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    • 5. 发明申请
    • MINI-ADENOVIRAL VECTOR SYSTEM FOR VACCINATION
    • 用于疫苗接种的微型腺病毒载体系统
    • WO0208436A3
    • 2003-02-27
    • PCT/US0123005
    • 2001-07-20
    • GENSTAR THERAPEUTICS CORPZHANG WEI-WEIALEMANY RAMONDAI YIFANJOSEPHS STEVENBALAGUE CRISTINAAYARES DAVIDSCHNEIDERMAN RICHARD
    • ZHANG WEI-WEIALEMANY RAMONDAI YIFANJOSEPHS STEVENBALAGUE CRISTINAAYARES DAVIDSCHNEIDERMAN RICHARD
    • A61K39/00A61K48/00C07K14/755C12N15/12C12N15/16C12N15/19C12N15/861
    • C12N15/86A61K39/00A61K48/00A61K2039/5256C07K14/755C12N2710/10343C12N2800/108C12N2800/30C12N2810/859C12N2830/008C12N2830/42C12N2830/85C12N2840/203C12N2840/206
    • The principle of this invention is to produce mini-Ad vectors using packaging-attenuated and replication-defective helper Ad and an E1-complementing Ad helper cell line. Since the essential cis-acting elements for Ad DNA replication and packaging are located at the ends of the viral genome (ITRs plus the packaging signal, less than 1 kb), the backbone of the mini-Ad vectors is trimmed down to contain only the essential cis -elements. The remainder of the Ad genome is to be replaced by non-viral DNA for gene transfer, retention, and expression. The capacity of the mini-Ad vectors may be up to 36 kb. The viral proteins required for DNA replication and encapsidation of the mini-Ad vectors are designed to be provided in trans from the helper Ad ( trans )-complementation) and the helper cell line. In order to generate relatively pure preparations of the mini-Ad virions, packaging of the helper Ad genome is controlled by selective attenuation of the packaging signal while that of the mini-Ad vector genome proceeds normally. In the absence of mini-Ad vector genome, the helper Ad does replicate and is propagated in an inefficient manner. In this system, the Ad helper cell line is necessary for the E1-complementation of the helper virus and may also provide other functions for packaging attenuation of the helper viral genome and enhancement of mini-Ad vector replication. If the preparation of mini-Ad vectors is not sufficiently pure, biochemical or other physical methods will be utilized to achieve further purification.
    • 本发明的原理是使用包装减毒和复制缺陷型辅助Ad和E1互补的Ad辅助细胞系来生产小型Ad载体。 由于用于Ad DNA复制和包装的基本顺式作用元件位于病毒基因组的末端(ITRs加上包装信号小于1kb),微型Ad载体的主链被修剪成仅包含 重要的顺式元件。 Ad基因组的其余部分将被用于基因转移,保留和表达的非病毒DNA替代。 小型Ad载体的容量可达36kb。 设计mini-Ad载体的DNA复制和壳化所需的病毒蛋白质是从辅助Ad(反式) - 互补)和辅助细胞系反向提供的。 为了产生相对纯的微型Ad病毒粒子的制剂,通过选择性衰减包装信号来控制辅助Ad基因组的包装,而迷你Ad载体基因组的包装正常进行。 在没有mini-Ad载体基因组的情况下,辅助Ad不会复制并以低效的方式传播。 在该系统中,Ad辅助细胞系对辅助病毒的E1互补是必需的,并且还可以提供用于包装辅助病毒基因组衰减和增强小型Ad载体复制的其他功能。 如果微型Ad载体的制备不够纯,将采用生物化学或其他物理方法来进一步纯化。