX2015012865A patent/MX2015012865A/es unknown
- 2014-03-14 DK DK14714150.1T patent/DK2970955T3/en active
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- 2014-03-14 JP JP2016502790A patent/JP6586075B2/ja active Active
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- 2014-03-14 WO PCT/US2014/028441 patent/WO2014152966A1/en active Application Filing
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- 2014-03-14 CN CN201480013665.4A patent/CN105051190A/zh active Pending
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- 2014-03-14 CA CA2902892A patent/CA2902892A1/en not_active Abandoned
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- 2015-07-29 IL IL240215A patent/IL240215A0/en unknown
-
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- 2018-03-26 US US15/936,289 patent/US10876104B2/en active Active
- 2018-08-20 JP JP2018154032A patent/JP6941084B2/ja active Active
-
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- 2020-11-24 US US17/103,439 patent/US20210324368A1/en active Pending
-
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- 2021-09-03 JP JP2021143908A patent/JP7166407B2/ja active Active
-
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- 2022-06-08 US US17/835,725 patent/US11692189B2/en active Active
- 2022-06-08 US US17/835,719 patent/US20220348898A1/en active Pending
- 2022-06-08 US US17/835,710 patent/US11820977B2/en active Active
- 2022-06-08 US US17/835,738 patent/US20220348899A1/en active Pending
- 2022-10-25 JP JP2022170547A patent/JP2023012495A/ja active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998005673A1 (en) * | 1996-08-01 | 1998-02-12 | Megabios Corporation | Methods for purifying nucleic acids |
| EP0853123A1 (de) * | 1997-01-10 | 1998-07-15 | Roche Diagnostics GmbH | Reinigung von DNA durch Cross-Flow-Filtration |
| WO2001007599A1 (en) * | 1999-07-23 | 2001-02-01 | Genentech, Inc. | Method for rnase- and organic solvent-free plasmid dna purification using tangential flow filtration |
| WO2002042317A2 (en) * | 2000-11-21 | 2002-05-30 | Glaxo Group Limited | Method of separating extra-chromosomal dna from other cellular components |
| WO2012077080A1 (en) * | 2010-12-10 | 2012-06-14 | Tracy Thompson | Compositions for separation methods |
Non-Patent Citations (1)
| Title |
|---|
| FERNANDEZ等: "Cross flow filtration of RNA by Hollow filber membrane", 《ACTA BIOTECHNOL》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112930396A (zh) * | 2018-08-24 | 2021-06-08 | 川斯勒佰尔公司 | 用于纯化信使rna的方法 |
| CN112930396B (zh) * | 2018-08-24 | 2024-05-24 | 川斯勒佰尔公司 | 用于纯化信使rna的方法 |
| CN113166790A (zh) * | 2018-11-08 | 2021-07-23 | 川斯勒佰尔公司 | 信使rna纯化的方法和组合物 |
| CN114127283A (zh) * | 2019-05-15 | 2022-03-01 | 川斯勒佰尔公司 | 用于纯化信使rna的方法 |
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