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    • 5. 发明申请
    • TISSUE CULTURE SYSTEM FOR PRODUCTION OF HEPATITIS C VIRUS
    • 生产丙型肝炎病毒的组织培养系统
    • WO2008024413A2
    • 2008-02-28
    • PCT/US2007/018591
    • 2007-08-22
    • NOVARTIS AGCHOO, Qui-LimHAN, JangHOUGHTON, MichaelKWON, TaewooSONG, Hyun ChulZHU, Yifei
    • CHOO, Qui-LimHAN, JangHOUGHTON, MichaelKWON, TaewooSONG, Hyun ChulZHU, Yifei
    • C12N15/861
    • C12N7/00A61K2039/525C07K14/005C07K2319/61C12N2760/16022C12N2770/24243G01N2333/186G01N2500/10
    • A tissue culture system for production of infectious hepatitis C virus is described. In particular, the invention provides recombinant monocistronic and bicistronic genomic constructs for production of virus, including constructs for production of wild-type HCV type 2a strain JFH1 and constructs for production of chimeric viruses comprising HCV proteins from strain JFH1 and a second HCV isolate (e.g., type 1a H77, type 1b Con1, or type 3a NZ1 or 452). Constructs of the invention also include a reporter gene to facilitate measurement of RNA replication and viral infectivity in cultures. The cell culture system may also include various factors that improve viral replication or infectivity, such as an RNA silencing suppressor (e.g., an influenza virus NS1 polypeptide) to inhibit host defense pathways or CD81 to facilitate viral entry into host cells. In addition, a neutralization assay using HCV grown in cell culture is described and neutralization titers measured by this assay have been found to correlate well with actual protection from HCV infection in vaccinated animals.
    • 描述了用于产生传染性丙型肝炎病毒的组织培养系统。 具体而言,本发明提供了用于产生病毒的重组单顺反子和双顺反子基因组构建体,包括用于生产野生型HCV 2a型菌株JFH1的构建体和用于产生包含来自菌株JFH1和第二HCV分离株(例如, ,类型1a H77,类型1b Con1或类型3a NZ1或452)。 本发明的构建体还包括报道基因以促进测量培养物中的RNA复制和病毒感染性。 细胞培养系统还可以包括改善病毒复制或感染性的各种因子,例如抑制宿主防御途径的RNA沉默抑制子(例如流感病毒NS1多肽)或促进病毒进入宿主细胞的CD81。 另外,描述了使用在细胞培养物中生长的HCV的中和测定法,并且发现通过该测定法测量的中和滴度与在接种疫苗的动物中实际保护免受HCV感染相关联。
    • 6. 发明申请
    • MODIFIED SMALL INTERFERING RNA MOLECULES AND METHODS OF USE
    • 修饰的小分子干扰RNA分子及其使用方法
    • WO2006039656A2
    • 2006-04-13
    • PCT/US2005/035493
    • 2005-09-30
    • CHIRON CORPORATIONHAN, JangHOUGHTON, Michael
    • HAN, JangHOUGHTON, Michael
    • C12N15/113
    • C12N15/1131A61K38/00C12N2310/14C12N2310/322C12N2310/334C12N2310/335C12N2310/3515Y02A50/463
    • The present invention provides double-stranded RNA molecules that mediate RNA interference in target cells, preferably hepatic cells. The invention also provides double-stranded RNA (dsRNA) molecules that are modified to be resistant to nuclease degradation, which inactivates a virus, and more specifically, hepatitis C virus (HCV). The invention also provides a method of using these modified RNA molecules to inactivate virus in mammalian cells and a method of making modified small interfering RNAs (siRNAs) using human Dicer. The invention provides modified RNA molecules that are modified to include a dsRNA or siRNA wherein one or more of the pyrimidines in the RNA molecule are modified to include 2'-Fluorine. The invention also provides dsRNA or siRNA in which all pyrimidines are modified to include a 2'-Fluorine. The invention provides that the 2'-Fluorine dsRNA or siRNA molecule is further modified to include a two base deoxynucleotide "TT" sequence at the 3' end of the molecule.
    • 本发明提供了在靶细胞,优选肝细胞中介导RNA干扰的双链RNA分子。 本发明还提供经修饰以抵抗核酸酶降解的双链RNA(dsRNA)分子,其使病毒失活,并且更具体地使丙型肝炎病毒(HCV)失活。 本发明还提供了使用这些修饰的RNA分子使哺乳动物细胞中的病毒失活的方法以及使用人类切丁酶制备修饰的小干扰RNA(siRNA)的方法。 本发明提供了修饰的RNA分子,其被修饰以包括dsRNA或siRNA,其中RNA分子中的一个或多个嘧啶被修饰以包括2'-氟。 本发明还提供了其中所有嘧啶被修饰为包含2'-氟的dsRNA或siRNA。 本发明提供了将2'-氟dsRNA或siRNA分子进一步修饰以包括双碱基脱氧核苷酸“TT” 序列在分子的3'末端。